International Review of Cytology: A Survey of Cell Biology, Volume 180

VOLUME 180

SERIES EDITORS Geoffrey H. Bourne James F. Danielli Kwang W. Jeon Martin Friedlander Jonathan Jarvik

1949-1 988 1949-1 984 19671984-1 992 1993-1 995

EDITORIAL ADVISORY BOARD Aimee Bakken Eve Ida Barak Rosa Beddington Howard A. Bern Robert A. Bloodgood Dean Bok Stanley Cohen Rene Couteaux Marie A. DiBerardino Charles J. Flickinger Hiroo Fukuda Elizabeth D. Hay P. Mark Hogarth Anthony P.Mahowald

M. Melkonian Keith E. Mostov Andreas Oksche Vladimir R. Pantic L. Evans Roth Jozef St. Schell Manfred Schliwa Wilfred 0. Stein Ralph M. Steinman M. Tazawa Yoshio Watanabe Donald P. Weeks Robin Wright Alexander L. Yudin

Edited by

Kwang W. Jeon Department of Biochemistry University of Tennessee Knoxville, Tennessee

VOLUME 180

ACADEMIC PRESS San Diego London Boston New York

Sydney Tokyo Toronto

Fronr cover photogrph: The cytoplasm of mouse basophils shows large numbers of perigranular vesicles. (For more details, see Chapter 3, Figure 6a.)

This book is printed o n acid-free paper.

@

Copyright 0 1998 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (222 Rosewood Drive, Danvers, Massachusetts 01923), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-1998 chapters are as shown on the title pages. If no fee code appears on the title page, the copy ee is the same as for current chapters. 0074-7696/98 $25.00

Academic Press u division of Hurcourt Bruce & Company

525 B Street, Suite 1900, San Diego, v92101-4495, USA http://www.apnet.com Academic Press Limited 24-28 Oval Road, London NWI 7DX, UK http://www.hbuk.co.uk/ap/ International Standard Book Number: 0-12-364584-0 PRINTED IN THE UNTIED STATES OF AMEIUCA 98 99 0 0 0 1 02 0 3 E B 9 8 7 6

5

4

3 2 1

Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii

Gene Expression during Amphibian Limb Regeneration Jacqueline Geraudie and Patrizia Ferretti I. II. 111. IV. V.

.................................. Introduction .......... Molecules Regulated duri .............................. Growth Control in the Blastema . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genes Controlling Morphogenesis in the Regenerating Limb ..................... Concluding Remarks ..................................... References ....................................

1 4

18

23

Biochemistry of the Extracellular Matrix of Volvox Manfred Sumper and Armin Hallmann I. 11. 111. IV. V. VI.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ultrastructure of the Volvox ECM . . . . . . . . . Biochemical Characterization of ECM Components ECM Biogenesis and Remodeling , ..................................... ................ Relationship to Higher Plant ECMs . . . . . . . .

.................................................... ....................................

51 54 56 62 74

75 79

Cell Biology of the Basophil Ann M. Dvorak I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Basophil Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

87 90

vi

CONTENTS

Ill. IV. V. VI. VII. VIII. IX. X. XI. XII.

Basophils in Disease . . . . . . . . . . . ................................... Basophils as Secretory Cells . . . . ......... ............... Development of Tools for Advanced Cell-Biological Studies of Basophils , , , , , , , , , , , Vesicles as Prominent Transport Organelles in the Cytoplasm of Basophils . . . . . . . . . Proof of a Degranulation Model Identified in Human Basophils in 1975 . . . . . . Charcot-Leyden Crystal Protein Distributionin Actively Degranulating Human Basophils Histamine Distribution in FMLP4timulated Human Basophil Granules . . . . . . . . . . . . . .............. Recovery of Basophils from Secretion . . Morphometric Analysis of Basophil Degra Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

97 98 110 122

157 192

212 214

Membrane Receptors for Endocytosis in the Renal Proximal Tubule Erik Its0 Christensen, Henrik Birn, Pierre Verroust, and S ~ r e nK. Moestrup I. 11. 111. IV. V. VI. VII.

Introduction , . , . . , . . , , , , , , , , . , . , , . , . , , , , , , . , , , , . , , , , . , , , , . , , . , . , , . , . , , . Ultrastructure of the Endocytic Apparatus in the Proximal Tubule . . . . . . . . . . . . . . . . . Megalin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . gp28Ollntrinsic Factor Receptor (IFR) . . . . . . . . . . . . . . . . IGF-IIIMan-6-P Receptor., , , . Folate Receptor. . . . . . . . . . . . . . . . . . ............. Concluding Remarks . . . . . . . . ....................... References . . . . . . . . . . . . . . . . . . . ...............................

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

237 239 243

272 272

285

CONTRIBUTORS

Number in parentheses indicate the pages on which the authors' contributions begin.

Henrik Birn (237), Department of Cell BioIogy, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark Erik Its0 Christensen(237), Department of CeII Biology, Institute ofAnatomy, University of Aarhus, DK-8000 Aarhus C, Denmark Ann M. Dvorak (87), Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 022 15 Patrizia Ferretti (1), Developmental Biology Unit, Institute of Child Health, London WClN IEH, United Kingdom Jacqueline Geraudie (1), Laboratoire de Biologie du Developpement, Universite Paris 7, Denis Diderot Case 7077, 75251 Paris Cedex 05, France Arrnin Hallman (51), Lehrstuhl Biochemie I, Universitat Regensberg, 0-93053 Regensberg, Germany Ssren K. Moestrup (237), Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark Manfred Sumper (51), Lehrstuhl Biochemie I, Universitat Regensberg, 0-93053 Regensberg, Germany Pierre Verroust (237), INSERM U64, Hljpital Tenon4, F-75970 Paris Cedex20, France

vii

This Page Intentionally Left Blank

Gene Expression during Amphibian Limb Regeneration‘ Jacqueline Geraudie* and Patrizia Ferrettit *Laboratoirede Biologie du DCveloppement, UniversitC Pans 7, Case 7077 75251 Paris Cedex 05, France; and tDevelopmenta1 Biology Unit, Institute of Child Health, UCL, London WClN IEH, United Kingdom

Limb regeneration in adult urodeles is an important phenomenon that poses fundamental questions both in biology and in medicine. In this review, we focus on recent advances in the characterization of the regeneration blastema at cellular and molecular levels and on the current understanding of the molecular basis of limb regeneration and its relationship to development. In particular, we discuss (i) the spatiotemporal distribution of genes and gene products in the mesenchyme and wound epidermis of the regenerating limb, (ii) how growth is controlled in the regeneration blastema, and (iii) molecules that are likely to be involved in patterning the regenerating limb such as homeobox genes and retinoids. KEY WORDS: Blastema, Regeneration, Limb, Urodeles, Extracellular matrix, Growth factors, Cytoskeleton, Homeobox, Retinoids, Wound epidermis.

1. Introduction Limb regeneration involves a series of hierarchical events leading to the replacement of the missing part and can be described as a developmental event induced within the context of an adult animal. It was Spallanzani who, in 1768, brought to the attention of the scientific community his striking observations on the regenerative ability of the legs of aquatic salamanders and asked the fundamental question, “how comes it to pass that other land animals are not endowed with the same power?” Despite the many approaches that have been taken to examine and manipulate the system since Spallanzani’sinitial observations, the regenerative process has

’ This work is dedicated to the memory of Michele Gtraudie. Infernorional Review of Cvrology, Vol. 180

0074-76%/98$25.00

1

Copyright 8 1998 by Academic Press. All rights of reproduction in any form reserved.

2

JACQUELINE GERAUDIE AND PATRlZlA FERRETTI

retained much of its mysteries. It is hoped that these will be unveiled in a not-too-distant future thanks to the multidisciplinary approach and new methodologies in cell and molecular biology that are now applied to the study of this remarkable phenomenon. The stereotyped sequence of morphological and cytological events that give rise to the newly formed limb has been widely reviewed (Singer, 1952; Thornton, 1968; Iten and Bryant, 1973; Wallace, 1981; Sicard, 1985; Tsonis, 1991,1996; Stocum, 1995) and will be briefly reiterated here. In this review, we will be mainly concerned with recent advances in the characterization of the regeneration blastema at the molecular level and the current understanding of the molecular basis of limb regeneration. The molecular characterization of regeneration blastemas started about 14 years ago when the then novel monoclonal antibody (mAb) technology was used to produce a number of valuable reagents directed against developmentally regulated antigens in the mesenchymal progenitor cells of regenerating limbs (blastemal cells) and in the specialized overlying wound epidermis (Kintner and Brockes, 1984; Tassava et al., 1986). Subsequently, construction and screening of several limb blastema libraries has allowed identification and analysis of the spatiotemporal pattern of expression of several genes. Novel approaches to elucidate the functional significance of some of these genes (Schilthuis et al., 1993; Pecorino et al., 1994, 1996), together with the attempt to identify useful mutants (Del Rio-Tsonis et al., 1992) and produce transgenic urodeles (Y. Le Parco, personal communication), have been recently developed. Analysis of the expression and regulation of genes and gene products associated with limb regeneration, and its comparison with limb bud development, will help to answer a question central to the phenomenon of limb regeneration: To what extent are the molecular signals governing limb development and morphogenesis reiterated during limb regeneration and to what extent does regeneration set its own developmental program? Regeneration of an adult limb is mainly studied using aquatic urodeles (Duellman and Trueb, 1986), such as newts (Notophthalmus, Pleurodeles, and Cynops) and axolotls (Ambystoma),because they display the highest regenerative capability. Amputation or severe tissue damage are the injuries that trigger regeneration and initiate regrowth and morphogenesis, leading to replacement of the lost part (Singer, 1952). The basic events that occur following amputation are epithelialization of the cut surface and formation of a specialized wound epidermis, accumulation of blastemal cells by dedifferentiation of stump tissue cells, proliferation, redifferentiation, and morphogenesis. Closure of the wound is the first process that occurs following limb amputation. Initially, wound healing of the cut surface of the stump is accomplished by migration of stump keratinocytes from the edges of the

GENE EXPRESSION DURING AMPHIBIAN LIMB REGENERATION

3

wound toward its center (Hay and Fishman, 1961; Repesh and Oberpriller, 1980), as observed following skin injury of other body parts. However, wound healing in the stump skin, besides occurring without any scarring, is not simply another example of skin repair. The wound epidermis of regenerating limbs does not rapidly form a basement membrane, like in injured skin in other vertebrates, but rather maintains a direct contact with the underlying mesenchyme (Salpeter and Singer, 1960). This is believed to be important for the extensive remodeling, growth, and patterning of the underlying mesenchymal tissues observed during regeneration. Identification and characterization of the molecular mechanisms controlling formation and maintenance of the limb wound epidermis is important in order to understand how the wound epidermis affects, and is affected by, the underlying mesenchyme. It may also help to gain insights into why skin injury induces scarring in certain parts of the urodele body but not in the regenerating limb. Following reepitheliahation of the wound, a limited histolysis of the stump tissues is observed. This is carried out through activation of several enzymes and remodeling of the extracellular matrix architecture, which modifies the local organization of the tissues (Schmidt, 1968). White blood cells (granulocytes, lymphocytes, and macrophages) invade the injured region and are involved in clearing cell debris and, as a consequence, edema formation can occur. Blastemal cells start to be released from the stump at this stage. The origin of the progenitor cells has been widely studied, and it has been shown that, with the exception of the epidermis, all stump tissues, including dermis, muscle, bone cartilage and nerve, are a source of blastemal cells (Wallace, 1981; Liversage, 1991; Ferretti and Brockes, 1991). Accumulation of blastemal cells is believed to occur through a process of "dedifferentiation" whereby mature differentiated cells lose their differentiated phenotype and reenter the cell cycle. Alternatively, reserve cells present in the stump might be recruited to form a blastema following amputation. However, there is no clear evidence for the existence of such a cell population in stump tissues. A possible exception is the muscle, in which cells with myogenic potential located outside the muscle basement membrane, the postsatellite cells, have been described (Cameron et al., 1986). However, the issue of the origin of progenitor cells in the adult limb, and of how they are activated and released to form the blastema, has not been fully elucidated, although the use of modern methodology has allowed progress toward this goal in the past few years (Brockes, 1994, 1998). Blastemal cells start to proliferate about 3 or 4 days after amputation and their division is under the control of growth factors originating from the peripheral nervous system, the wound epidermis, and possibly the blastemal cells themselves. The blastema develops steadily through stages defined as

4

JACQUELINE GERAUDIE AND PATRlZlA FERRETTI

early, medium, and late bud (Singer, 1952). Cell differentiation begins after the late bud stage and proceeds in a proximal to distal direction through palette (or paddle), notch, and digit stage (Fig. 1). Complete morphological and functional reconstruction of the missing part is achieved between 6 and 10 weeks postamputation, depending on the species, the age of the animal, and the temperature at which the animal is kept. For clarity of presentation, we will discuss changes in gene expression in the wound epidermis (WE) and mesenchyme separately. However, it is important to remember that the functions of WE and blastemal cells are closely integrated (Fig. 2).

II. Molecules Regulated during Regeneration

A. Wound Epidermis The WE of the regenerating adult limb, like that of the developing limb in urodeles, does not form a prominent ridge. In this respect, it is different from the apical ectodermal ridge of the chick limb bud. However, in common with the apical ectodermal ridge is the fact that both epithelia do not have a basal membrane, and both are in direct contact with the underlying mesenchyme. One day after amputation, cells in all layers of the WE contain [3H]thymidine (Riddiford, 1960; Hay and Fishman, 1961), a feature not found in normal epidermis in which stem cells are located in the basal (germinative) layer (Barrandon, 1993). Thymidine incorporation, however,

FIG. 1 Successive stages of regeneration of forelimbs of Ambystoma maculatum larvae amputated at the mid-humerus level and analyzed in longitudinal tissue sections (A-F) and whole mount skeletal preparations stained for cartilage with methylene blue (G and H). The rate of regeneration depends on the temperature, on the urodele species studied and is faster in larvae than in adults. The sequence of events during limb regeneration, however, is essentially the same in all urodeles. (A) Within 5 days after amputation, a few blastemal cells have accumulated beneath the WE at the tip of the damaged stump tissues. (B) Early bud blastema stage. ( C ) Medium bud blastema stage. (D) Late bud stage. (E) Early redifferentiation stage: Note that cartilage is differentiating at the tip of the humerus and cartilage condensation of radius and ulna is beginning. (F) Notch stage (two digits). The carpals and the first two fingers have started to differentiate. A basement membrane has become visible between the epidermis covering the regenerate and the underlying tissues. (G) Three-finger stage. X, unsegmented carpal mesenchyme; d l and d2, carpals articulating with digits 1 and 2. (H) Four-finger stage. All carpals have differentiated. r, carpal radiale; u, carpal ulnare; cl, carpal centrale; d3 and d4, carpals articulating with digits 3 and 4. The first three digits are already fully shaped (photographs provided by D. Stocum).

-_

,/a....

1-3 days preblastema

WE4 (actin-binding protein) WE6 (cytoskeletal protein) 9G 1 collagen XI1 fibronectin tenascin

__ -

metalloproteinases GAGS hyaluronate 22/18 (cytoskeletal protein) 22/31 (vimentin) Hox sonic hedgehog -bandedhedgehog hyaluronidase collagen laminin ST1 -CRABP I same expression as at 1-3d WE3 (actin-binding protein) NvKlI (type I1cytokeratin) KGFR 1 17C-I neuropepbdes

-

4-7 days early blastema

same expression as at 1-3d NvK8 (cytokeratin) NvK18 (cytokeratin) NCAM tenascin fibronectin type I and XI1 collagen FGFRl

-

8-20 days blastemdcone FIG. 2 Drawing summarizing changes in gene and protein expression in the WE and blastemal cells of regenerating urodele limbs at different times after amputation. The day range indicated at each stage reflects the differences in rate of regeneration between species and serves only as a rough guide.

GENE EXPRESSION DURING AMPHIBIAN LIMB REGENERATION

7

is very seldom observed at later stages of regeneration in the thickened WE of the limb. The WE has phagocytic activity and also has a role in the cytolysis of the stump tissues, which may favor the release of blastema cells (Singer and Salpeter, 1961; Thornton, 1968; Stocum, 1985). It is well established that inhibition of the regenerative process can be produced by removal of the WE, by covering the cut surface with a skin flap of normal epidermis, or by sawing the skin edges at the amputation site (Stocum, 1985). This is similar to the effect induced by stripping the apical ectodermal ridge of chick and frog limb buds in that it impairs limb growth and produces truncated limbs (Saunders, 1948; Tschumi, 1957). However, whereas the apical ectodermal ridge does not regenerate, the WE regrows very rapidly following its removal. The presence of this specialized epidermis and an adequate nerve supply provide the conditions necessary for initiating the process of recruitment of progenitor cells from the mature tissue of the stump and their proliferation. Changes in the morphology of the WE are reflected by significant changes in the molecular composition of the cells in its different layers. Early cytochemical studies by Schmidt (1968) have demonstrated that the WE synthetizes a variety of enzymes such as alkaline and acid phosphatases, nucleoside phosphatases, lactate dehydrogenase, and esterases. A significant increase in our knowledge of the molecular differences between normal epidermis, epidermis of the developing limb buds, and WE, however, was obtained only when specific antibodies and cDNA/RNA probes were developed. The molecular differences between the WE and the normal epidermis are summarized in Table I, and their possible role in the regenerate outgrowth is discussed below. Homeobox-containing genes expressed in the epidermis, such as members of the dlx family, and some of the molecules that are upregulated both in the WE and in blastemal cells, such as NCAM, tenascin, RARS1, will be discussed later (Fig. 2). The antigen identified by mAb WE3 is expressed in a few small round cells in the epidermis, in cells of the integumentary glands, and in some internal tissues of the adult Nofophfhalmusviridescens (Tassava et aZ., 1986; Goldhamer et aZ., 1989). WE3 is first detected in the basal layers of the WE during the second week after amputation (Tassava etal., 1986). Reactivity of mAb WE3 is not species specific because a positive signal is also observed in the WE of regenerating Pleurodeles walfl limbs (Tassava et al., 1993). Upregulation and maintenance of expression of WE3 are correlated with the increase in mitotic activity of the underlying progenitor cells: Its expression is high during the period of blastemal cells accumulation and growth and is downregulated when the regenerate reaches the digit stage (Fig. 1). WE3 reactivity, which is initially granular in appearance, becomes filamentous and associated with the cytoskeleton during blastemal growth (Goldhamer et aZ., 1989). It has been shown that this mAb recognizes a

8

JACQUELINE GERAUDIE AND PATRIZIA FERRETTI

TABLE I Molecules Expressed in the Limb Wound Epidermis (WE) in the Newt, Nofophthalmus viridescens (Unless Otherwise Specified)

Molecule

Detection

Normal limb epidermis

Reference

-

mAb WE3 Actin-binding glycoprotein WE3 (putative carbonic anhydrase)

Some round cells Goldhamer et al. (1989), Tassava et al. (1993), (particularly mitochondriaTassava and Acton (1989) rich cells) Negative

Castilla and Tassava, (1992), Tassava et a/. (1993)

mAb WE6 Cytoskeletonassociated protein WE6 9G1, intracellular mAb 9G1 (axolotl) component (not yet characterized)

Some cells

Estrada et al. (1992)

Not reported

Onda and Tassava (1991)

NvKII type I1 epidermal keratin

Negative

Ferretti et al. (1989, 1993), Ferretti and Ghosh (1997)

mAb 117C1 117C intracellular component (not yet characterized)

Very low levels

Koshiba et al. (1994)

KGFR (variant of FGFR2)

KGFR riboprobe

Very low levels

Poulin et a/. (1993), Poulin and Chiu (1995)

Substance P

Polyclonal antiserum Not reported

Globus and Alles (1990)

Neurotensin

Polyclonal antiserum Not reported

Globus and Alles (1990)

P-Endorphins

Polyclonal antiserum Not reported

Vethamany-Globus (1987)

N-CAM Type XI1 collagen

Polyclonal antiserum Absent mAb MT2 Absent IS-2 riboprobe NvTN.l riboprobe Present (low mAb MT1 levels in basal layer) mAb 55C12 (axolotl)

Maier and Miller (1992) Klatt et al. (1992), Wei et al. (1995)

Fibronectin

Polyclonal antiserum Absent

Repesh et al. (1982). Gulati etal. (1983) Nace and Tassava (1995)

RARSl

mAb MT4 Polyclonal antisera (FW6 and RP8)

Actin-binding protein WE4

Tenascin

mAb WE4

mAb LPlK NvKII riboprobes

Many nuclei

Onda et al. (1990) Onda et al. (1991) Koshiba et al. (1994)

Hill et al. (1993)

GENE EXPRESSION DURING AMPHIBIAN LIMB REGENERATION

9

43- or 44-kDa doublet (Castilla and Tassava, 1992) identified as an actinbinding protein(s) (Tassava et af., 1993). WE3 is coexpressed with the enzyme carbonic anhydrase and it has been proposed that both molecules could be involved in controlling the osmotic regulation and the ionic composition of the blastema (Goldhamer et af., 1989). For example, they could regulate the pH of the intercellular fluid,whose variation might then activate the metabolism of progenitor cells, and possibly their reentry into the cell cycle. Further studies will be needed to clarify whether WE3 itself is a carbonic anhydrase, a possibility supported by its expression in cells of the unamputated limb and in cells of the gastrointestinal tract with secretory/ ion transport function. In addition, this antigen is upregulated by retinoic acid (RA), which has, among many other effects that will be discussed later, the well-known property of inducing secretory differentiation (Tassava, 1992). This strengthens the possibility that WE3 may have a role in secretory/transport function in the adult epidermis. Finally, WE3 is absent in the ectoderm of the limb bud (Tassava and Acton, 1989), in which either a different or no secretory/transport activity is required. The WE3 doublet eluted from a denaturating gel was used as an immunogen for the production of monoclonal antibodies (Castilla and Tassava, 1992). One of the mAbs that was generated, WE4, presented a pattern of reactivity partly overlapping with that of mAb WE3. The two antigens are probably physiologically related because they are coexpressed in many cell types and are both actin-binding proteins (Tassava et af., 1993). However, there are also some significant differences in their expression pattern. Unlike WE3, WE4 is not present in normal limb epidermis of N. viridescens. In addition, WE4, unlike WE3, is expressed in the WE as early as 2 days after amputation and also in the germinative layer of the stump epidermis. Therefore, WE4 expression appears to be induced in response to amputation and is maintained up to the digit stage, longer than WE3 expression. WE4 pattern of expression in P. waltl is very similar to that described in N. viridescens, except for the normal limb epidermis that is WECpositive in P. waltf (Tassava et af., 1993). In order to identify molecules regulated by RA in the WE, Estrada et a2. (1993) immunized mice with the WE of newts that had been treated with RA. One of the mAbs they isolated, WE6, recognizes a cytoskeleton protein that is detected as early as 1 day after amputation in all layers of the WE and does not undergo a transition from a granular to a filamentous form as does WE3. In contrast to WE3 however, WE6 is not regulated by RA and is found in epidermal cells of the stump adjacent to the lesion site but not in the epidermis of the proximal region of the stump tissues. Another antigenic determinant expressed in all cells of the WE has been identified by using the 9G1 mAb (Onda and Tassava, 1991). 9G1 mAb recognizes an intracellular antigen(s), and its reactivity is diffuse and granu-

10

JACQUELINE GERAUDIE AND PATRlZlA FERRETTI

lar in appearance. In young axolotl larvae, but not in adult newts, 9G1 is expressed at most stages of limb regeneration but is absent in normal skin epidermis. However, 9G1 reactivity is not restricted to the WE; it is also found in dedifferentiating chondrocytes and blastemal cells, in which a filamentous network is observed. 9G1 is also induced in the basal layer of WE in the flank and in the underlying mesenchymal cells. Therefore, its expression is associated with wound healing rather than specifically with limb regeneration. Their presence could be related both to specific functions of these molecules during healing and to the age of the experimental animals. Because 9G1 expression was studied in premetamorphosis axolotls, it cannot be ruled out that regulation of this molecule is under the control of thyroid hormones (Onda and Tassava, 1991). In denervated early blastemas, when proliferation of blastemal cells is nerve dependent, 9G1 expression is abolished in the WE but not in blastemal cells (Onda and Tassava, 1991). This suggests that the nerve controls, either directly or indirectly, the phenotype of the cells in the WE. NvKII is a WE-associated keratin that was originally identified as one of the two proteins recognized by the mAb LPlK in the newt, N . viridescens (Ferretti et al., 1989,1991,1993; Ferretti and Ghosh, 1997). To date, NvKII is the only WE marker that has been cloned. NvKII shares a high percentage of amino acid homology with human keratin 5 (K5) and 6 (K6), which are markers of different states of epidermal differentiation. Interestingly, K6 is induced in response to skin injury and in highly proliferative epithelia. Notwithstanding the sequence homology between NvKII and K6 there are some important differences in the expression and regulation of these keratins in newts and humans. NvKII expression is not induced simply in response to injury, as is K6, because it is not detected following injury of the newt flank skin. In addition, it is not expressed in regenerating upper and lower jaws, but it is expressed in cells of the WE of regenerating limbs, in which it is first detected around the time when proliferation of blastemal cells begins (4 or 5 days after amputation). Staining with mAb LPlK does not show any reactivity in the ectoderm of the developing newt limb (Ferretti et al., 1989). Both K6 and NvKII respond to RA treatment, but whereas K6 is upregulated by RA, NvKII is dramatically downregulated. Its transcript levels are higher in distal than in proximal blastemas, but the apparent difference observed in its expression in proximal and distal normal limbs appears to be due to the blastema-like quality of the fingertips, which express many regeneration-associated antigens (Ferretti and Ghosh, 1997). The need for such a specific expression of this cytokeratin in the limb WE is not understood, but the facts that NvKII is higher in distal than in proximal blastemas and is regulated by RA suggest that it may be expressed in cells that play a role in specifying positional information.

GENE EXPRESSION DURING AMPHIBIAN LIMB REGENERATION

11

Strong reactivity with the 117C1 mAb is observed in the cytoplasm of WE and blastema cells, but only faint staining is detectable in epidermis, dermis, muscle, perichondrium, and cartilage of normal limbs (Koshiba et al., 1994). The molecules recognized by the 117C1 mAb have molecular weights of approximately 130,200, and 240 kDa, as established by Western blotting, but their exact nature is not known. The WE has been shown to contain neuropeptides (Vethamany-Globus, 1987; Globus and Alles, 1990). Among these, there is substance P (SP), an undecapeptide that functions as a neurotransmitter in the central and peripheral nervous system and appears to have a mitogenic effect on the blastemal cells in newt limbs and in planaria (Globus et al., 1983; Salo and Baguna, 1986). Reactivity with an antibody to SP is observed in the suprabasal layers of the WE, but it has not been shown whether similar reactivity is present in normal skin. By radioimmunoassay high levels of SP are also detected in the blastema mesenchyme, and this discrepancy has not yet been convincingly explained (Globus and Alles, 1990). It will be important to establish whether, as suggested, SP is present in different forms in the mesenchyme (soluble form) and in the WE (bound to membrane receptors). Immunoreactivity for endogenous opioid peptides (P-EP, P-endorphin-like molecule), which are known to induce analgesia in man, has also been observed in the suprabasal layers of the WE cells of the blastema (Vethamany-Globus, 1987) and may be important for reducing the local pain stimuli induced by amputation. High plasma P-EP levels are maintained during limb regeneration, possibly to reduce pain perception in the amputated animal. In contrast to SP and P-EP, neurotensin-like immunoreactivity is detected only in the basal layer of the WE, and low levels of expression of other peptides belonging to the tachykinin family have been reported in limb blastemas (Globus and Alles, 1990). The precise role of the neuropeptides found in the WE is unknown, and it is also unknown whether their expression reflects a general response to injury or is specific to limb regeneration. The information so far available about the molecular composition of the WE (Fig. 2) reveals that whereas some of the dramatic changes induced by amputation are equivalent to those induced by skin wounding in other body parts, others are intimately linked to the process of limb regeneration. Furthermore, the fact that expression of some of these antigens recapitulates their developmental regulation in the limb bud supports the view that the ectoderm of the developing bud and the WE may play similar roles in the ontogenesis of embryonic and adult limbs. In conclusion, the WE is a very complex structure that encompasses features of both embryonic ectoderm and injured adult skin (9G1) and also appears to have some traits of its own (WE3 and NvKII). It is interesting that both WE-specific molecules (WE3 and NvKII), a cytoskeleton-associated molecule, and a cytoskeletal

12

JACQUELINE GERAUDIE AND PATRIZIA FERRETTI

protein, respectively, are regulated by RA. Because RA affects growth and patterning of the regenerating limb, this suggests that the cytoskeleton may play an important role in controlling the physiological state of the cells of the WE and possibly epithelial-mesenchymal interactions. Analysis of potential molecular mechanisms underlying the permissive role of the WE on growth and patterning of blastemal cells is in progress, and recent works from Bryant’s, Tassava’s, and Boilly’s laboratories suggest that fibroblast growth factor (FGF) is present in the WE and may play an equivalent role in limb development and regeneration (see Section 111).

B. Blastemal Mesenchyme 1. Preblastema We use the term “preblastema” to refer to the regenerative phase between the time of amputation and the onset of cell division, which includes closing of the wound, and the initial phase of formation blastemal cells and their accumulation beneath the wound epidermis. Significant changes in the connective tissues at the level of amputation are apparent within 24-48 h after surgery. As mentioned in the Introduction, the first event is degradation of certain components of the extracellular matrix (ECM), upregulation of others, and partial histolysis of the distal stump tissues to release the progenitor cells that will form the blastema. One of the molecules that is rapidly degraded following amputation is collagen, which cannot be detected by biochemical assays at the early stages of regeneration (Grillo et al., 1968; Dresden and Gross, 1970). Collagen belongs to a large multigene family consisting of at least 15 different species (types I-XV collagen). Only some of the newt collagen types have been identified and studied in the regenerating limb. Type I collagen distribution has been studied by transmission electron microscopy (TEM), given its characteristic periodic cross-striation (GCraudie and Singer, 1981). The nucleotide sequence of a1 chain of type XI1 collagen was isolated by using the MT2 mAb, and its pattern of expression was examined by both immunocytochemistry and in situ hybridization (Klatt et al., 1992; Wei et al., 1995). In normal limbs, type XI1 collagen is expressed in tendons and perichondrium but is not expressed in the mesenchyme at the site of amputation. However, it is upregulated in the cells of the basal layer of the WE in 3day regenerates (Wei et al., 1995). It has been shown that collagenolytic enzymes are activated in the stump upon amputation, at the time of histolysis and dedifferentiation (Grillo et al., 1968;Dresden and Gross, 1970). Recently, several enzymes of the matrix metalloproteinases (MMPs) family, which include collagenases, gelatinases,

GENE EXPRESSION DURING AMPHIBIAN LIMB REGENERATION

13

and stromelysins, have been identified in newt and axolotl limbs (Yang and Bryant, 1994; Miyazaki et al., 1996). The MMP clones isolated in the newt encode for a collagenase, a gelatinase also isolated in axolotl, and a stromelysin. None of these enzymes are detected in normal limbs, but they are all upregulated as early as 2 days after amputation, although their later time course of expression differs slightly. Another ECM molecule that disappears following amputation is laminin, a complex glycoprotein that is present in basement membranes, provides a site of attachment for epithelial cells, and plays a role in the maintenance of tissue integrity (Timpl, 1989). This is of great functional importance because, as discussed previously, epithelial-mesenchymal interactions are indispensable for the progression of regeneration and are impaired by the presence of a basement membrane. Fibronectin is an ECM glycoprotein that in normal limbs colocalizes with laminin and type IV collagen in basement membranes. Unlike laminin, fibronectin is detected in the WE 24 h after amputation, during the initial phase of histolysis of stump tissues (Repesh et aL, 1982; Gulati et al., 1983; Nace and Tassava, 1995), whereas the fibronectin transcript is localized in the basal cells of the WE, the protein is detected in the acellular space beneath it. The presence of fibronectin in the WE is interesting because normally this protein is produced by mesenchymal cells and is not expressed in normal epidermis (Nace and Tassava, 1995). It cannot be ruled out that some of the fibronectin observed at this stage is due to deposition of blood fibronectin. Fibronectin may play a role in the migration of dedifferentiating cells, and later of blastemal cells, because receptors for its RGDS cell binding motif are present on epidermal cells (Donaldson and Mahan, 1987). An ECM component that appears to be different from any of those already characterized in the newt and is downregulated following amputation is identified by the mAb Stump 1(ST1) (Yang et aL, 1992). This reagent was obtained by immunizing mice with newt homogenates of regenerating retinas and lenses. ST1 is expressed in the epimysium, perimysium, perineurium, tendons, blood vessels, and around the epidermal glands and skeletal tissues of normal limbs, but it is downregulated in the distal stump region and is not detectable in the early blastema. Hyaluronate and chondroitin sulfate are the major large hydrophilic polyanionic glycosaminoglycans of the ECM associated with blastema formation and cell differentiation in the regenerating limb (Toole and Gross, 1971; Mescher and Munaim, 1986; Mescher and Cox, 1988). Hyaluronate is first synthesized at the time of dedifferentiation, when concomitantly hyaluronidase activity disappears (Smith et aL, 1975; Mescher and Munaim, 1986). It has been suggested that this polysaccharide, which induces tissue hydration, provides an environment suitable for cell migration (Toole et al., 1984) and could facilitate the migration of progenitor cells from the

14

JACQUELINE GERAUDIE AND PATRIZIA FERRETTI

stump by promoting expansion of the distal portion of the stump (Mescher and Cox, 1988). Together, these changes in the extracellular environment are thought to be fundamental for the initiation of blastema formation, but their precise role in the cascade of events leading to the release of blastemal cells from the stump has yet to be elucidated. Cell migration of stump dermal fibroblasts, and of cells that are released from the cut stump myofibers, cartilage, and myelin, occurs early after amputation (Hay and Fishman, 1961; Gardiner et al., 1986; Kintner and Brockes, 1985; Muneoka et al., 1986). The mesenchymal progenitor cells that can be observed within 24 h after amputation at the stump tip express vimentin and another cytoskeletal protein, 22/18, which is not detectable in cryostat sections of normal limbs (Kintner and Brockes, 1984, 1985; Gordon and Brockes, 1988). 22/18 appears to identify a conformational change in an intermediate filament component, demonstrating that a significant reorganization of the cytoskeleton occurs during formation of blastemal cells (Ferretti and Brockes, 1990), possibly related to modifications in cell-extracellular matrix interactions. Interestingly, 22/18 is expressed in blastemal cells whose division depends on the presence of the nerve, but mesenchymal cells of the limb bud, whose development is nerve independent, do not express it (Fekete and Brockes, 1987). However, induction of 22/18 expression in the regenerate is not under nerve control, confirming that initial formation of blastemal cells and their release from the stump tissues do not require innervation (Tassava and Olsen, 1985). In the distal portion of the stump, 22/18 reactivity is also observed in some mononucleated cells at the cut surface of the muscle and of the nerve, which appear to be Schwann cells being released from the myelin sheath because they also express the Schwann cell marker Leu-7 (Gordon and Brockes, 1988). The existence of Leu-7-22/18-positive cells migrating from the stump supports previous morphological observations that Schwann cells contribute to blastema formation (Maden, 1977). Expression of the myelin protein PO is also briefly observed in the Schwann cells moving away from the nerve stump but is rapidly downregulated (Kintner and Brockes, 1985). Other genes that are upregulated within 24 h after amputation and are important for patterning, such as Hox genes, will be discussed in Section IV. How blastemal cells originate from the stump tissues, and the molecular mechanism governing this process, is still unclear. Two possible origins of blastemal cells are dedifferentiation of mature tissues of the stump, which would regress to an undifferentiated state and reenter the mitotic cycle, and activation of populations of normally quiescent stem cells in the different stump tissues. The two possibilities are not mutually exclusive, and different tissues may contribute cells to the blastema through different mechanisms. In either case, changes in the phenotype of cells at the cut

GENE EXPRESSION DURING AMPHIBIAN LIMB REGENERATION

15

surface of the stump occur, as reflected by the appearance of 22/18 reactivity, and they may be triggered by the changes in their extracellular environment discussed previously. To date, the only tissue in which convincing evidence of dedifferentiation has been presented is the muscle. However, this is also the only tissue in which the presence of muscle progenitor cells (postsatellite cells), which may be similar to the satellite cells of the mammalian muscle, has been shown in in v i m studies (Popiela, 1976; Cameron et al., 1986; Carlson, 1988; Lyons and Buckingham, 1992). It is conceivable that there are two different modes of rebuilding muscle depending on the type of damage. Also, whereas postsatellite cells may be used in muscle repair, the process of dedifferentiation may be required only for muscle regeneration after limb amputation (Carlson, 1979; Griffin et al., 1987; Ferretti and Corcoran, manuscript in preparation). The occurrence of muscle dedifferentiation was first observed by Hay (Hay, 1959; Hay and Fishman, 1961) in TEM sections of stump muscle, in which single nuclei of the severed myofiber appeared to pinch off from the degenerating fiber and migrate away as mononucleated cells. This interpretation has been supported by double-labeling experiments in which some blastemal cells have been shown to coexpress 22/18 and the muscle marker 12/101 (Kintner and Brockes, 1984) and recently by elegant tracing experiments (Lo et af., 1993). Ten days after implantation in the blastema of myotubes prelabeled with [3H]thymidine and rhodamine-dextran in culture, mononucleated cells containing the labels are detected in viva These cells seem to have the ability to fuse because some labeled myotubes are present in the blastemas. These results indicate that the implanted myotubes have indeed undergone a process of dedifferentiation, which results in the production of mononucleated myogenic progenitor cells. Interestingly, a few of these cells appear to be able to differentiate into chondrocytes, supporting the view that at least some transdifferentiation can occur during limb regeneration, as also suggested by the use of tissue-specific hypomethylation sites as lineage markers (Casimir et al., 1988; Ferretti and Brockes, 1991). Other tissues contributing cells to the blastema are skeletal tissue and fibroblasts. The contribution of periosteal cells to blastema formation has been demonstrated by histological analysis and grafting experiments reviewed elsewhere (Wallace, 1981). However, skeletal tissues seem to contribute only 2% cells, whereas dermal fibroblasts contribute approximately 48% (Namenwirth, 1974; Muneoka et al., 1986). 2. Blastema

The blastemal cells that accumulate at the distal tip of the stump begin to proliferate between 3 and 5 days after amputation while release of blastemal

16

JACQUELINE G6RAUDIE AND PATRlZlA FERRET1

cells from the stump is still occurring. At this stage expression of other two cytoskeletal proteins, the keratins 8 and 18 now cloned in the newt (Ferretti et al., 1993; Corcoran and Ferretti, 1997), is upregulated in blastemal cells. Activation of these genes in the mesenchyme is rather surprising because they are normally restricted to simple epithelia. Their expression might be simply due to the need for a more rigid skeleton in the growing blastema than in limb bud development, where NvK8 and NvK18 are not observed. However, recent experiments have shown that antisense oligonucleotides to K8 and K18 inhibit cell division in cultured blastemal cells, suggesting a causal relationship between keratin expression and blastema growth (Corcoran and Ferretti, 1997). Different regulation by RA of these keratins in myogenic cells cultures originating either from normal limb or regeneration blastema suggests two possible origins of myogenic cells; one from satellite cells, which would be activated for muscle repair, and one from dedifferentiation of mature muscle, which would occur in the process of epimorphic regeneration (Carlson, 1979; Ferretti and Corcoran, manuscript in preparation). Complex functional links between the organization of cytoskeletal elements, cell surface molecules, and extracellular matrix components appear to control changes in cell shape, movement, and fate (Daniels and Solursh, 1991).The identification of numerous changes in the cytoskeleton of blastema1 cells suggeststhat such changes may be causally related to dedifferentiation, growth, and redifferentiation of blastemal cells. Therefore, it will be important to further investigate the role of cytoskeletal proteins during regeneration. It has been shown that myosin larval forms are temporarily expressed in early blastemas, and that they are replaced by adult isoforms at the onset of differentiation (Saadi et al., 1993). The changes in myosin isoforms do not follow the pattern observed during limb bud development, possibly because this is controlled by thyroid hormone in the embryonic limb, but not in the adult regenerate. Recent analysis of the expression of some of the helix-loop-helix transcription factors that control myogenesis during development, such as Myf5 and MRF-4, has revealed that Myf-5, which is the first of the myogenic genes expressed during embryogensis, is detected by Northern blotting throughout the regenerative process (Simon et al., 1995). However, it is unknown whether Myf-5 is expressed in a discrete population of blastemal cells or in all of them. This information would be very valuable because it would allow one to gain some insight in cell lineages in the blastema. In contrast, the MRF-4 transcript is not detected in the blastema but is abundant in adult myofibers (Simon et al., 1995). It has been proposed that there is a causal relationship between MRF-4 downregulation in the blastema and myofiber dedifferentiation in the stump. However, it is also possible

GENE EXPRESSION DURING AMPHIBIAN LIMB REGENERATION

17

that its downregulation, like that of the 12/101 protein, is the consequence and not the cause of dedifferentiation. MRF-4 reexpression follows the pattern of expression of the muscle-specific myosin gene observed in the late regenerates, at the time of muscle cell differentiation. The extracellular environment of the blastema is also highly dynamic. In early blastemas, type XI1 collagen is expressed not only in the WE but also in the mesenchyme (Klatt et al., 1992; Wie et al., 1995). The spatiotemporal distribution of this molecule suggests that it could be secreted by the W E into the mesenchymal compartment before mesenchymal cells begin to synthesize it themselves. The level of expression of type XI1 collagen decreases in the WE of late blastemas, whereas at this stage it is still high in the mesenchyme. With the progression of differentiation, however, collagen XI1 is also downregulated in the mesenchyme, with the exception of the perichondrium. Another type of collagen detected in the mesenchyme of early blastemas is type I collagen, and its expression is maintained throughout regeneration (GCraudie and Singer, 1981). Increased activity of prolyl hydroxylase, an enzyme involved in hydroxylation of the prolyl residues present in collagen molecules (Colquhoun and Dresden, 1983), and massive collagen biosynthesis are detected at the onset of digit regeneration (Mailman and Dresden, 1976). Fibronectin, like type XI1 collagen, begins to be synthesized by mesenchyma1 cells at the early bud stage and is released in the extracellular space (Nace and Tassava, 1995), as during development of the chick limb bud (Tomasek et al, 1982). During muscle differentiation, blastemal cells with myogenic potential are surrounded by fibronectin, align along the longitudinal axis of the regenerate, and fuse; after cell fusion, fibronectin immunoreactivity decreases. Finally, fibronectin is also transiently present in differentiating chondroblasts and hypertrophic chondrocytes. Like fibronectin, laminin is detected during redifferentiation in regenerating myotubes and around the chondrocyte lacunae, where laminin is also normally present (Gulati ef al., 1983). However, neither laminin nor ST1 are detected in the undifferentiated blastema, but both are reexpressed at the time of cell differentiation. The sequence of expression of glycosaminoglycans in the course of limb regeneration parallels that described in the avian developing limb bud (Toole, 1973). Whereas high levels of hyaluronate are detected at all blastema stages, hyaluronidase levels are extremely low until the onset of differentiation. Increase in hyaluronidase activity, and consequent decrease in hyaluronate levels, might favor the onset of vascularization of the blastema observed in late buds (Peadon and Singer, 1966; West er al., 1985). Chondroitin sulfate, which is initially very low and slowly increases during blastema growth, becomes the primary GAG present at the onset of chon-

18

JACQUELINE GERAUDIE AND PATRlZlA FERRETTI

drification of the regenerating skeleton (Toole and Gross, 1971; Smith et al., 1975). Although it is known that heparan sulfate is expressed both in the blastema mesenchyme and in the W E (Boilly et al., 1995), detailed analysis of the time course of its expression during regeneration is still missing. Immunocytochemical studies have shown that the ECM glycoprotein tenascin is first detected at the WE/mesenchyme borders and in a few mesenchymal cells beneath it 5 days after amputation, although tenascin mRNA is already transcribed in the W E at 2 days (Onda et a/., 1990, 1991; Koshiba et al., 1994). Transcription in the distal portion of the WE, particularly in the basal cells, is maintained throughout the regenerative process, and it is therefore likely that the tenascin detected at the epithelialmesenchymal interface is mainly synthetized by the WE. Intracellular tenascin-positive reactivity is detected in some cells in the wound epidermis. In the blastema mesenchyme tenascin is uniformly distributed and is lost with the progression of differentiation in a proximal to distal fashion. Work by Koshiba et al. (1994) has suggested that tenascin reactivity is initially reduced in retinoid-treated blastemas and is later found in areas where active proliferation takes place. Although numerous studies have investigated changes in the ECM during regeneration, little is known about the expression and role of cell-cell adhesion molecules. N-CAM, which functions as a homophilic ligand in cell-cell interactions and serves as an adhesive substrate for guidance of growth cones, has been detected in the WE and mesenchyme of the blastema and in cultured blastemal cells, but it is not known when it is first expressed (Maier et al., 1986; Ferretti and Brockes, 1990). Interestingly, infusion of anti-N-CAM Fab fragments in the blastema in vivo retards its growth, as observed after partial denervation (Maier et al., 1986). The fact that growth inhibition is not total may be due to technical reasons, such as the infusion protocol, the stability of the antibody, and its distribution in the blastema. Why anti-N-CAM infusion induces growth retardation must still be clarified, but it may be the consequence of either inhibition of blastemal cell interactions or inhibition of axonal growth in the blastema. Whereas N-CAM is widely expressed in the blastema, the distribution of other adhesion molecules that contain the L1 and L2 carbohydrate epitopes and are known to be involved in neural cell-cell interactions is more restricted. In fact, an anti-Ll antibody and the monoclonal antibodies HNK-1/Leu-7 (anti-L2) label only a subpopulation of blastemal cells that are believed to originate from dedifferentiation of the Schwann cells of the nerve stump (Gordon and Brockes, 1988; Maier and Miller, 1992). The distribution of other molecules involved in adhesion mechanisms, such as integrins, cadherins, or selectins, during limb regeneration has not been thoroughly investigated. Results from Tsonis (1996) and our laboratories (J. GCraudie and P. Ferretti, unpublished results) have indicated that

GENE EXPRESSION DURING AMPHIBIAN LIMB REGENERATION

19

a3, a6, av, and 0 3 are upregulated in the blastema, whereas a l , a4, and

01 integrin subunits are downregulated. Whereas a 4 was detected in muscle, chondrocytes, and skin but not in limb blastemal cells in axolotl, the newt tail blastema appeared to express this integrin subunit. It is not clear whether this reflects a difference in reactivity across species or differences between regenerating limbs and tails. It is apparent from the studies discussed here and summarized in Table I1 and Fig. 2 that many ECM and membrane proteins with a wide range of adhesion properties are expressed in a coordinated spatiotemporal fashion during regeneration. This is not surprising because changes in cell interactions and cell migration are certainly key factors in blastema formation. Attempting to understand the role played by different molecules in cell-cell and cell-matrix interactions during limb regeneration is going to be an exciting but formidable task.

111. Growth Control in the Blastema As mentioned in the Introduction, early regeneration blastemas require the presence of both the WE and an adequate level of nervous supply for their outgrowth (Todd, 1823; Singer, 1974; Tassava and Mescher, 1975; Carlone and Mescher, 1985; Sicard, 1985; Brockes, 1987; Fekete et al., 1987; Singer and GCraudie, 1991). In addition, the “hormonal milieu” appears to be important for blastema growth, but it is difficult to define the specific role of individual hormones (Tassava, 1969, 1983; Sicard, 1985). Because of the dramatic growth arrest induced by denervating the limb, which can be reversed by treatment with neural extracts, much experimental work on growth control in the blastema in vivo has focused on the comparison between normal and denervated blastemas. When a limb is denervated prior to amputation, wound healing and accumulation of blastemal cells occur normally, but the blastemal cells do not proliferate and regeneration is inhibited (Singer, 1952,1974; Thornton, 1970). On the contrary, if the limb is denervated after a blastema has formed, regeneration proceeds, but the regenerated limb is smaller in size. Therefore, regeneration depends on the presence of the nerve only during the phase of rapid proliferation of blastemal cells (Singer and Craven, 1948). The nervous system is believed to control cell growth predominantly during the G1 phase of the cell cycle (Goldhamer and Tassava, 1987), and it has been postulated that this action is exercised either directly or indirectly by neurotrophic factors secreted by the nerves (Singer, 1974; Singer et al., 1976; Jabaily and Singer, 1977; Brockes, 1987; Ferretti and Brockes, 1991; Dinsmore and Mescher, 1998).

TABLE I1 Gene Expression in the Limb Blastema Mesenchyme

Molecule ECM molecules Metalloproteinases Type XI1 collagen Type I collagen Laminin Fibronectin

Tenascin GAGS

Surface molecules N-CAM L2 epitope (HNKl)* L1 PO" Cytoskeleton 22/18 intermediate filament component Keratin 8

Detection Riboprobes Biochemical IS-2 riboprobe mAb MTZ TEM Polyclonal antiserum

Yang and Bryant (1994), Miyazaki et al. (1996) Klatt et al. (1992) Wei et al. (1995) Gtraudie and Singer (1981) Gulati et al. (1983), Maier and Miller (1992) Repesh et al. (1982), Gulati er Polyclonal antiserum mAb MT4 al. (1983), Maier and Miller (1992), Nace and Tassava (1995) NvTN.l riboprobe mAb MTl Onda et al. (1990, 1991). mAb 55C12 (axolotl) Koshiba et al. (1994) Biochemical Toole and Gross (1971), Mescher and Munaim (1986), Mescher and Cox (1988), Boilly et al. (1995) Polyclonal antiserum mAb Leu-7 Polyclonal antiserum mAbs 3013, 30114, 30/26 mAb 22/18

Vimentin

NvK8 riboprobe mAbs LPlWLE41 NvKlS riboprobe mAbs RGE53, CK18.2 mAb 22131

Keratin 14 GFAP Myosin

mAb Polyclonal antiserum Riboprobe

Keratin 18

Cell membrane receptors Riboprobes FGFRl bek (variant of FGFR2) Riboprobes Insulin receptor Others Sarcoplasmic reticulum protein" CRABP

Reference

Maier et al. (1986) Fekete and Brockes (1987), Gordon and Brockes (1988) Maier and Miller (1992) Kintner and Brockes (1985) Kintner and Brockes (1985), Ferretti and Brockes (1990) Ferretti et al. (1989, 1993) Corcoran and Ferretti (1997) Ferretti et al. (1989, 1993) Corcoran and Ferretti (1997) Kintner and Brockes (1985), Fekete and Brockes (1987), Tsonis et al. (1992) Maier and Miller (1992) Casimir et al. (1985)

Polyclonal antiserum

Poulin et al. (1993) Poulin et al. (1993), Poulin and Chiu (1995) Foty and Liversage (1993)

mAb 12/101

Kintner and Brockes (1985)

Biochemical

Keeble and Maden (1986), McCormick et al. (1988) . ,

"Detected in a few cells at early stages of regeneration; 12/101 is reexpressed in a few blastemal cells at the bud stage.

GENE EXPRESSION DURING AMPHIBIAN LIMB REGENERATION

21

In the early blastema axotomy induces a transient outburst in DNA, RNA, and protein synthesis; this is followed by a 40-50% decrease in their levels within 48 h and growth arrest of the blastema (Dresden, 1969; Lebowitz and Singer, 1970; Singer and Caston, 1972; Smith et al., 1975; GCraudie and Singer, 1978; Mescher and Cox, 1989). Recently, expression of some molecules involved in the control of the cell cycle, such as the protooncogenes c-myc (Geraudie et ul., 1990), Ki-ras (see AndCol et ul., 1990; Y. AndCol and J. Geraudie, unpublished results), rus (Tsonis, 1991), ski (Ludolph et al., 1995), and the marker of S phase proliferating cell nuclear antigen (PCNA), has been examined in regenerating limbs. It has been shown that both c-myc (GCraudie et al., 1990) and p53 (see Tchang et al., 1993; F. Tchang and J. GCraudie, unpublished results) are upregulated in regenerating limbs of Xenopus froglets compared to controls, whereas PCNA levels do not change significantly (Lemaitre et al., 1992). Expression of ski has been reported in different stages of axolotl limb blastemas by Northern analysis (Ludolph et ul., 1995). However, because this protooncogene appears to be expressed at high levels in differentiated muscle, cellular localization will be necessary to rule out the possibility that its expression is due to contamination with muscle tissue at the early stages of regeneration and to muscle redifferentiation at the later ones. Denervation results in significant changes in the expression of many of these molecules in the regenerating limbs. As demonstrated by Northern blot, Xenopus c-myc (Gtraudie et al., 1990) and Ki-rm (Y. And601 and J. Geraudie, unpublished results) transcripts are downregulated in denervated limbs. However, a transient accumulation of the c-myc protein in the regenerate is observed. Furthermore, PCNA is significantly upregulated for at least 4 days after denervation (Lemaitre et al., 1992); the significance of this accumulation is unclear, and it will be important to examine changes in PCNA expression in normal and denervated blastemas in species with a higher regenerative capability than Xenopus. Altogether, the upregulation of the c-myc and PCNA proteins has led to the proposal that nervous system normally exerts a negative control on their expression (Lemaitre et al., 1992). This suggests that the nerve might control proliferation of blastema1 cells through both positive and negative activities. Identification of the neurotrophic molecule(s) postulated by Singer (1974) has proved difficult, but during the past few years it has become apparent that both mitogenic and trophic factors are required for blastema growth. Molecules that seem to satisfy at least some of the properties expected of a “neurotrophic factor” (Brockes, 1984) are members of the FGF fibroblast growth factor and neuregulin/glial growth factor (GGF) families and the iron transport protein transferrin. Also, insulin appears to be important in limb regeneration (Foty and Liversage, 1993), and factors such as EGF and substance P, but not PDGF and NGF, have been shown

22

JACQUELINE GERAUDIE AND PATRlZlA FERRETTI

to be mitogenic for blastemal cells in vitro (Mescher and Loh, 1981; Smith and Globus, 1989; Smith et al., 1995). Whereas relatively little information is available on the GGF-like activity demonstrated in regeneration blastemas (Brockes and Kintner, 1986), more is known about FGFs and transferrin in regenerating limbs, and it is possible that these are indeed the key mitogenic and trophic factors required for nerve-dependent blastemal cell proliferation. The mitogenic effect of FGF was initially suggested by the fact that infusion of crude FGF preparations in denervated limb blastema could reverse growth arrest (Mescher and Gospodarowicz, 1979; Gospodarowicz and Mescher, 1980). The presence of both acidic (FGF-1) and basic FGF (FGF-2) in the limb blastema was later shown by binding assays, Western blot analysis, and immunocytochemistry (Albert et al., 1987; Boilly and Albert, 1990; Boilly et al., 1991; Mullen et al., 1996; Zenjary et al., 1996). Strong FGF-1 and FGF-2 immunoreactivity is detected in the wound epidermis, but only FGF-1 reactivity seems to be present in the blastema mesenchyme (Mullen et af., 1996; Zenjary et al., 1996). As mentioned earlier, lack of a wound epidermis, as well as removal of the apical ectodermal ridge in developing limb buds, inhibits limb outgrowth. In the limb bud this effect can be reverted by FGF application (Niswander et al., 1993; Fallon et al., 1994), and FGF can also induce “regeneration” of the embryonic limb (Taylor et al., 1994; Kostakopoulou et al., 1996). It will be important to establish whether, in a similar fashion, FGF can compensate for the lack of a WE in regenerating limbs; however, given the fast regrowth of the WE after its removal, it may not be a trivial task to address this issue. Recent work has shown that FGF-1 is also transcribed in the cell bodies of the neurones innervating the regenerating limb (Dinsmore and Mescher, 1998), and that the FGF-2 protein is present in the nerves in which its levels, like those in the wound epidermis, are significantly decreased following denervation (Mullen et a/.,1996). Furthermore, implantation of a FGF-2 bead rescues, at least in part, the growth inhibition induced by severing the nerve. These results point to a causal link between FGF-2 mitogenic activity and nerve-dependent growth in limb blastemas. Also, FGF-1 appears to control blastemal cell proliferation, as suggested by in vivo experiments using neutralizing antibodies and molecules that inhibit growth factor binding (Zenjary et al., 1996), but whether FGF-1 activity and nerve dependency are linked has yet to be elucidated. The presence of other members of the family, such as FGF-4 and FGF-8, that appear to be involved in limb patterning during development (Niswander et al., 1993; Crossley et al., 1996) has not yet been reported in the regenerating newt limb. FGF signaling is mediated by a family of tyrosine kinase transmembrane receptors, the FGF receptors (FGFRs). Distinct spatiotemporal expression of FGFR-1 and two variants of FGFR-2 (bek and KGFR) has been observed

GENE EXPRESSION DURING AMPHIBIAN LIMB REGENERATION

23

by in situ hybridization in newt blastemas (Poulin et al., 1993, Poulin and Chiu, 1995). FGFR-1 transcription is observed in blastemal cells during the early stages of regeneration, whereas FGFR-2 message is detected in the basal layer of the WE and in periosteal cells of the bone stump, and its expression is maintained in regenerating cartilage. In conclusion, although other mitogenic factors may be involved in blastemal cell growth, the pattern of expression of FGFs and FGFRs together with the functional experiments carried out so far strongly suggest that these molecules play an important role in limb outgrowth during regeneration. It is unclear, however, whether they also play a role in patterning of the regenerating limb, as they do during limb development (Izpis6a-Belmonte et al., 1992; Laufer et al., 1994; Niswander et al., 1994; Ferretti and Tickle, 1997). The intracellular events involved in the transduction of mitogenic signals have not been thoroughly investigated. However, the fact that FGFRs can activate phospholipase C in other species, and that nerve extracts and substance P induce formation of inositol phosphates (Smith et al., 1995), indicates that the inositol phospholipid signaling pathways is involved in mitogenic signal transduction in the blastema. A list of urodele genes encoding for soluble signaling molecules that have been detected in limb blastemas is given in Table 111. As mentioned previously, another factor that appears to play a key role in keeping the blastemal cells cycling is transferrin. In fact, both chelation of ferric ions and the use of an antitransferrin antibody prevents the mitogenic effect of neural extracts (Munaim and Mescher, 1986; Albert and Boilly, 1988; Dinsmore and Mescher, 1998). Transferrin is abundant in Schwann cells and in peripheral nerves, where it is transported both anterogradely and retrogradely. A significant decrease in transferrin levels is observed in

TABLE Ill

Urcdele Genes Encoding for Soluble Signaling Molecules Gene

Urodele amphibian

Reference Takabatake et al. (1996), Imokawa and Yoshizato (1997)

sonic hedgehog

Cynops

banded hedgehog

Notophthalmus viridescens Stark et al. (manuscript in preparation)

FGF

Cynops

Imokawa ef al. (manuscript in preparation)

Wnt-?

Cynops

Imokawa et al. (manuscript in preparation)

Wnt-5"

Axolotl

Busse and Seguin (1993) Caubit et al. (1996)

Wnt-SA," -5B,a -7A" Pleurodeles wait1 a

Genes whose expression in limb regeneration blastemas has not been studied.

24

JACQUELINE GERAUDIE AND PATRlZlA FERRET1

the growth-arrested denervated blastemas (Mescher and Munaim, 1984; Kiffmeyer et al., 1991; Mescher and Kiffmeyer, 1992), and, at least a short time after denervation, application of transferrin significantly increases [3H]thymidine incorporation in blastemal cells in vivo (Mescher and Kiffmeyer, 1992). It will be important to establish in long-term experiments whether application of FGF and transferrin can fully substitute for the nerve and results into normal regeneration of denervated blastemas. The recent advancements in the identification of mitogenic and trophic factors required for blastemal growth discussed previously, the increasing understanding of the molecular mechanisms controlling reentry into the cell cycle following amputation (Tanaka et ul., 1997), and the analysis of the “immortal” nature of blastemal cells in culture (Ferretti and Brockes, 1988; Powell et a1.,1997) are currently very exciting and promising areas of research in the regeneration field. In fact, the regenerating limb provides one of the very few opportunities to study the issues of reversal of the differentiated state, reentry into the cell cycle, and control of proliferation in an adult organism in a nononcogenic context.

IV. Genes Controlling Morphogenesis in the Regenerating Limb

In previous sections we discussed the changes in the phenotype of blastemal and WE cells and some of the differences in gene expression in adult limb blastemas and in developing limb buds. These molecular differences, together with the fact that cell division is under nerve control during regeneration but not during development, are likely to reflect the different origin of limb progenitor cells in embryos and adults. Therefore, at least in regard to limb progenitors, regeneration does not simply recapitulate development. In contrast, patterning of the regenerating limb is likely to be governed by the same set of genes that have been shown to play an important role during development. This section will review the information currently available on such genes in the regenerating limb. Among the molecules that appear to play a key role in patterning of the limb are retinoids and their receptors, segment polarity genes (sonic hedgehog, wnt, and engruiled), FGFs and their receptors (FGFRs), bone morphogenetic proteins (bmp), and homeobox-containing genes (e.g., hoxa and hoxd clusters, msx-1 and -2, dlx). The homologs of many of these genes have now been cloned in urodeles (Table IV), although much information on their expression in regenerating limbs, especially at the cellular level, is still missing. The emerging picture, as outlined in more detail below, is that expression of all the genes believed to play a role in patterning of the limb bud studied so far is either maintained in the adult urodele limb or

25

GENE EXPRESSION DURING AMPHIBIAN LIMB REGENERATION

TABLE IV Homeobox-Containing Genes and Retinoid Acid Receptors Gene

Urodele amphibian

Reference

HOXa-4, -S, -7, -9, -10, -11, -13

Axolotl

Gardiner et al. (1995)

Hoxa-4, -9 Hox-~II

Pleurodeles waltl

Nicolas et al. (1995)

Notophthalmus viridescens

Beauchemin et al. (1994) Beauchemin and Savard (1993) Gardiner et al. (1995) Nicolas et al. (1995)

Hox-~~ H o x - ~ -66 ~,

Notophthalmus viridescens

Hoxb-3, -9

Axolotl Pleurodeles waltl

HOXC-6,-I0

Notophthalrnus viridescens

HOXC-12

Pleurodeles waltl

HOXC-13 Hoxd-10, -I1

Axolotl Notophthalmus viridescens

H o x ~ - 8 ,-10, -I1

Axolotl

Hoxd-I3

Pleurodeles waltl

MSX-1

Notophthalmus viridescens

Msx-I and Msx-2

Axolotl

dlx-related genes

Notophthalmus viridescens

Dlx-3 DLU-3'

Axolotl Pleurodeles waltl Notophthalmus viridescens

T-box (TBX) RARa, RARP, RARS ~~

Notophthalmus viridescens

Savard et al. (1988), Tabin (1989), Simon and Tabin (1993), Savard and Tremblay (1995) Nicolas et 01. (1995) Gardiner et al. (1995) Brown and Brockes (1991), Simon and Tabin (1993) Gardiner et al. (1995) Nicolas et al. (1995) Crews et al. (1995), Simon et al. (1995) Gardiner et al. (1995) Beauchemin and Savard (1992, 1993), Gardiner et al. (1993) Mullen et al. (1996) Nicolas et al. (1996) Simon et al. (1997) Giguere et al. (1989), Ragsdale et at. (1989, 1992a,b)

~

Expression in regeneration blastemas has not been studied.

reinduced following amputation. However, it appears that the pattern of expression of at least some of these genes is somehow different from that observed in the developing bud.

A. Retinoids and Their Receptors Retinoids (vitamin A and its derivatives) appear to be required for normal limb development and when administered exogenously can severely affect

26

JACQUELINE GERAUDIE AND PATRlZlA FERREnl

patterning of developing and regenerating limbs. Therefore, effects of retinoids on developing and regenerating limbs have been the focus of many studies and reviews (Brockes, 1990; Stocum and Maden, 1990; Stocum, 1991; Bryant and Gardiner, 1992; Mendelsohn et al., 1992; Lohnes et al., 1994; Tickle and Eichele, 1994). In developing chick limb buds, beads soaked with all-trans-RA can mimic the effect of the zone of polarizing activity (ZPA) when implanted in the anterior margin of the bud and induce anteroposterior mirror-image duplications (Tickle et al., 1982;Tickle, 1991). A similar effect can also be induced by R A in developing anurans limbs (Migliorini Bruschelli and Rosi, 1971; Niazi and Saxena, 1978), but there is no evidence that such an effect can be induced in developing urodeles. During regeneration exogenous retinoids can affect patterning of all the three limb axes (Stocum, 1991), besides temporarily delaying blastema growth and inducing apoptosis in the mesenchymal cells of the blastema (Geraudie and Ferretti, 1997) and the appearance of ciliated and secretory cells in the WE (Maden, 1983; Scadding, 1989). However, the most striking effect of retinoids on limb regeneration in urodeles is the dose-dependent induction along the proximodistal axis of supernumerary structures, which are more proximal in identity than the amputated ones (Maden, 1982; Thomas and Stocum, 1984;Stocum, 1991). Because normally only structures distal to the amputation plane regenerate, the effect induced by retinoids along the proximodistal axis of the limb is often described as a “proximalization” of the positional identity of the blastema (Fig. 3). It has been reported that implantation of RA-soaked silastin blocks in regenerating axolotl limbs induces formation of supernumerary limbs (Maden et al., 1985). From this study, though, it is not clear whether this is due to localized toxicity, which would induce destruction of the tissue surrounding the RA-soaked implant and trigger formation of an accessory blastema, or to a specific effect of R A on the anteroposterior axis. This is a possibility because using surgically made half limbs it has been demonstrated that retinoic acid can affect the anteroposterior and dorsoventral axes of regenerating limbs (Kim and Stocum, 1986; Ludolph et al., 1990; Monkemeyer et al., 1992). In axolotl, equal concentrations of exogenously administered retinoids have apparently different effects on developing and regenerating limbs because the developing limb does not show duplications along the proximodistal or anteroposterior axis but is hypomorphic (Scadding and Maden, 1986). This may simply be due to differences in sensitivity to the toxic effects of exogenously administered retinoids on progenitor cells of developing and regenerating limbs. Such a possibility is consistent with the differences in cell phenotype and control of cell division in developing limb buds and regeneration blastemas, which were discussed previously. However, because RA-induced duplications along the proximodistal axis of developing limbs have not been observed in other species, it is also possible that such

GENE EXPRESSION DURING AMPHIBIAN LIMB REGENERATION

27

FIG. 3 RA effects on proximodistal duplication in the regenerating limb are dose dependent. Axolotl forelimbs were amputated at the wrist level as indicated by the line, and the skeletal pattern was assessed in whole mount limb preparations stained with methylene blue. (A) Control regenerate (injected intraperitoneally 4 days postamputation with the RA vehicle, DMSO); note that the eight carpals and four digits that had been removed have regenerated. (B) Low dose of RA; note the duplication of a third of the radius and ulna. (C) Intermediate dose of RA; note the duplication of complete radius and ulna. (D) Dose of RA inducing maximal duplication (100-150 pg RA/g body wt); note the complete duplication of humerus, radius and ulna and the partial shoulder girdle (arrow). u, ulna; r, radius (photographs provided by D. Stocum; from Stocum and Crawford (1987).

a response cannot be evoked in the developing urodele limb. In contrast, the lack of duplications along the anteroposterior axis may indeed be explained by the fact that the treatment by immersion used by Scadding and Maden (1986) does not allow a sufficient increase in R A concentration specifically in the anterior margin of the limb bud when used at low doses. Treatment with RA at higher concentrations, however, could have a generalized toxic effect resulting in hypomorphic limbs. Although the exact relationship between RA-induced effects during development and regeneration has not yet been fully elucidated, it is evident that exogenous retinoids can dramatically affect patterning of regenerating limbs. This raises the question of whether endogenous retinoids play a role in limb regeneration as suggested in limb development. In order to address this issue, various groups have taken complementary approaches and started to assess the presence of retinoids, of their cytoplasmic binding proteins, and of nuclear receptors in the limb blastema. The levels of various retinoids in the limb regeneration blastema of axolotl have been measured by using high-performance liquid chromatography (Scadding and Maden, 1994). This study has demonstrated that en-

28

JACQUELINE GERAUDIE AND PATRIZIA FERRETTI

dogenous all-trans-RA is indeed present in a gradient in regenerating limb blastemas and that it is three- to fivefold more concentrated posteriorly than anteriorly. In contrast, no significant anteroposterior gradient of either retinol, although its levels are slightly higher posteriorly, or 3,4dehydroretinol have been observed. The levels of another naturally occurring retinoid, 3,4-didehydroretinoic acid, which is a powerful morphogen in the chick wing bud (Thaller and Eichele, 1990; Eager e f al., 1991), have not been measured in this study. Although currently there are no data available on the concentrations of RA, retinol, and 3,4-dehydroretinol in axolotl limb buds, the RA gradient measured in the regeneration blastema parallels that observed in the chick wing bud (Eichele and Thaller, 1987), and therefore it has been suggested that endogenous RA may play the same role in developing and regenerating limbs at least on patterning of the anteroposterior axis (Scadding and Maden, 1994). Scadding and Maden (1994) have also compared retinoid levels in blastema1 cells and wound epidermis and found that RA and retinol levels in the mesenchyme and WE are very similar, whereas 3,4-dehydroretinol concentration is much higher in the mesenchyme than in the epidermis. It is not yet known, however, whether this vitamin A metabolite may also play a role in limb patterning. In contrast, another naturally occurring isomer of RA, 9-cis-retinoic acid, which is about 25 time more potent than RA in inducing anteroposterior duplications in the chick wing bud (Thaller et al., 1993), is synthesized and secreted by the WE of the newt limb blastema (Vivian0 et at., 1995).Its levels in the WE appear to be significantly higher than those of RA, and 9-cis-retinoic acid synthesis and release by the WE not only may be important for the maintenance of the WE but also may affect the behavior of the underlying mesenchymal cells. In fact, it has been shown that 9-cis-retinoic acid is more potent than RA in proximalizing regenerating axolotl limbs (Tsonis et al., 1994). Retinoid-binding proteins are cytoplasmic proteins that may not only translocate retinoids to the nucleus but also play an important role in controlling the amount of free retinoids present in the cell and, as a consequence, the level of activation of their nuclear receptors. Two retinoic acid-binding proteins, CRABP-I and CRABP-11, and two retinol-binding proteins, CRBP-I and CRBP-11,have been identified in birds and mammals, and their distribution during development has been thoroughly studied (Maden, 1991; Donovan et al., 1995). It has been shown that a CRABP is present in limb regeneration blastemas (Keeble and Maden, 1986; McCormick et al., 198S), and that its concentration is about fourfold higher in regenerating than in normal limbs of axolotl, by binding assays using [11,123H]all-truns-RA (Keeble and Maden, 1986). Ludolph et al. (1993) isolated a partial DNA sequence encoding for the axolotl CRABP-I, and Northern blot analysis indicates that CRABP-I is not expressed at detectable levels

GENE EXPRESSION DURING AMPHIBIAN LIMB REGENERATION

29

in the regeneration blastema, but it is present in unamputated limbs. Therefore, it is likely that the CRBP, which has been shown to be upregulated in the blastema by Keeble and Maden (1986), is the axolotl homolog of CRABP-11. It is not known whether the CRABP expressed in the regenerating axolotl limb is upregulated by RA as is CRABP-I1 in other species (Astrom et al., 1992), and whether it is asymmetrically distributed in the blastema. In chick limb buds CRABP is more concentrated anteriorly than posteriorly (Maden et al., 1988),whereas RA levels are higher posteriorly (Eichele and Thaller, 1987). It has therefore been suggested that CRABP might be involved in steepening the gradient of RA along the anteroposterior axis by sequestering it and decreasing its availability to the receptors (Maden et al., 1988). However, much of the recent work has undermined the RA gradient hypothesis and supported an involvement of RA in the establishment and maintenance of the zone of polarizing activity (Brockes, 1990; Noji et al., 1991; Wanek et al., 1991). Furthermore, the fact that significant differences in the level of different retinoids and of CRABPs (Scott et al., 1994) have been found in the developing limb of different species, together with the variable phenotype in CRABP transgenic mice (Wei and Chen, 1991), makes it very difficult to build a clear and solid picture of the developmental role of these molecules. Currently, one should neither assume that their role in limb development in different species is absolutely equivalent nor too easily extrapolate from limb development to regeneration. Much more information on urodele CRABPs and CRBPs, their regulation, and distribution will be needed in order to assess their possible role in modulating retinoid-induced effects in regenerating limbs. Whereas only one of the retinoid-binding proteins has been cloned in an urodele species and the function of this protein family has yet to be studied thoroughly in the limb regeneration blastema, more information concerning retinoid nuclear receptors and how they may mediate the multiple effects of retinoids in different systems has been accumulated in the past few years (Means and Gudas, 1995). The retinoid receptors (RARs and RXRs) belong to the steroidkhyroid hormone receptor superfamily and activate transcription by binding either as homodimers or as heterodimers to specific response elements of target genes. Three RARs, a,l3, and y, and three RXRs, a,p and y, with multiple spliced variants have been identified in mammals (Leid et al., 1992; Mangelsdorf et al., 1993). RA has been shown to have a high affinity for the RARs but low affinity for RXRs. The retinoid 3,4-didehydroretinoic acid, which can induce digit duplications in chick limb buds, also displays low affinity for RXR (Eager et al., 1991). In contrast, 9-cis-retinoic acid has been shown to bind with an affinity 40-fold higher than RA to the RXRs (Mangelsdorf et al., 1992). The fact that RARs and RXRs have different activities and distinct patterns of expression

30

JACQUELINE GERAUDIE AND PATRlZlA FERRET1

in the developing mammalian limb has raised the question of their role in the patterning of limb regenerates. Five RARs have so far been identified in the newt, N. viridescens, and three of them are clearly the newt homologs of mammalian RARal, RARa2, and RARP (Giguere et al., 1989; Ragsdale et al., 1989, 1992a,b). In contrast, no urodele RXR has yet been cloned. Full sequences of the newt R A R d and RARa2 are available (Ragsdale et aL, 1989, 1992a), but only the partial sequence of RARP has been published (Giguere etal., 1989).The other two newt RARs, although related to mammalian RARy, have been named RAR61 and RARE? because of significant differences in the amino acid sequence of regions A (N terminus) and F (C terminus), which in the case of the other RARs are highly conserved across species (Ragsdale et al., 1989, 1992b). RARSl contains two methionine initiators, and analysis with a panel of polyclonal antibodies specific for different regions of the protein suggests that they are both used in the limb regeneration blastema and produce 61a and 61b receptors (Hill et al., 1993). RARal, RARp,and RARS;! have also been detected in the blastema, but the levels of these transcripts appear to be lower than those of RAR61 (Giguere et al., 1989; Ragsdale et al., 1989, 1992b). The high levels of RARSl in limb and limb blastemas and its unique sequence characteristics in the A region, compared to all the other vertebrate RARs, have raised the question of whether expression of this receptor might be of particular significance within the context of regeneration and prompted its thorough characterization. Reactivity on blastema sections with anti-61 antibodies has shown that RARG is expressed in about 50% of the nuclei in the epidermis and mesenchyme of both normal and regenerating limbs. No differences were observed between proximal and distal blastemas, but a higher percentage of blastema cells expressing RAR61 (70-80%) was observed just beneath the WE (Hill e f al., 1993). Furthermore, no significant change in RARS1 expression was induced following injection of a proximalizing dose of RA. The lack of a gradient of RAR61 and of RA-induced changes in its levels and distribution observed by immunocytochemistry is consistent with the analysis of its expression carried out by RNAase protection (Ragsdale et al., 1992b). Because of the wide tissue distribution of RARG1 and its significant upregulation in the blastema compared to the normal limb, Hill et al. (1993) suggested that its expression may be causally linked to the ability of RARSpositive cells to be recruited into the blastema following amputation. If this were the case one would expect that the majority of cells throughout the blastema, and not just a small population of cells beneath the wound epidermis, would express this gene. The possible significance of this pattern of RAR6 expression has yet to be clarified.

GENE EXPRESSION DURING AMPHIBIAN LIMB REGENERATION

31

No information about the cellular distribution of RARa in normal and regenerating limbs is available, and the RARP transcript appears to be expressed throughout the blastema, without any apparent anteroposterior gradient or proximodistal gradient (Giguere et aZ., 1989). Analysis of RARG1 expression by immunocytochemistry, in conjunction with analysis of the expression of RARa and RARM by RNAase protection in cultured blastemal cells, has demonstrated that different types of receptors are coexpressed with RARG1 at least in subpopulations of these cells (Ragsdale et aZ., 1992a,b). In order to tackle the complex issue of the role of retinoids in limb regeneration and of which RARs may mediate the different responses to RA in this system, Brockes and colleagues have constructed a series of chimeric receptors and introduced them either into cultured cells (COS cells and limb blastema cells) or in the regenerating limb in v i v a Cultured blastemal cells were transfected either by microinjecting the plasmids into the nucleus or by using a biolistic particle delivery system (Brockes, 1994). In some experiments the transfected cells were reimplanted into the regenerating limb (Brockes, 1992). In vivo transfections were carried out by using the biolistic particle delivery system (Brockes, 1994; Pecorino et a[., 1994,1996). This experimental approach of combining the use of chimeric receptors and transfection in vitro and in vivo is proving very valuable for elucidating the role of retinoids and their receptors in the regenerating limb. Cultured newt limb cells transfected with a CAT reporter gene carrying the RA-responsive element of RARP respond to RA in a dose-dependent fashion and are diagnostic of environmental differences in proximal and distal blastemas (Brockes, 1992). In fact, when they are implanted at different axial levels in the regenerating limb of a newt injected with a proximalizing dose of RA, the level of normalized CAT activity is higher proximally than distally, suggesting the existence of a proximodistal gradient of RA. However, the nature of the environmental differences revealed by these experiments is not yet known. Because RA inhibits blastema growth for about a week after a single injection in vivo and dramatically decreases [3H]thymidine incorporation in cultured blastemal cells, RARa1-thyroid hormone T3 receptor a (TRa) and RAR61-TRa chimeras have been used to establish which of these RARs mediates RA-induced growth inhibition (Schilthuis et aZ., 1993). These studies have shown that stimulation of chimeric receptors with T3 can induce growth inhibition equivalent to that induced by RA in cultured blastemal cells transfected with the RARal-TRa receptor but not in cells transfected with RARG1-TRa. When WE cells are transfected in vivo with RARG1-TRa, T3 treatment can induce expression of the antigen WE3, a marker of secretory differentiation specific for the WE that is upregulated by RA (Tassava, 1992; see Section Il), in the transfected cells. In contrast,

32

JACQUELINE GERAUDIE AND PATRlZlA FERRETTI

no WE3 expression has been detected in WE cells transfected with the chimeric receptor RARal-TRa (Pecorino et al., 1994). Therefore, RARa1 is involved in growth inhibition but not in the regulation of WE3, and the opposite is true in the case of RARG1. However, RARG1 is unable to change the proximodistal identity of blastemal cells in the limb, whereas RAR62, although expressed at lower levels in limb blastemas, can mediate the proximalizing effect of RA (Pecorino et al., 1994, 1996). In conclusion, the ability of RA to affect limb patterning, its presence in regenerating limbs (Scadding and Maden, 1994; Vivian0 et al., 1995), and the demonstration that different retinoic acid receptors are expressed in the regenerate and mediate different functions (Schilthuis et al., 1993; Pecorino et al., 1994, 1996) support the view that endogenous retinoids play important and varied roles in limb regeneration. With the powerful tools now available and judiciously designed experiments, such as those discussed previously, it should be possible to definitely clarify this issue in the near future.

8. Homeobox-Containing Genes Developing limb buds of birds and mammals express numerous genes of the hox complex, which consists of four clusters, hoxu, hoxb, hoxc, and hoxd. A similar organization of the hox genes clusters has been recently demonstrated in urodeles (Belleville et ul., 1992). In higher vertebrates, genes of the hoxa and hoxd cluster are expressed in a coordinated fashion in specific overlapping domains along the proximodistal and anteroposterior axis, respectively, during limb development. This pattern of expression has led to the idea that they may encode position in the limb bud, and for this reason these hox gene clusters have been more thoroughly studied in developing limbs. Their possible role in setting the proximodistal and anteroposterior axes has been supported by a large bulk of experiments in chick and mouse in which overexpression and knockout of these genes have resulted in patterning abnormalities (IzpisuaBelmonte and Duboule, 1992; Morgan et al., 1992; Doll6 et al., 1993; Small and Potter, 1993; Davis and Capecchi, 1994; Davis et al., 1995; Favier et al., 1995; Yokouchi et al., 1995). Genes of the hoxb and hoxc clusters are also expressed during limb development, but no coordinated pattern of expression of the genes in developing limbs has so far become apparent. Nonetheless, interesting expression patterns in limbs of wild-type mice, and the limb phenotypes in mice transgenic for members of these clusters, suggest that hoxb and hoxc may also be pivotal to correct limb patterning. Hoxb genes may be important in inducing limb outgrowth at the appropriate level along the body axis, and it has been recently suggested that hoxc

GENE EXPRESSION DURING AMPHIBIAN LIMB REGENERATION

33

genes may be important in specifying the proximal region of the limb (humerus and femur) (Davis et al., 1995). It is therefore becoming apparent that different combinations of hox genes have a role in determining different regions of the limb during development (Ferretti and Tickle, 1997). Furthermore, some hox genes contain RAREs and are regulated by RA both in vitro and in vivo (Boncinelli et al., 1991; Marshall et al., 1994; Ogura and Evans, 1995). Because of their important role in limb development and because of their responsiveness to retinoids, a major effort in identifying urodele homologs of homeoboxcontaining genes, and in assessing their potential role in limb regeneration, has been undertaken. A list of homeobox-containing genes and of other genes believed to play a significant role in limb patterning that have been cloned in urodeles is given in Table IV. Nvhoxcd (former hvHbox I ) was the first hox gene to be isolated and characterized in urodeles (Savard et al., 1988). In species that cannot regenerate their limbs in adulthood, including Xenopus, hoxcd is expressed in developing but not in adult limbs. In contrast, in adult newts this gene is expressed in both normal and regenerating forelimbs and hindlimbs in a proximodistal gradient (Savard et al., 1988; Tabin, 1989; Savard and Tremblay, 1995). It has therefore been proposed that its expression may be causally related to the regenerative ability of the adult urodele limb. Another gene of the same cluster identified in N. viridescens is Nvhoxc10 (former hox-3.6) (Simon and Tabin, 1993). Nvhoxc-I0 mRNA is expressed in the normal limb and is upregulated during regeneration, reaching its highest level in the undifferentiated mid-bud blastema. No significant difference between proximal and distal blastemas is observed, and expression is restricted to the mesenchyme. With the progression of regeneration, Nvhoxc-10 expression is gradually downregulated to the levels detected in the normal limb. Nvhoxc-10, like a spliced from of Nvhoxcd (Savard et al., 1988; Savard and Tremblay, 1995), is also expressed in normal and regenerating tails but, unlike Nvhoxc-6, is not detected in either normal or regenerating forelimbs (Simon and Tabin, 1993). This parallels the pattern of expression of hoxc genes in developing mouse limbs. In fact, hoxc-ll is expressed in hindlimb but not in forelimb mesenchyme (Peterson et al., 1994). In contrast, hoxc-8 (former hox-3.1) appears to be expressed in both developing hindlimbs and forelimbs, as revealed by analysis of a reporter gene carried out in hoxc-8 null mutations obtained through homologous recombination (Le Mouellic et al., 1992). Because expression of both newt hoxc genes studied so far is maintained in the adult limb, it will be interesting to establish whether this property extends to other members of this cluster and could indeed be causally related to the regenerative capability of the urodele limb.

34

JACQUELINE GERAUDIE AND PATRlZlA FERRETTI

Numerous urodele genes belonging to the hoxa and hoxd clusters have been cloned (Table IV), but full DNA sequences are currently available only for hoxa-9 and hoxa-13, which have been isolated from an axolotl limb blastema cDNA library (Gardiner et al., 1995), and hoxa-11, hoxd-10, and hoxd-11, which have been cloned in the newt, N. viridescens (Brown and Brockes, 1991; Simon and Tabin, 1993; Beauchemin et al., 1994). The level of expression of the hoxd genes, like that of Nvhoxc-6, is higher in proximal than distal blastemas. Like the hoxc genes, hoxa and hoxd genes are also expressed in the undifferentiated mesenchyme but not in the WE of the regenerating limb. However, in contrast to Nvhoxc-6 and Nvhoxc10, most of these genes are not expressed in normal adult limbs but are induced following amputation. Only the newt hoxa-11 seems to depart from this behavior, and it is detected in the normal limb, primarily in muscle and bone. It will be interesting to establish whether the axolotl hoxa11 behaves like its newt counterpart because this would confirm that its regulation in the adult limb is different from that of other members of the same cluster. The fact that hoxa genes in the adult limb may not be expressed colinearily as during limb development is also supported by the work of Gardiner and colleagues (1995). Analysis of hoxa-9 and hoxa-11 transcripts in the axolotl regeneration blastemas by whole mount in situ hybridization has shown that, in most limbs, both genes are expressed beneath the WE 24 hours after amputation,independently from the level at which the limb was cut. Nonetheless, a detailed time course of the expression of hoxa genes between 0 and 48 h after amputation will have to be carried out to clearly establish that their activation in regenerating limbs does not follow the colinearity rule. The cellular localization of the hoxd genes and their spatiotemporal pattern of expression early after limb amputation are not yet known. However, analysis of hoxd-11 and hoxd-12 in regenerating zebrafish fins suggests that although expression of genes of this cluster is reinduced following amputation, their spatial pattern of expression in the regenerate is different (J. GCraudie et al., unpublished results) from that observed during development (Sordino et al., 1995). In addition, they both are detected at 24 h after amputation. Therefore, it appears that, although the same set of genes expressed during development is used to rebuild the missing part of a lower vertebrate appendage, the role they play in the process may be, at least at the early stages of regeneration, somehow different. Partial sequences of genes of the hoxb cluster have been isolated from regenerating axolotl limbs, but no information on their spatiotemporal pattern of expression during regeneration, or on whether they are expressed in normal limbs, is available (Gardiner et al., 1995). In the mouse, hoxb-8 (former hox-3.1) is expressed initially in the lateral plate mesoderm posterior to somite 9 and then in the posterior part of the mouse limb bud in a

GENE EXPRESSION DURING AMPHl6lAN LIMB REGENERATION

35

region corresponding to the ZPA (CharitC et al., 1994). On the basis of this pattern of expression and of the anteroposterior limb duplication observed in transgenic embryos ectopically expressing hoxb-8, it has been suggested that hoxb-8 may have a role in the establishment of the ZPA (Charitt el al., 1994). It will therefore be important to look into the expression of this neglected gene cluster in the limb blastema because it may prove very informative regarding the establishment of limbness and early patterning mechanisms in development and regeneration. Because of the patterning effects induced by R A on regenerating limbs, and the fact that R A has been shown to regulate expression of certain hox genes (Boncinelli et al., 1991; Marshall et al., 1994; Ogura and Evans 1995), much work has been aimed at assessing its effects on hox genes in regenerating limbs. Most of these studies have been carried out by RNAase protection because, until recently, there have been technical problems with the detection of hox transcripts by in situ hybridization in urodeles. Analysis of hox transcript by RNAase protection in the newt has been carried out, at the earliest, 5 days after R A injections. Because the initial effect induced by R A is inhibition of blastema growth, it is difficult to collect a sufficient amount of material that is not contaminated by stump tissues for RNA analysis at earlier stages. Under the experimental conditions used, hoxd10 expression appears to be upregulated by R A (Simon and Tabin, 1993), whereas the more 5' hoxd-11 is not (Brown and Brockes, 1991). In contrast, in axolotl limb blastemas analyzed by in situ hybridization, the more 5' hoxa-13, but not hoxa-9, appears to be downregulated by R A (Gardiner et al., 1995). In order to confirm that there is indeed a difference in the regulation of hox genes along the complex it will be important to examine the response to R A of other members of these two clusters, preferably in the same species. Besides the hox gene family, other families of homeobox-containing genes, such as the msx family (related to Drosophila msh) and the dlx family (related to Drosophila distal-less), appear to play important roles in the development of limbs and face, in which epithelial-mesenchymal interactions are pivotal to the developmental process. Genes belonging to the msx family are expressed both in the ridge and in the mesenchyme of developing appendages (limbs and fins) and are believed to be important in maintaining cells in an undifferentiated state in the progress zone (Hill et al., 1989; Robert et al., 1989, 1991; Davidson et al., 1991; Song et al., 1992; Vogel et al., 1995; Akimenko et al., 1994a). Interestingly, in higher vertebrates high levels of msx-1 in the fingertip of developing digits appear to correlate with significant regenerative capability (Reginelli et al., 1995). Only the newt msx-1 has been studied in detail during limb regeneration (Crews et al., 1995; Simon et al., 1995), but a partial sequence of axolotl

36

JACQUELINE GERAUDIE AND PATRlZlA FERRETTI

msx-2 has been isolated (see Table IV). Parallel to the pattern of expression observed in other vertebrates during limb development, the newt msx-1 is found both in the blastema mesenchyme and in the WE (Crews et aL, 1995). These authors, unlike Simon et al., (1995), detect comparable levels of msx2 in unamputated limbs by both Northern blot and RNAase protection analysis, and surprisingly the exposure times required to detect msx-l in the two studies are significantly different (14 days and 2 or 3 days, respectively). Whether these discrepancies could be partly due to the different probes used to detect the transcript 5' coding region in the case of Crews et al. and 3' untranslated region in the case Simon et al. and/or to the presence/ absence in the normal limb samples of the fingertips, which appear to be blastema-like (Ferretti and Ghosh, 1997), is currently unclear. Finally, functional studies have indicated that in urodeles, unlike in mammals, msxI does not seem to affect either proliferation or differentiation of newt myogenic cells in culture (Crews et al., 1995). More work will be required to clarify the different expression results obtained and to establish which role msx-I might play in limb regeneration. On the basis of much work carried out in Drosophila and expression in the apical ectodermal ridge of developing limbs and fins (Akimenko et al., 1994b), genes of the dlx family are thought to play a role in limb outgrowth and in the establishment of proximodistal pattern (Cohen et al., 1989; Doll6 et al., 1992; Bulfone et al., 1993; Simeone el aL, 1994; Diaz-Benjumena et al,, 1994; Morass0 et al., 1995). Two newt dlx genes have been identified, NvHBox-4 and NvHBox-5 (Beauchemin and Savard, 1992); the homeodomain of NvBox-4 is identical at the amino acid level of that of mouse, axolotl, and Pleurodeles dlx-3 (Mullen et al., 1996; Nicolas et al., 1996). Analysis by Northern blot of newt dlxs has shown that both genes are ubiquitously expressed in the adult skin, and that their levels in normal and regenerating limbs, at different axial levels, are comparable. In contrast, the axolotl dlx-3 has recently been shown to be upregulated in the WE of regenerating limbs by both Northern blot and in situ hybridization (Mullen et al., 1996). Furthermore, its levels are higher in the WE of distal blastemas than in that of proximal blastemas. In denervated blastemas both regeneration and expression of dlx-3 are inhibited, but both effects can be reversed by FGF-2-soaked beads. Dlx-3 is also downregulated by R A treatment, but by the time this compound has cleared from the limb and growth resumes, dlx-3 has returned to normal levels. Together, these results suggest that expression of dlx-3 and limb outgrowth may be linked. If NvHBox-4 and axolotl dlx-3 are truly homologous, it is difficult to understand why their patterns of expression and regulation appear to be rather different in two related species with comparable regenerative capabilities.

GENE EXPRESSION DURING AMPHIBIAN LIMB REGENERATION

37

Recently, by using a differential display technique, a newt gene belonging to the T-box family of transcription factors, which includes brachyury, has been isolated (Simon et al., 1997). This gene appears to be upregulated in forelimb but not in hindlimb blastemas. Given the proposed important role of T-box genes in the evolution of fin and limb morphogenesis (GibsonBrown et al., 1996),the newt T-box gene identified might play an important role in controlling forelimb “identity” in the regenerate. C. Segment Polarity Genes

Although some of the urodele segment polarity genes have been cloned (Table 111), information about their distribution and possible role in the regenerating limbs is still minimal. At the last International Conference on Limb Development and Regeneration (1996), it was shown that two hedgehog genes sonic hedgehog (shh) and banded hedgehog (bhh), are expressed in developing and regenerating limbs. Whereas bhh expression is clearly detected throughout developing and regenerating limb buds (D. Stark, P. Gates, J. Brockes, and P. Ferretti, unpublished results), it has been suggested that shh has a posterior domain of expression in the regenerating blastema as well as in the developing limb bud (Imokawa and Yoshizato, 1997; Endo et al., 1997). If this is indeed the case, shh distribution in newt limb blastemas would be different from that observed in another regenerating appendage, the zebrafish fin. In fact, although shh is expressed in posterior mesenchyme of early fin buds (Akimenko and Ekker, 1995), no posterior localization is observed when its expression is reinduced following fin amputation (Laforest et al., manuscript in preparation). Because indiun hedgehog, the mammalian homolog of bhh, is not expressed in early limb buds, it is conceivable that expression of bhh in the developing and regenerating newt limbs may be related to the regenerative capability in this species. Bhh may either play a role in dedifferentiation, or be implicated in the establishment of positional identity since it is significantly upregulated by RA. It is not clear how expression of hedgehog genes in the blastema is regulated. It will be important to establish whether in the regenerating limb there is a loop including hedgehog genes, FGF, and RA that links outgrowth with patterning as suggested in the chick limb bud (Izpisua-Belmonte et al., 1992; Laufer er al., 1994; Niswander er al., 1994; Stratford et aL, 1996). If this is the case, application of both FGF and RA to digit stumps in mammals may constitute a first step toward inducing regeneration in higher vertebrates. In summary, the bulk of data currently available on patterning of the regenerating limb support the view that the same key molecules are used to build both embryonic and adult limbs. However, there are indications

38

JACQUELINE GERAUDIE AND PATRlZlA FERRET1

that at the early bud stages they may be used in a slightly different fashion, and this might be due to differences in the origin of the progenitor cells during development and regeneration and to the presence of positional cues in the stump of the adult newt limb.

V. Concluding Remarks Much information on the molecular basis of regeneration has been gained since Spallanzani’s (1768) first report of this extraordinary phenomenon. In the past decade in particular, the application of the most advanced experimental tools has given great impetus to the molecular analysis of epimorphic regeneration. In this article we have attempted to give a comprehensive overview of the expression patterns and possible roles of molecules, a few specific to regeneration and many common to development, that have been studied within the context of limb regeneration in urodeles. Although much work has yet to be done if we are to induce significant regeneration in mammals, the rapidly growing understanding of this phenomenon in amphibians is shifting this prospect from the realm of science fiction to that of possibility. Acknowledgments The authors thank all the colleagues for providing data prior to their publication and Dr. D. Stocum for supplying Figs. 1 and 2. JG was partly supported by Laboratoire de Biologie du DCveloppement des Poissons (Professor J. M. Vernier), Universite Paris-Sud XI and UA CNRS 1134. PF was supported by The Wellcome Trust and the MRC.

References Akimenko, M. A,, and Ekker, M. (1995). Anterior duplication of the Sonic hedgehog expression pattern in the pectoral fin buds of zebrafish treated with retinoic acid. Dev. Biol. 170,243-247. Akimenko, M. A., Johnson, S. L., Westerfield, M., and Ekker, M. (1994a). Differential induction of four msx homeobox genes during fin development and regeneration in zebrafish. Development 121,347-357. Akimenko, M. A., Ekker, M., Wegner, J., Lin, W., and Westerfield, M. (1994b). Combinatorial expression of three zebrafish genes related to distal-less: Part of a homeobox gene code for the head. .I. Meurosci. 14, 3475-3486. Albert, P., and Boilly, B. (1988). Effect of transferrin on amphibian limb regeneration: A blastema cell culture study. Roux’s Arch. Dev. Biol. 197, 193-196. Albert, P., Boilly, B., Courty, J., and Barritault, D. (1987). Stimulation in cell culture of mesenchymal cells of newt limb blastemas by EDGF I or I1 (basic or acidic FGF). Cell Differ. 21, 63-68. AndCol, Y., Gusse, M., and MCchali, M. (1990). Characterization and expression of a Xenopus ras during oogenesis and development. Dev. Biol. 139,24-34.

GENE EXPRESSION DURING AMPHIBIAN LIMB REGENERATION

39

Astrom, A., Pettersson, U., and Voorhees, J. J. (1992). Structure of the human cellular retinoic acid-binding protein I1 gene. Early transcriptional regulation by retinoic acid. J. Biol. Chem. 267,25251-25255. Barrandon, Y . (1993). The epidermal stem cell: An overview. In “Epithelial Stem Cells. Part 1. Keratinocytes. Seminars in Developmental Biology” (C. Potten, Ed.). Academic Press, New York. Beauchemin, M., and Savard, P. (1992). Two distal-less related homeobox-containing genes expressed in regeneration blastemas of the newt. Dev. B i d . 154,55-65. Beauchemin, M., Noiseux, N., Tremblay, M., and Savard, P. (1994). Expression of Hox A l l in the limb and the regeneration blastema of adult newt. Int. J. Dev. Bid. 38,641-649. Belleville, S., Beauchemin, M., Tremblay, M., Noiseux, N., and Savard, P. (1992). Homeoboxcontaining genes in the newt are organized in clusters similar to other vertebrates. Gene 114, 179-186. Boilly, B., and Albert, P. (1988). Blastema cell proliferation in vitro: Effects of limb amputation on the mitogenic activity of spinal cord extracts. Biol. Cell 62, 183-187. Boilly, B., and Albert, P. (1990). in vitro control of blastema cell proliferation by extracts from epidermal cap and mesenchyme of regenerating limbs of axolotls. Roux’s Arch. Dev. Biol. 198, 443-441. Boilly, B., Cavanaugh, K. P., Thomas, D., Hondermarck, H., Bryant, S. V., and Bradshaw, R. A. (1991). Acidic fibroblast growth factor is present in regenerating limb blastemas of axolotls and binds specifically to blastema tissue. Dev. B i d . 145, 302-310. Boilly, B., Oudghir, M., Deudon, E., Boilly-Marer, Y., and Hondermarck, H. (1995). Nerve dependent sulphated glycosaminoglycan synthesis in limb regeneration of the newt Pleurodeles wall. Roux’s Arch. Dev. Biol. 204, 509-512. Boncinelli, E., Simeone, A., Acampora, D., and Mavilio, F. (1991). Hox gene activation by retinoic acid. Trends Genet. 7,329-334. Brockes, J. P. (1984). Mitogenic growth factors and nerve dependence of limb regeneration. Science 225, 1280-1287. Brockes, J. P. (1987). The nerve dependence of amphibian limb regeneration. J. Exp. B i d . 132,79-91. Brockes, J . P. (1989). Retinoids, homeobox genes, and limb morphogenesis. Neuron 2,12851294. Brockes, J . P. (1990). Retinoic acid and limb regeneration. J. Cell Sci. Suppl. 13, 191-198. Brockes, J. P. (1992). Introduction of a retinoid reporter gene into the urodele limb blastema. Proc. Natl. Acad. Sci. USA 89, 11386-11390. Brockes, J. P. (1994). New approaches to amphibian limb regeneration. Trends Genet. 10, 169-173. Brockes, J. P. (1998). Progenitor cells for regeneration: Origin by reversal of the differentiated state. In “Cell and Molecular Biology of Regeneration” (P. Ferretti and J. GCraudie, Eds.), pp. 63-77. Wiley, Chichester, UK. Brockes, J. P., and Kintner, C . R. (1986). Glial growth factor and nerve-dependent proliferation in the regeneration blastema of urodele amphibians. Cell 45,301-306. Brown, R., and Brockes, J. P. (1991). Identification and expression of a regeneration-specific homeobox gene in the newt limb blastema. Development 111,489-496. Bryant, S. V., and Gardiner, D. M. (1992). Retinoic acid, local cell-cell interactions, and pattern formation in vertebrate limbs. Dew. Biol. 152, 1-25. Bulfone, A,, Kim, H. J., Puelles, L., Porteus, M. H., Grippo, J. F., and Rubenstein, J. L. (1993). The mouse Dlx-2 (Tes-I) gene is expressed in spatially restricted domains of the forebrain, face and limbs in midgestation mouse embryos. Mech. Dev. 40,129-140. Busse, U., and Seguin, C. (1993). Isolation of cDNAs for two closely related members of the axolotl Wnr family, Awnt-SA and Awnt-SB, and analysis of their expression during development. Mech. Dev. 40,63-72.

40

JACQUELINE GERAUDIE AND PATRlZlA FERRETTI

Cameron, J. A., Hilgers, A. R., and Hintenberger, T. J. (1986). Evidence that reserve cells are a source of a regenerated adult newt muscle in vitro. Nature (London) 321, 607-610. Carlone, R. L., and Mescher, A. L. (1985). Trophic factors from nerves. In “Regulation of Vertebrate Limb Regeneration” (R. E. Sicard, Ed.), pp. 93-105. Oxford Univ. Press, London. Carlson, B. M. (1979). The relationship between the tissue and epimorphic regeneration of muscle. In “Muscle Regeneration” (A. Mauro, Ed.), pp. 57-71. Raven Press, New York. Carlson, B. M. (1988). Some aspects of regeneration in skeletal and cardiac muscle. In “Recent Trends in Regeneration Reseach” (V. Kiortsis, S. Koussoulakos, and H. Wallace, Eds.), NATO AS1 Series, Life Sciences, Vol. 12, pp. 147-157. Plenum. Casimir, C. M., Gates, P. B., Patient, R. K., and Brockes, J. P. (1988).Evidence for dedifferentiation and metaplasia in amphibian limb regeneration from inheritance of DNA methylation. Development 104,657-668. Castilla, M., and Tassava, R. A. (1992). Extraction of the WE3 antigen and comparison of reactivities of mAb WE3 and WE4 in adult newt regenerate epithelium and body tissues. In “Monographs in Developmental Biology” (H. W. Sauer, Ed.), Vol. 23, pp. 116-130. Karger, Basel. Caubit, X., Nicolas, S., Shi, D. L., and Le Parco, Y. (1997). Reactivation and graded expression pattern of Wnt-ZOagene during early regeneration stages of adult tail in Amphibian urodele Pleurodeles waltl. Dev. Dyn.208, 139-148. CharitC, J., de Graaff, W., and Deschamps, J. (1994). Ectopic expression of Hoxb-8 causes duplication of the ZPA in the forelimb and homeotic transformation of axial structures. Cell 78, 589-601. Choo, A. F., Logan, D. M., and Rathbone, M. (1978). Nerve trophic effects: An in vitro assay for factors involved in regulation of protein synthesis in regenerating amphibians limbs. J. EXP.2001.206,347-354. Cohen, S. M., Bronner, G.,Kuttner, F., Jurgens, G., and Jackle, H. (1989). Distal-less encodes a homeodomain protein required for limb development in Drosophila. Nature 338,432-434. Colquhoun, J. E., and Dresden, M. H. (1983). Prolyl hydroxylase activity during newt forelimb regeneration. Dev. Biol. 95, 193-199. Corcoran, J. P. T., and Ferretti, P. (1997). Keratin 8 and 18 expression in mesenchymal progenitor cells of regenerating limbs is associated with cell proliferation and differentiation. Dev. Dyn. 210,355-370. Crews, L., Gates, P. B., Brown, R., Joliot, A., Foley, C., Brockes, J. P., and Gann, A. A. (1995). Expression and activity of the newt Msx-I gene in relation to limb regeneration. Proc. R. SOC. London B. Biol. Sci. 259,161-171. Crossley, P. H., Minowada, G. Macarthur, C. A., and Martin, G. R. (1996). Roles for FGF8 in the induction, initiation, and maintenance of chick limb development. Cell 84, 127-136. Daniels, K., and Solursh, M. (1991). Modulation of chondrogenesis by the cytoskeletal and extracellular matrix. J. Cell Sci. 100,249-254. Davidson, D. R., Crawley, A., Hill, R. E., and Tickle, C. (1991). Position-dependentexpression of two related homeobox genes in developing vertebrate limbs. Nature (London) 352, 429-431. Davis, A. P., and Capecchi, M. R. (1994). Axial homeosis and appendicular skeleton defects in mice with a targeted disruption of hoxd-21. Development 1243,2187-2198, Davis, A. P., Witte, D. P., Hsieh-Li, H. M., Potter, S. S., and Capecchi, M. (1995). Absence of radius and ulna in mice lacking hoxa-11 and hoxd-11. Nature (London) 375,791-795. Del Rio-Tsonis, K., Washabaugh, C. H., and Tsonis, P. A. (1992). The mutant axolotl short toes exhibits impaired limb regeneration and abnormal basement membrane formation. Proc. Natl. Acad. Sci. USA 89,5502-5506. Diaz-Benjumena,F. J., Cohen, B., and Cohen, S. M. (1994). Cell interaction between compartments establishes the proximal-distal axis of Drosophila legs. Nature 372, 175-179.

GENE EXPRESSION DURING AMPHIBIAN LIMB REGENERATION

41

Dinsmore, C. E., and Mescher, A. L. (1998). The role of the nervous system in regeneration. In “Cell and Molecular Biology of Regeneration” (P. Ferretti and J. GCraudie, Eds.), pp. 79-108. Wiley, Chichester, UK. DollC, P., Price, M., and Duboule, D. (1992). Expression of the murine DLx-I homeobox gene during facial, ocular and limb development. Differentiation 49, 93-99. DollC, P., Dierich, A., LeMeur, M., Schimmang,T., Schuhbaur,B., Chambon, P., and Duboule, D. (1993). Disruption of the Hoxd-13 gene induces localized heterochrony leading to mice with neotenic limbs. Cell 75,431-441. Donaldson, D. J., and Mahan, J. T. (1987). Newt epidermal cell migration in vitro and in vivo appears to involve Arg-Gly-Asp-Ser receptors. J. Cell Sci. 87,525-534. Donovan, M., Olofsson, B., Gustafson, A. L., Dencker, L., and Eriksson, U. (1995). The cellular retinoic acid binding proteins. J. Steroid Biochem. Mol. Biol. 53, 459-465. Dresden, M. H. (1969). Denervation effects on newt limb regeneration: DNA, RNA and protein synthesis. Dev. Biol. 19,311-320. Dresden, M. H., and Gross, J. (1970). The collagenolytic enzyme of the regenerating limb of the newt Triturus viridescens. Dev. Biol. 22, 129-137. Duellman, W. E., and Trueb, L. (1986). “Biology of amphibians.” McGraw-Hill, New York. Eager, N. S., Rowe, A., and Brickell, P. M. (1991). A member of the chicken RXR family of nuclear receptors activates transcription in response to retinoic acid. FEBS Lett. 292, 103-106. Eichele, G., and Thaller, C. (1987). Characterization and concentration gradients of a morphogenetically active retinoid in the chick limb bud. J. Cell Biol. 105, 1917-1923. Endo, T., Yokoyama, H., Tamura, K., and Ide, H. (1997). Shh expression in developing and regenerating limb buds of Xenopus laevis. Dev. Dyn. 209,227-232. Estrada, C. M., Park, C. D., Castilla, M., and Tassava, R. A. (1993). Monoclonal antibody WE6 identifies an antigen that is up-regulated in the wound epithelium of newts and frogs. In “Limb Development and Regeneration, Part A” (J. F. Fallon, P. F. Goetinck, R. 0. Kelley, and D. L. Stocum, Eds.), pp. 271-282. Wiley, New York. Fallon, J. F., Lopez, A., Ros, M., Savage, M. P., Olwin, B. B., and Simandl, B. K. (1994). FGF2: Apical ectodermal ridge growth signal for chick limb development. Science 264,104-107. Favier, B., LeMeur, M., Chambon, P., and Doll& P. (1995). Axial skeleton homeosis and forelimb malformations in Hoxd-11 mutant mice. Proc. Natl. Acad. Sci. USA 92,310-314. Fekete, D., and Brockes, J. P. (1987). A monoclonal antibody detects a differencein the cellular composition of developing and regenerating limbs of newts. Development 99, 589-602. Fekete, D., Ferretti, P., Gordon, H., and Brockes, J. P. (1987). Ontogeny of nerve dependence of amphibian limb regeneration. In “From Message to Mind Directions in Developmental Neurobiology” (S. S. Easter, K. F. Barrald, and B. M. Carlson, Eds.), pp. 29-42. Sinauer, Sunderland, MA. Ferretti, P., and Brockes, J. P. (1988). Culture of newt cells from different tissues and their expression of a regeneration associated antigen. J. Exp. Zool. 247, 77-91. Ferretti, P., and Brockes, J. P. (1990). The monoclonal antibody 22/18 recognizes a conformational change in an intermediate filament of the newt, Notophthalmur viridescens, during limb regeneration. Cell Tissue Res. 259, 483-493. Ferretti, P., and Brockes, J. P. (1991). Cell origin and identity in limb regeneration and development. Glia 4,214-224. Ferretti, P., and Ghosh, S. (1997). Expression of regeneration-associatedcytoskeletalproteins reveals differences and similarities between regenerating organs. Dev. Dyn. 210,288-304. Ferretti, P., and Tickle, C. (1997). The limbs. In “Embryos, Genes and Birth Defects” (P. Thorogood, Ed.), pp. 101-132. Wiley, Chichester, UK. Ferretti, P., Fekete, D. M., Patterson, M., and Lane, E. B. (1989). Transient expression of simple epithelial keratins by mesenchymal cells of regenerating newt limb. Dev. Biol. 133,415-424.

42

JACQUELINE GERAUDIE AND PATRlZlA FERRETTI

Ferretti, P., Brockes, J. P., and Brown, R. (1991). A new type I1 keratin restricted to normal and regenerating limbs and tails is responsive to retinoic acid. Development 111,497-507. Ferretti, P., Corcoran, J. P. T., and Ghosh, S. (1993). Expression and regulation of keratins in the wound epithelium and mesenchyme of the regenerating newt limb. Prog. Clin. Biol. Res. 383A, 261-269. Foty, R. A., and Liversage, R. A. (1993). Detection of insulin receptors in newt liver and forelimb regenerates and the effects of local insulin deprivation on epimorphic regeneration. J. EXP. ZOO^. 266,299-311. Gardiner, D. M., Muneoka, K., and Bryant, S. V. (1986). The migration of dermal cells during blastema formation in axolotls. Dev. Biol. 118,488-493. Gardiner, D. M., Blumberg, B., and Bryant, S. V. (1993). Expression of homeobox genes in limb regeneration. Prog. Clin. Biol. Res. 383A, 31-40. Gardiner, D. M., Blumberg, B., Komine, Y., and Bryant, S. V. (1995). Regulation of HoxA expression in developing and regenerating axolotl limbs. Development U1, 1731-1741. Geraudie, J., and Ferretti, P. (1997). Correlation between RA-induced apoptosis and patterning defects in regenerating fins and limbs. In/. J. Dev. Biol. 41, 529-532. Gkraudie, J., and Singer, M. (1978). Nerve-dependent macromolecular synthesis in the epidermis and blastema of the adult newt limb regenerate. J. Exp. Zool. 203,455-460. Geraudie, J., and Singer, M. (1981). Scanning electron microscopy of the normal and denervated limb regenerate in the newt, Notophthalmus, including observations on embryonic amphibian limb-bud mesenchyme and blastema of the fish-fin regenerate. Am. J. Anat. 162,7347. GCraudie, J., Hourdry, J., Vriz, S., Singer, M., and Mechali, M. (1990). Enhanced c-myc gene expression during forelimb regeneration outgrowth in the young Xenopus luevis. Proc. Nutl. Acad. Sci. USA 87,3797-3801. Gibson-Brown, J. J., Agulnik, S. I., Chapman, D. L., Alexiou, M., Garvey, N., Silver, L. M., and Papaioannou, V. E. (1996). Evidence of a role for T-box genes in the evolution of limb morphogenesis and the specification of forelimb/hindlimb identity. Mech. Dev. 56, 93-101. Giguere, V., Ong, E. S.,Evans, R. M., and Tabin, C. J. (1989). Spatial and temporal expression of the retinoic acid receptor in the regenerating amphibian limb. Nature (London) 337, 566-569. Globus, M., and Alles, P. (1990). A search for immunoreactive substance P and other neural peptides in the limb regenerate of the newt, Notophthalmus viridescens. J. Exp. Zool. 254, 165-176. Globus, M., Vethamany-Globus, S., Kesik, A., and Milton, G. (1983). Roles of neural peptide substance P and calcium in the newt Notophthalmus viridescens. In “Limb Development and Regeneration” (J. F. Fallon and A. I. Caplan, Eds.), pp. 513-523. A. R. Liss, New York. Goldhamer, D. J., and Tassava, R. A. (1987). An analysis of proliferative activity in innervated and denervated forelimb regenerates of the newt, Notophthalmus viridescens. Development 100,619-628. Goldhamer, D. J., Tomlinson, B. L., and Tassava, R. A. (1989). A developmentally regulated wound epithelial antigen of the newt limb regenerate is also present in a variety of secretory/ transport cell types. Dev. B i d . 135, 392-404. Gordon, H., and Brockes, J. P. (1988). Appearance and regulation of an antigen associated with limb regeneration in Nofophthulmus viridescens. J . Exp. Zool. 247, 232-243. Gospodarowicz, D., and Mescher, A. L. (1980). Fibroblast growth factor and the control of vertebrate regeneration and repair. Ann. N. Y. Acad. Sci. 339, 151-174. Griffin, K. J. P., Fekete, D. M., and Carlson, B. M. (1987). A monoclonal antibody stains myogenic cells in regenerating newt muscle. Development 101, 267-277. Grillo, H. L., Lapikre, C. M., Dresden, M. H., and Gross, J. (1968). Collagenolytic activity in regenerating forelimbs of the adult newt (Triturus viridescens). Dev. Biol. 17, 571-583. Gulati, A. K., Zalewski, A. A., and Reddi, A. (1983). An immunofluorescent study of the distribution of fibronectin and laminin during limb regeneration in the adult newt. Dev. Biol. 46,355-365.

GENE EXPRESSION DURING AMPHIBIAN

LIMB REGENERATION

43

Hay, E. D. (1959). Microscopic observations of muscle dedifferentiation in regenerating Ambiystoma limbs. Dev. Biol. 1, 555-585. Hay, E. D., and Fishman, D. A. (1961). Origin of the blastema in regenerating limbs of the newt Triturus viridescens. Dev. Biol. 3,26-59. Hill, D. S., Ragsdale, C. W., Jr., and Brockes, J. P. (1993). Isoform-specific immunological detection of newt retinoic acid receptor d l in normal and regenerating limbs. Development 117, 937-945. Hill, R. E., Jones, P. F., Rees, A. R., Sime, C. M., Justice, M. J., Copeland, N. G., Jenkins, N. A., Graham, E., and Davidson, D. R. (1989). A new family of mouse homeobox containing genes: Molecular structure, chromosomal location and developmental expression of Hox7.1. Genes Dev. 3, 26-37. Hinds, P. W., and Weinberg, R. A. (1994). Tumor suppressor genes. Curr. Opin. Gene Dev. 4,135-141. Imokawa, Y., and Yoshizato, K. (1997). Expression of Sonic hedgehog gene in regenerating newt limb blastemas recapitulates that in developing limb buds. Proc. Nutl. Acud. Sci. USA 94, 9159-9164. Izpisua-Belmonte. J. C., and Duboule, D. (1992). Homeobox and pattern formation in the vertebrate limb. Dev. Biol. 152,26-36. Izpisua-Belmonte, J. C., Brown, J. M., Duboule, D., and Tickle, C. (1992). Expression of hox-4 genes in the chick wing links pattern-formation to the epithelial mesenchymal interactions that mediate growth. EMBO J. 11, 1451-1457. Iten, L. E., and Bryant, S. V. (1973). Forelimb regeneration from different levels of amputation in the newt, Notophthalmus viridescens: Length, rate and stages. Rou’s Arch. Dev. Biol. 173,263-282. Jabaily, J., and Singer, M. (1977). Neurotrophic stimulation of DNA synthesis in the regenerating forelimb of the newt, Triturus. J. Exp. Zool. 199,251-256. Keeble, S., and Maden, M. (1986). Retinoic acid-binding protein in the axolotl: Distribution in mature tissue and time of appearance during limb regeneration. Dev. Biol. 117,435-441. Kiffmeyer, W. R., Tomusk, E. V., and Mescher, A. L. (1991). Axonal transport and release of transferrin in nerves of regenerating amphibian limbs. Dev. Biol. 147,392-402. Kim, W. S., and Stocum, D. L. (1986). Retinoic acid modifies positional memory in the anteroposterior axis of regenerating axolotl limbs. Dev. Biol. 114, 170-179. Kintner, C., and Brockes, J. P. (1984). Monoclonal antibodies identify blastemal cells derived from dedifferentiating muscle in newt limb regeneration. Nature (London) 308,67-69. Kintner, C., and Brockes, J. P. (1985). Monoclonal antibodies to the cells of a regenerating limb. J. Embryol. Exp. Morphol. 89, 37-51. Klatt, K. P., Yang, E. V., and Tassava, R. A. (1992). Monoclonal antibody MT2 identifies an extracellular matrix glycoprotein that is colocalized with tenascin during adult newt limb regeneration. Differentiation 50, 133-140. Koshiba, K., Tamura, K., and Ide, H. (1994). Expression of regeneration-associated antigens in normal and retinoid-treated regenerating limbs of Ambystoma mexicanum. Dev. Growth Differ. 36, 357-364. Kostakopoulou, K., Vogel, A,, Brickell, P., and Tickle, C. (1996). “Regeneration” of wing bud stumps of chick embryos and reactivation of Msx-1 and Shh expression in response to FGF-4 and ridge signals. Mech. Dev. 55, 119-131. Laufer, E., Nelson, C. E., Johnson, R. L., Morgan, B. A,, and Tabin, C. (1994). Sonic hedgehog and fgf-4 act through a signaling cascade and feedback loop to integrate growth and patterning of the developing limb bud. Cell 79, 993-1003. Lebowitz, P., and Singer, M. (1970). Neurotrophic control of protein synthesis in the regenerating limb of the newt, Triturus. Nature (London) 225, 824-827. Leid, M., Kastner, P., and Chambon, P. (1992). Multiplicity generates diversity in the retinoic acid signalling pathways. Trends Biochem. Sci. 17,427-433.

44

JACQUELINE GERAUDIE AND PATRIZIA FERRE?TI

Lemaitre, J. M., Mkchali, M., and GCraudie, J. (1992). Nerve-dependent expression of c-myc protein during forelimb regeneration of Xenopus laevis froglets. Inf.J. Dev. Biol. 36,483-489. Le Mouellic, H., Lallemand, Y . ,and BrOlet, P. (1992). Homeosis in the mouse induced by a null mutation in the Hox 3.1 gene. Cell 69, 251-264. Liversage, R. A. (1991). Origin of the blastema cells in epimorphic regeneration of urodele appendages: A history of ideas. In “A History of Regeneration Research” (C. E. Dinsmore, Ed.), pp. 179-199. Cambridge Univ. Press, Cambridge, UK. Lo, D. C., Allen, F., and Brockes, J. P. (1993). Reversal of muscle differentiation during urodele limb regeneration. Proc. Natl. Acad. Sci. USA 90,7230-7234. Lohnes, D., Mark, M., Mendelsohn, C., DollC, P., Dierich, A,, Gorry, P., Gansmuller, A,, and Chambon, P. (1994). Function of the retinoic acid receptors (RARs) during development. (I). Craniofacial and skeletal abnormalities in RAR double mutants. Development 120, 2723-2748. Ludolph,D. C.,Cameron, J. A.,andStocum,D. L. (1990).Theeffect ofretinoicacidonpositional memory in the dorsoventral axis of regenerating axolotl limbs. Dev. Biol. 140,41-52. Ludolph, D. C., Cameron, J. A,, Neff, A. W., and Stocum, D. L. (1993). Cloning and tissuespecific expression of the axolotl cellular retinoic acid-binding protein. Dev. Growth Differ. 35,341-347. Ludolph, D. C., Neff, A. W., Parker, M. A., Mescher, A. L., Smith, R. C. and Malacinski, G. M. (1995). Cloning and expression of the axolotl proto-oncogene ski. Biochem. Biophys. Acta 1260, 102-104. Lyons, G. E., and Buckingham, M. (1992). Developmental regulation of myogenesis in the mouse. Sem. Dev. Biol. 3,243-253. Maden, M. (1977). The role of Schwann cells in paradoxical regeneration in the axolotl. J. Embryol. Exp. Morphol. 41, 1-13. Maden, M. (1982). Vitamin A and pattern formation in the regenerating limb. Nature (London) 295,672-675. Maden, M. (1983). The effect of vitamin A on the regenerating axolotl limb. J. Embryol. Exp. Morphol. 77,273-295. Maden, M. (1991). Retinoid-binding proteins in the embryo. Sem. Dev. Biol. 2, 161-170. Maden, M., Keeble, S., and Cox, R. A. (1985). The characteristics of local application of retinoic acid to the regenerating axolotl limb. R o u ’ s Arch. Dev. Biol. 194,228-235. Maden, M., Ong, D. E., Summerbell, D., and Chytil, F. (1988). Spatial distribution of cellular protein-binding to retinoic acid in the chick limb bud. Nature (London) 335, 733-735. Maier, C. E., and Miller, R. H. (1992). In vifro and in vivo characterization of blastemal cells from regenerating newt limbs. J. Exp. Zool. 262, 180-192. Maier, C. E., Watanabe, M., Singer, M., McQuarrie, I. G., Sunshine, J., and Rutishauser, U. (1986). Expression and function of neural cell adhesion molecule during limb regeneration. Proc. Natl. Acad. Sci. USA 86, 8395-8399. Mailman, M. L., and Dresden,M. H. (1976).Collagen metabolism in the regenerating forelimb of Nofophthalmus viridescens: Synthesis, accumulation and maturation. Dev. Biol. 50,378-394. Mangelsdorf, D. J., Borgmeyer, U., Heyman, R. A., Yang Zhou, J., Ong, E. S., Oro, A. E., Kakizuka, A,, and Evans, R. M. (1992). Characterization of three RXR genes that mediate the action of 9-cir retinoic acid. Genes Dev. 6,329-344. Mangelsdorf, D. J., Kliewer, S. A., Kakizuka, A., Umesono, K., and Evans, R. M. (1993). Retinoid receptors. Recent Prog. Hormone Res. 48,99-121. Marshall, H., Studer, M., Popperl, H., Aparicio, S., Kuroiwa, A., Brenner, S., and Krumlauf, R. (1994). A conserved retinoic acid response element required for early expression of the homeobox gene Hoxb-I. Nature (London) 370,567-571. McCormick, A. M., Shubeita, H. E., and Stocum, D. L. (1988). Cellular retinoic acid binding protein: Detection and quantitation in regenerating axolotl limbs. J. Exp. Zool. 245,270-276. Means, A. L., and Gudas, L. J. (1995). The role of retinoids in vertebrate development. Annu. Rev. Biochem. 64.201-233.

GENE EXPRESSION DURING AMPHIBIAN LIMB REGENERATION

45

Mendelsohn, C., Ruberte, E., and Chambon, P. (1992). Retinoid receptors in vertebrate limb development. Dev. Biol. 152, 50-61. Mescher, A. L. (1993). Development and regeneration of limbs in the Short toes axolotl mutant. In “Limb Development and Regeneration, Part A ’ (J. F. Fallon, P. F. Goetinck, R. 0. Kelley, and D. L. Stocum, Eds.), pp. 181-191. Wiley, New York. Mescher, A. L., and Cox, C. A. (1988). Hyaluronate accumulation and nerve-dependent growth during regeneration of larval Ambystoma limbs. Differentiation 38, 161-168. Mescher, A. L., and Gospodarowicz, D. (1979). Mitogenic effect of a growth factor derived from myelin on denervated regenerates of newt forelimbs. J. Exp. 2001.207,497-503. Mescher, A. L., and Kiffmeyer, W. R. (1992). Axonal release of transfemn in peripheral nerves of axolotl during regeneration. Monogr. Dev.B i d . 23, 100-109. Mescher, A. L., and Loh, J.-J. (1981). Newt forelimb regeneration blastemas in vitro: Cellular response to explantation and effects of various growth factors. J. Exp. 2001.216,235-245. Mescher, A. L., and Munaim, S. I. (1984). “Trophic” effect of transferrin on amphibian limb regeneration blastemas. J. Exp. 2001. 230,485-490. Mescher, A. L., and Munaim, S. I. (1986). Changes in the extracellular matrix and glycosaminoglycan synthesis during the initiation of regeneration in adult newt forelimbs. Anat. Rec. 214,424-431. Migliorini Bruschelli, G., and Rosi, G. (1971). Polymelia induced by vitamin A in embryos of Bufo vulgaris. Rev. Biol. 64, 277-283. Miller, S. D., Farmer, G., and Prives, C. (1995). p53 inhibits DNA replication in vitro in a DNA binding dependent manner. Mol. Cell. B i d . 15, 6554-6560. Miyazaki, K., Uchiyama, K., Imokawa, Y., and Yoshizato,K. (1996). Cloning and characterization of cDNAs for matrix metalloproteinase of regenerating newt limbs. Proc. Natl. Acad. Sci. USA 93,6819-6824. Monkemeyer, J., Ludolph, D. C., Cameron, J. A., and Stocum, D. L. (1992). Retinoic acidinduced change in anteroposterior positional identity in regenerating axolotl limbs is dosedependent. Dev. Dyn. 193,286-294. Morasso, M. I., Mahon, K. A., and Sargent, T. D. (1995). A Xenopus distal-less gene in transgenic mice. Conserved regulation in distal limb epidermis and other sites of epithelialmesenchymal interaction. Proc. Natl. Acad. Sci. USA 92, 3968-3972. Morgan, B. A., Izpisua-Belmonte, J. C., Duboule, D., and Tabin, C. J. (1992). Targeted misexpression of hox-4.6 in the avian limb bud causes apparent homeotic transformations. Nature (London) 358,236-239. Mullen, L. M., Bryant, S. V., Torok, M. A., Blumberg, B., and Gardiner, D. M. (1996). Nerve dependency of regeneration: The role of Distal-less and FGF signaling in amphibian limb regeneration. Development 122,3487-3497. Munaim, S. I., and Mescher, A. L. (1986). Transferrin and the trophic effect of neural tissue on amphibian limb regeneration blastemas. Dev. Biol. 116, 138-142. Muneoka, K., Fox, W., and Bryant, S. V. (1986). Cellular contribution from dermis and cartilage to the regenerating limb blastema in axolotl. Dev. Biol. 116,256-260. Nace, J. D., and Tassava, R. A. (1995). Examination of fibronectin distribution and its sources in the regenerating newt limb by immunocytochemistry and in situ hybridization. Dev. Dyn. 202,153-164. Namenwirth, M. (1974). The inheritance of cell differentiation during limb regeneration in the axolotl. Dev. Biol. 41, 42-56. Niazi, I. A., and Saxena, S. (1978). Abnormal hindlimb regeneration in tadpole of the toad, Bufo andersonii, exposed to excess vitamin A. Folia Biol. (Krakow) 26,3-11. Nicolas, S., Massacrier, A., Caubit, X., Cau, P., and Le Parco, Y. (1996). A distal-less-like gene is induced in the regenerating central nervous system of the urodele Pleurodeles waltl. Mech. Dev. 56,209-220. Niswander, L., Tickle, C., Vogel, A., Booth, I., and Martin, G. L. (1993). FGF-4 replaces the apical ectodermal ridge and directs outgrowth and patterning of the limb. Cell 75,579-587.

46

JACQUELINE GERAUDIE AND PATRlZlA FERRET1

Niswander, L., Jeffrey, S., Martin, G. R., and Tickle, C. (1994). A positive feedback loop coordinates growth and patterning in the vertebrate limb. Nature (London) 371,609-612. Noji, S.,Nohno, T., Koyama, E., Muto, K., Ohyama, K., Aoki, Y., Tamura, K., Ohsugi, K., Ide, H., and Taniguchi, S. (1991). Retinoic acid induces polarizing activity but is unlikely to be a morphogen in the chick limb bud. Nature (London) 350,83-86. Ogura, T., and Evans, R.M. (1995). Evidence for two distinct retinoic acid response pathways for HOXBl gene regulation. Proc. Natl. Acad. Sci. USA 92, 392-396. Onda, H.,and Tassava, R. A. (1991). Expression of the 9G1 antigen in the apical cap of axolotl regenerates requires nerves and mesenchyme. J. Exp. 2001.257, 336-349. Onda, H., Goldhamer, D. J., and Tassava, R. A. (1990). An extracellular matrix molecule of newt and axolotl regenerating limb blastemas and embryonic limb buds: Immunological relationship of MT1 antigen with tenascin. Development 108, 657-668. Onda, H., Poulin, M. L., Tassava, R. A., and Chiu, I. M. (1991). Characterization of a newt tenascin cDNA and localization of tenascin mRNA during newt limb regeneration by in situ hybridization. Dev. B i d . 148, 219-232. Peadon, A. M., and Singer, M. (1966). The blood vessels of the regenerating limbs of the adult newt, Triturus. J. Morphol. 118, 79-90. Pecorino, L.T., and Lo, D. C. (1992). Having a blast with gene transfer. Curr. Biol. 2,31-32. Pecorino, L.T., Lo, C. D., and Brockes, J. P. (1994). Isoform-specific induction of a retinoidresponsive antigen after biolistic transfection of chimaeric retinoic acidlthyroid hormone receptors into a regenerating limb. Development 120,325-333. Pecorino, L.T.,Entwistle, A,, and Brockes, J. P. (1996). Activation of a single retinoic acid receptor isoform mediates proximodistal respecification. Curr. Biol. 6, 563-569. Peterson, R. L., Papenbrock, T., Davda, M. M., and Awgulewitsch, A. (1994). The murine Hoxc cluster contains five neighboring AbdB-related Hox genes that show unique spatially coordinated expression in posterior embryonic subregions. Mech. Dev.47,253-260. Popiela, H.(1976). Muscle satellite cells in urodele amphibians: Facilitated identification of satellite cells using ruthenium red staining. 1. Exp. Zool. 198,57-64. Poulin, M. L.,and Chiu, I. M. (1995). Re-programming of expression of the KGFR and bek variants of fibroblast growth factor receptor 2 during limb regeneration in newts (Notophthalmus viridescens). Dev. Dyn. 202,378-387. Poulin, M. L.,Patrie, K. M., Botelho, M. J.,Tassava, R. A., and Chiu, I. M. (1993). Heterogeneity in the expression of fibroblast growth factor receptors during limb regeneration in newts (Notophthalmus viridescens). Development 119,353-361. Powell, A. J., Gates, P. B., Wylie, D., Velloso, C. P., Brockes, J. P., and Jat, P. S. (1997). Immortalization of rodent embryo fibroblasts by a 3’ untranslated region from the newt limb blastema. Submitted for publication. Ragsdale, C. W., Jr., Petkovich, M., Gates, P. B., Chambon, P., and Brockes, J. P. (1989). Identification of a novel retinoic acid receptor in regenerative tissues of the newt. Nature (London) 341, 654-657. Ragsdale,C. W.,Jr.,Gates,P. B.,andBrockes,J.P.(1992a).Identification andexpressionpattern of a second isoform of the newt alpha retinoic acid receptor. Nucleic Acids Res. 20,5851. Ragsdale, C. W., Jr., Gates, P. B., Hill, D. S., and Brockes, J. P. (1992b). Delta retinoic acid receptor isoform 6 is distinguished by its exceptional N-terminal sequence and abundance in the limb regeneration blastema. Mech. Dev. 40,99-112. Reginelli, A.D., Wang, Y. Q., Sassoon, D., and Muneoka, K. (1995). Digit tip regeneration correlates with regions of Msxl (Hox-7)expression in fetal and newborn mice. Development 121, 1065-1076. Repesh, L. A., and Oberpriller, J. C. (1980). Ultrastructural studies on migrating epidermal cells during the wound healing stage of regeneration in the adult newt, Norophthalmus viridescens. Am. 1.Anat. 159, 187-208. Repesh, L. A,, Fitzgerald, T. J., and Furtch, L. T. (1982). Changes in the distribution of fibronectin during limb regeneration in newts using immunocytochemistry. Differentiation 22,229-240.

GENE EXPRESSION DURING AMPHIBIAN LIMB REGENERATION

47

Riddiford, L. M. (1960). Autoradiographic studies of tritiated thymidine infused into the blastema of the early regenerate in the adult newt, Triturus. J. Exp. 2001.144, 25-32. Robert, B., Sassoon, D., Jacq, B., Gehring, W., and Buckingham, M. (1989). Hox-7, a mouse homeobox gene with a novel pattern of expression during embryogenesis. EMBO J. 8,91-100. Robert, B., Lyons, G., Simandl, B. K., Kuroiwa, A., and Buckingham, M. (1991). The apical ectodermal ridge regulates Hox-7 and Hox-8 gene expression in developing chick limb buds. Genes Dev. 5,2363-2374. Saadi, A., Gallien, C. L., Guyot-Lenfant, M., and Chanoine, C. (1993). A new approach of urodele amphibian limb regeneration: Study of myosin isoforms and their control by thyroid hormone. Mech. Dev. 43,49-56. Salo, E., and Baguna, J. (1986). Stimulation of cellular proliferation and differentiation in the intact and regenerating planarian Dugesia ( G ) figrina by the neuropeptide substance P. J. EXP.2001.237,129-135. Salpeter, M., and Singer, M. (1960). The fine structure of mesenchymatous cells in the regenerating forelimb of the adult newt Triturus. Dev. B i d . 2,516-534. Saunders, J. W., Jr. (1948). The proximo-distal sequence of origin of the parts of chick wing and the role of the ectoderm. J . Exp. 2001.108,363-403. Savard, P., and Tremblay, M. (1995). Differential regulation of Hox C6 in the appendages of adult urodeles and anurans. J. Mol. Biol. 249,879-889. Savard, P., Gates, P. B., and Brockes, J. P. (1988). Position dependent expression of a homeobox gene transcript in relation to amphibian limb regeneration. EMBO J. 7,4275-4282. Scadding, S. R. (1989). Histological effects vitamin A on limb regeneration in the larval axolotl, Ambystoma mexicanum. Can. J. 2001.68, 159-167. Scadding, S . R., and Maden, M. (1986). Comparison of the effects of vitamin A on limb development and regeneration in the axolotl, Ambystoma mexicanum. J. Embryol. Exp. Morphol. 91,19-34. Scadding, S . R., and Maden, M. (1994). Retinoic acid gradients during limb regeneration. Dev. Biol. 162,608-617. Schilthuis, J. G., Gann, A. A,, and Brockes, J. P. (1993). Chimeric retinoic acidlthyroid hormone receptors implicate RAR-alpha 1 as mediating growth inhibition by retinoic acid. EMBO J. 12,3459-3466. Schmidt, A. J. (1968). “Cellular Biology of Vertebrate Regeneration and Repair,” pp. 420. Univ. of Chicago Press, Chicago. Scott, W. J., Jr., Walter, R., Tzimas, G., Sass, J. O., Nau, H., and Collins, M. D. (1994). Endogenous status of retinoids and their cytosolic binding proteins in limb buds of chick vs mouse embryos. Dev. Biol. 165,397-409. Sicard, R. E. (1985). “Regulation of Vertebrate Limb Regeneration,” pp. 185. Oxford Univ. Press, Oxford, UK. Simeone, A., Acampora, D., Pannese, M., D’Esposito, M., Stornaiuolo, A., Gulisano, M., Mallamaci, A., Kastury, K., Druck, T., and Huebner, K. (1994). Cloning and characterization of two members of the vertebrate Dlx gene family. Proc. Nafl.Acad. Sci. USA 91,2250-2254. Simon, H. G., Kittappa, R., Khan, P. A,, Tsilfidis, C., Liversage, R. A., and Oppenheimer, S. (1997). A novel family of T-box genes in urodele amphibian limb development and regeneration: Candidate genes involved in vertebrate forelimb/hindlimb patterning. Development W, 1355-1366. Simon, H. G., and Tabin, C. J. (1993). Analysis of Hox-4.5 and Hox-3.6 expression during newt limb regeneration: Differential regulation of paralogous Hox genes suggest different roles for members of different Hox clusters. Development 111, 1397-1407. Simon, H.-G.,Nelson, C., Goff, D., Laufer, E., Morgan, B., and Tabin, C. J. (1995). Differential expression of myogenic regulator genes and msx-1 during dedifferentiation of regenerating amphibian limbs. Dev. Dyn. 202, 1-12. Singer, M. (1952). The influence of the nerve in regeneration of the amphibian extremity. Q. Rev. Biol. 27, 169-200.

48

JACQUELINE GERAUDIE AND PATRlZlA FERRE3lI

Singer, M. (1974). Neurotrophic control of limb regeneration in the newt. Ann. N. Y. Acud. Sci. 228,308-322. Singer, M., and Caston, J. D. (1972). Neurotrophic dependence of macromolecular synthesis in the early limb regenerate of the newt, Triturus. J. Embryol. Exp. Morphol. 28,l-11. Singer,M., and Craven, L. (1948).The growth and morphogenesis of the regenerating forelimb of adult Triturus following denervation at various stages of development. 1. Exp. Zool. 108,279-308. Singer, M., and GCraudie, J. (1991). The neurotrophic phenomenon: Its history during limb regeneration in the newt. In “A History of Regeneration Research. Milestones in the Evolution of a Science” (C. E. Dinsmore, Ed.), pp. 101-112. Cambridge Univ. Press, Cambridge, UK. Singer, M., and Salpeter M. M. (1961). Regeneration in vertebrates: The role of the wound epithelium. In “Growth of the Living Systems” (M. X. Zarrow, Ed.), pp. 277-311. Basic Books, New York. Singer, M., Maier, C. E., and McNutt, W. S. (1976). Neurotrophic activities of brain extracts in forelimb regeneration of the urodele, Trimrus. J. Exp. 2001.196,131-150. Small, K. M., and Potter, S. S. (1993). Homeotic transformations and limb defects in Hox A l l mutant mice. Genes Dev. 7,2318-2328. Smith, G. N., Toole, B. P., and Gross, J. (1975). Hyaluronidase activity and glycosaminoglycan synthesis in the amputated newt limb: Comparison of denervated, nonregenerating limbs with regenerates. Dev. B i d . 43,221-232. Smith, M. J., and Globus, M. (1989). Multiple interactions in juxtaposed monolayers of amphibian neuronal epidermal and mesodermal limb blastema cells. In Virro Cell Dev. Biol. 25, 849-856. Smith, M. J., Globus, M., and Vethamany-Globus, S. (1995). Nerve extracts and substance P activate the phosphatidylinositol signaling pathway and mitogenesis in newt forelimb regenerates. Dev. Biol. 167,239-251. Song, K., Wang, Y., and Sassoon, D. (1992). Expression of Hox-7.1 in myoblasts inhibits terminal differentiation and induces cell transformation. Nature 360,477-481. Sordino, P., Vanderhoeven, F., and Duboule, D. (1995). Hox gene expression in teleost fins and the origin of vertebrate digits. Nature (London) 375,678-681. Spallanzani, L. (1768). Prodromo di un opera da imprimersi sopra la riproduzioni animal. Giovanni Montanari, Modena. “An essay on Animal Reproduction” (Translated in 1769 by M. Maty). T. Becket and DeHondt, London. Stocum, D. L. (1985). The role of the skin in urodele limb regeneration. In “Regulation of Vertebrate Limb Regeneration” (R. E. Sicard, Ed.), pp. 32-53. Oxford Univ. Press, Oxford, UK. Stocum, D. L. (1991). Retinoic acid and limb regeneration. Sem. Dev. Biol. 2, 199-210. Stocum, D. L. (1995). “Wound Repair, Regeneration and Artificial Tissues.” R. G . Landes, Austin, TX. Stocum, D. L., and Maden, M. (1990). Regenerating limbs. Methods Enzymol. 190 (Part B), 189-200. Stratford, T., Horton, C., and Maden, M. (1996). Retinoic acid is required for the initiation of outgrowth in the chick limb bud. Curr. B i d . 6, 1124-1133. Tabin, C. J. (1989). Isolation of potential vertebrate limb-identity genes. Development 105, 813-820. Tanaka, E. M., Gann, A. A. F., Gates, P. B., and Brockes, J. P. (1997). Newt myotubes re-enter the cell cycle by phosphorylation of the retinoblastoma protein. J. Cell Biol. 136, 155-165. Tassava, R. A. (1969). Hormonal and nutritional requirements for limb regeneration and survival of adult newts. J. Exp. Zool. 170,33-54. Tassava, R. A. (1983). Limb regeneration to digit stages occurs in well-fed adult newts after hypophysectomy. J. Exp. 2001.225,433-441.

GENE EXPRESSION DURING AMPHIBIAN LIMB REGENERATION

49

Tassava, R. A. (1992). Retinoic acid enhances monoclonal antibody WE3 reactivity in the regenerate epithelium of the adult newt. 1. Morphol. 213, 159-169. Tassava, R. A., and Acton, R. D. (1989). Distribution of a wound epithelium antigen in embryonic tissues of newts and salamanders. Ohio J. Sci. 89, 12-15. Tassava, R. A., and Mescher, A. L. (1975). The role of injury, nerves, and wound epidermis during the initiation of amphibian limb regeneration. Differentiation 4, 23-24. Tassava, R. A., and Olsen, C. L. (1985). Neurotrophic influences on cellular proliferation in urodele limb regeneration: In vivo experiments. In “Regulation of Vertebrate Limb Regeneration” (R. E. Sicard, Ed.), pp. 81-91. Oxford Univ. Press, Oxford, UK. Tassava, R. A., Johnson-Wint, B., and Gross, J. (1986). Regenerate epithelium and skin glands of the adult newt react to the same monoclonal antibody. J. Exp. 2001.239,229-240. Tassava, R. A., Castilla, M., Arsanto, J. P., and Thouveny, Y. (1993). The wound epithelium of the regenerating limbs of Pleurodeles waltl and Notophthalmus viridescens: Studies with mAbs WE3 and WE4, phalloidin, and DNase 1. J. Exp. Zool. 267, 180-187. Taylor, G. P., Anderson, R., Reginelli, A. D., and Muneoka, K. (1994). FGF-2 induces regeneration of the chick limb bud. Dev. Biol. 163, 282-284. Tchang, F., Gusse, M., Soussi, T., and MBchali, M. (1993). Stabilization and expression of high levels of p53 during early development in Xenopus laevis. Dev. Biol. 159,163-172. Thaller, C., and Eichele, G. (1990). Isolation of 3,4-didehydroretinoic acid, a novel morphogenetic signal in the chick wing bud. Nature (London) 345, 815-819. Thaller, C., Hofmann, C., and Eichele, G. (1993). 9-cis-retinoic acid, a potent inducer of digit pattern duplications in the chick wing bud. Development 118,957-965. Thoms, S . D., and Stocum, D. L. (1984). Retinoic acid-induced pattern duplication in regenerating urodele limbs. Dev. Biol. 103, 319-328. Thornton, C. S. (1968). Amphibian limb regeneration. In “Advances in Morphogenesis” (M. Abercrombie, J. Brachet, and T. J. King, Eds.), Vol. 7, pp. 205-249. Academic Press, New York. Thornton, C. S. (1970). Amphibian limb regeneration and its relation to nerves. Am. Zool. 10,113-118. Tickle, C. (1991). Retinoic acid and chick limb development. Development (Suppl. l), 113-121. Tickle, C., and Eichele, G. (1994). Vertebrate limb development. Annu. Rev. Cell. Biol. 10, 121-152. Tickle, C., Alberts, B. M., Wolpert, L., and Lee, J. (1982). Local application of retinoic acid to the limb bud mimics the action of the polarizing region. Nafure (London) 2%, 564-565. Timpl, R. (1989). Structure and activity of basement membrane proteins. Eur. J. Biochem. 180,487-502. Todd, T. J . (1823). On the process of reproduction of the members of the aquatic salamander. Q. J. Sci. 16, 84-96. Tomasek, J. J., Mazurkiewicz, J. E., and Newman, S. A. (1982). Non-uniform distribution of fibronectin during avian limb development. Dev. Biol. 90, 118-126. Toole, B. P. (1973). Hyaluronate and hyaluronidase in morphogenesis and differentiation. Am. 2001.W, 1061-1065. Toole, B. P., and Gross, J. (1971). The extracellular matrix of the regenerating newt limb: Synthesis and removal of hyaluronate prior to differentiation. Dev. Biol. 25, 57-77. Toole, B. P., Goldberg, R. L., Chi-Rosso, G., Underhill, C. B., and Orkin, R. W. (1984). Hyaluronate cell-interactions. In “The Role of Extracellular Matrix in Development” (R. L. Trelstad, Ed.), A. R. Liss, New York. Tschumi, P. A. (1957). The growth of the hind limb bud of Xenopus Iaevb and its dependence upon the epidermis. J. Anat. 91,149-173. Tsonis, P. A. (1991). Amphibian limb regeneration. In vivo 5, 541-550. Tsonis, P. A. (1996). “Amphibian Limb Regeneration.” Cambridge Univ. Press, Cambridge, UK.

50

JACQUELINE GERAUDIE AND PATRlZlA FERREllI

Tsonis, P. A,, Washabaugh, C. H., and del Rio-Tsonis, K. (1994). Morphogenetic effects of 9-cis-retinoic acid on the regenerating limbs of the axolotl. Roux’ Arch. Dev. Biol. 203, 230-234. Vethamany-Globus, S . (1987). Hormone action in newt limb regeneration: Insulin and endorphins. Biochem. Cell Biol. 65, 730-738. Viviano, C. M., Horton, C. E., Maden, M., and Brockes, J. P. (1995). Synthesis and release of 9-cis retinoic acid by the urodele wound epidermis. Development 121,3753-3762. Vogel, A., Roberts-Clarke, D., and Niswander, L. (1995). Effect of FGF on gene expression in chick limb bud cells in vivo and in vim. Dev. Biol. 171, 507-520. Wallace, H. (1981). “Vertebrate Limb Regeneration.” Wiley, Chichester, UK. Wanek, N., Gardiner, D. M., Muneoka, K., and Bryant, S . V. (1991). Conversion by retinoic acid of anterior cells into ZPA cells in the chick wing bud. Nature (London) 380, 81-83. Wei, L. N., and Chen, G. J. (1991). Production and analyses of transgenic mice with ectopic expression of cellular retinoic acid-binding protein. Biochem. Biophys. Res. Commun. 179,210-216. Wei, Y., Yang, E. V., Klatt, K. P., and Tassava, R. A. (1995). Monoclonal antibody MT2 identifies the urodele a1 chain of Type XI1 collagen, a developmentally regulated extracellular matrix protein in regenerating newt limbs. Dev. Biol. 168, 503-513. West, D. C., Hampson, I. N., Arnold, F., and Kumar, S . (1985). Angiogenesis induced by degradation products of hyaluronic acid. Science 288, 1324-1326. Yang, E. V., Shima, D. T., and Tassava, R. A. (1992). Monoclonal antibody ST1 identifies an antigen that is abundant in the axolotl and newt limb stump but is absent from the undifferentiated regenerate. J. Exp. Zool. 264, 337-350. Yang, E. V., and Bryant, S . V. (1994). Developmental regulation of a matrix metalloproteinase during regeneration of axolotl appendages. Dev. B i d . 166,696-703. Yokouchi, Y., Nakazato, S., Yamamoto, M., Goto, Y., Kameda, T., Iba, H., and Kuroiwa, A . (1995). Misexpression of Hoxa-I3 induces cartilage homeotic transformation and changes cell adhesiveness in chick limb buds. Genes Dev. 9, 2509-2522. Zenjari, C., Boilly-Marer, Y., Desbiens, X., Oudghir, M., Hondermarck, H., and BoiIIy, B. (1996). Experimental evidence for FGF-1 control of blastema cell proliferation during limb . Biol. 40, 965-971. regeneration of the amphibian Pleurodeles waltl. Int. .IDev.

Biochemistry of the Extracellular Matrix of Volvox Manfred Sumper and Armin Hallmann Lehrstuhl Biochemie I, Universitat Regensburg, D-93053 Regensburg, Germany

The volvocine algae range in complexity from unicellular Cblamydomonas to multicellular organisms in the genus Volvox. The transition from unicellularity to multicellularity in the Volvocales is a recent event in evolution. Thus, these organisms provide a unique opportunity for exploring the development of a complex extracellular matrix (ECM) from the cell wall of a unicellular ancestor. The ECM of Volvox is divided into four main zones: The flagellar, boundary, cellular, and deep zones. Each zone is defined by ultrastructure and by characteristic ECM glycoproteins. Voivox ECM is modified under developmental control or in response to external stimuli, like the sexinducing pheromone or stress factors. The structures of more than 10 ECM glycoproteins from a single species of Volvox are now known in molecular detail and are compared to other algal and plant cell walllECM glycoproteins. Although usually classified as hydroxyproline-rich glycoproteins, the striking feature of all algal ECM glycoproteins is a modular composition. Rod-shaped hydroxyproline-rich modules are combined with hydroxyproline-free domains that meet the multiple functional requirements of a complex ECM. The algal ECM provides another example of the combinatorial advantage of shuffling modules that is so evident in the evolution of the metazoan ECMs. KEY WORDS: Extracellular matrix, Green algae, Volvocales, Volvox, Cell wall, Glycoproteins, Plant development.

1. Introduction The extracellular matrix (ECM) of a multicellular organism is a complex organelle that serves structural as well as nonstructural functions. It provides a scaffolding to create and to stabilize the physical structure of tissues. In addition, the ECM mediates many developmental responses of cells includInfemnfronal Review of Cyrology, Val. 180 0074-76%/98 $25.00

51

Copyright D 1998 by Academic Press. All rights of reproduction in any form reserved.

52

MANFRED SUMPER AND ARMlN HALLMANN

ing regulation of growth and differentiation, wound repair, and pathogen defense. It is now recognized that common principles operate in the design of ECMs in plants and animals: extended and usually crosslinked glycoproteins with repeating sequence motives provide tensile strength, and polypeptides organized in a modular fashion have been evolved to allow both specific targeting and controlled enzymatic actions within the ECM network. The development of a complex ECM from a simple cell wall was one of the prerequisites to promote the evolutionary transition from unicellularity to multicellularity. A family of organisms collectively referred to as the Volvocaceae provides the unique opportunity for exploring molecular genetic pathways that led from unicellularity to multicellularity. The volvocine algae range in complexity from unicellular Chlamydomonus through colonial genera (such as Gonium, Pandorim, and Eudorina) to multicellular organisms, with differentiated cells and complete division of labor, in the genus Volvox. The multicellular members in the order contain 2" Chlamydornonas-like cells (the maximum value of n being a species character) held together in a predictable pattern by a hydroxyproline-rich ECM. Volvox is among the simplest multicellular organisms and yet it shares many features that characterize the life cycles and developmental histories of much more complex organisms. The rRNA sequence data indicate that the transition from unicellularity to multicellularity within the volvocine algae probably happened within the past 50-75 million years and is therefore a very recent event in evolution (Rausch et al., 1989; Larson et al., 1992). The changes within genomes required to achieve this fundamental event may not yet be obscured by genetic drift and, therefore, it should be possible to analyze the evolutionary sequence from an ancestor resembling Chlamydomonas to the multicellular members of the Volvocales. Recent progress in the development of nuclear transformation (Kindle, 1990; Schiedlmeier et al., 1994), the introduction of selectable markers (Kindle et al., 1989; Debuchy et al., 1989; Stevens et af., 1996; Schiedlmeier et al., 1994; Hallmann and Sumper, 1996) and reporter genes (Davies et al., 1992; Hallmann and Sumper, 1994b), and gene replacement by homologous recombination in Chlamydomonas (Sodeinde and Kindle, 1993; Gumpel et al., 1994; Nelson and Lefebvre, 1995) as well as in Volvox carteri (Hallmann et al., 1997) has made it possible to apply the powerful strategies of molecular genetics. Cell walls and the ECM of the volvocine algae (Miller et al., 1974) are entirely assembled from glycoproteins with a high content of hydroxyproline (hydroxyproline-rich glycoproteins, HRGPs). The walls lack cellulose, hemicelluloses, pectins, and lignin (Adair et al., 1987). HRGPs also represent a main constituent of higher plant ECMs and much work has been done to analyze the structures of these proteins (Cooper et al., 1984; ShoWalter and Varner, 1989; Varner and Lin, 1989; Kieliszewski and Lamport,

BIOCHEMISTRY OF

THE Vo/vox ECM

53

1994). However, there are few examples in the literature where multiple HRGPs have been examined in molecular detail from a single species or from closely related species. This would allow a more integrated approach to elucidate the structure, assembly, and function of ECM proteins. In recent years, this type of analysis has been initiated with volvocine algae and the emerging picture will be summarized in this review. V. carteri f. nagariensis has become the standard subject of Volvox research, owing to its genetic accessibility. v.carteri is composed of only two cell types: 2000-4000 biflagellate Chlurnydornonas-like somatic cells are arranged in a monolayer at the surface of a hollow sphere and 16 much larger reproductive cells (“gonidia”) lie just below the somatic cell sheet (Starr, 1969) (Fig. 1). Eleven or twelve rapid and synchronous cleavage divisions of a gonidium generate all the cells of an adult organism. An asymmetric division of 16 cells at the stage of the 32-cell embryo delineates 16 new reproductive cells from the somatic cell initials which continue cleavage. At the termination of cell divisions, the embryo enters the process of inversion, thereby turning the embryo inside-out. After inversion, the somatic cells begin to secrete ECM material, causing each cell to move apart from its neighbors. The organism now grows in size but not cell

FIG. 1 Asexual spheroid of Vdvox carferi containing 16 large gonidia (asexual reproductive cells), which just have initiated embryogenesis. The small dots represent 2000-4000 terminally differentiated somatic cells. Magnification, 80X.

54

MANFRED SUMPER AND ARMIN HALLMANN

number. When the daughter spheroids are about a quarter of their final size they are released from the parent organism through holes formed by local enzymatic degradation of the ECM. A number of previous reviews describe main topics of Volvox biology in detail: a perfect introduction of the organism (Starr, 1970), genetic and biochemical aspects (Kirk and Harper, 1986), ontogenetic and phylogenetic aspects (Kirk, 1988; Schmitt et al., 1992), as well as morphological and physiological aspects (Desnitski, 1995; Nozaki, 1996).

II. Ultrastructure of the Volvox ECM The ECM of representatives of the genus Volvox consists of a large number of anatomically distinct structures arranged in a defined spatial pattern. Based on light- and electron-microscopic observations, David Kirk and co1986) proposed a highly useful system of nomenclature workers (Kirk et d., that greatly facilitates discussion of comparative morphology and phylogeny of the ECM. In this nomenclature the ECM is divided into four main zones: the flagellar zone (FZ), the boundary zone (BZ), the cellular zone (CZ), and the deep zone (DZ). In the stylized drawings of Figs. 2 and 3 these

cz

DZ

FIG. 2 Highly stylized cross section of a Volvox carteri spheroid emphasizing the major compartments of the ECM. CZ, cellular zone; DZ, deep zone; G, gonidium; S, somatic cell. For details, see Fig. 3.

BIOCHEMISTRY OF THE Volvox ECM

55

BZ

CZ

FIG. 3 Stylized drawing of a portion of a Volvox carteri spheroid illustrating ECM zones and subzones according to Kirk er al. (1986). FZ, flagellar zone; BZ, boundary zone; CZ, cellular zone. Subzones are as described in the text.

zones are defined in a cross section of V. carteri, the species from which most structural data are available. Each of these major zones is subdivided in a hierarchical fashion to define observable substructures. This terminology is entirely based on morphological criteria and therefore does not imply any similarities or differences with respect to the biochemical properties among the various compartments. The definitions of zones and subzones given by Kirk et al. (1986) are as follows.

A. The Flagellar Zone The FZ (Fig. 3) includes all ECM specializations seen only on or in the immediate vicinity of the flagellum. The FZ is further subdivided into three compartments: FZ1 contains all coatings and appendages of the flagellar

56

MANFRED SUMPER AND ARMlN HALLMANN

membrane. FZ2 includes all components that create the flagellar collar. FZ3 describes modifications of the boundary and, or, cellular zones in the region traversed by the flagellum.

B. The Boundary Zone The BZ (Fig. 3) includes those components of the ECM that, except in periflagellar regions, appear to be continuous over the surface of the organism but are not structurally continuous with deeper layers. Again, three subzones are distinguishable. Because BZ2 (tripartite layer) is highly conserved in all Volvocales examined to date, it was used as a reference point in numbering the components of the boundary zone. The tripartite layer corresponds to layers W2-W6 in the ECM terminology of Roberts (1974) and contains the crystalline layer. Therefore, the subzones BZ1 and BZ3 are those components external (BZ1) and internal (BZ3) to BZ2. C. The Cellular Zone

The CZ (Figs. 2 and 3) includes components lying internal to the boundary zone and exhibiting specializations around individual cells (in unicellular Volvocales the boundary zone and cellular zone are synonymous). The coherent meshwork of ECM filaments attached to the plasmalemma of each cell body is denoted as CZ1. CZ2 describes the relatively amorphous components filling all portions of the CZ not occupied by more highly structured components. The distinct fibrous material that creates chambers around individual cells is defined as CZ3.

D. The Deep Zone The DZ (Fig. 2) contains all ECM components internal to the cellular zone. More specifically, DZ1 is a fibrous layer enclosing DZ2. DZ2 appears as a relatively amorphous component filling the deepest regions of the spheroid and is by far the largest region of the spheroid in most Volvox species.

111. Biochemical Characterization of ECM Components

To some extent, the four ECM zones defined by morphological characteristics also behave as structural units during biochemical fractionation. For

BIOCHEMISTRY OF THE Volvox ECM

57

instance, treatment of Volvox spheroids with chaotropic agents selectively extracts main parts of the BZ. On the other hand, a very mild mechanical treatment that introduces slits into Volvox spheroids causes selective liberation of most of the highly viscous material of the DZ. Treatment of Volvox spheroids with hot SDS removes all soluble molecules, but it leaves the ECM appearing largely intact. Colorless Volvox “ghosts” are produced which mainly consist of covalently crosslinked components of the ECM. These “ghosts” exhibit typical morphological features of CZ. Finally, a number of procedures are known that selectively remove all the flagella from a Volvox spheroid (Snell, 1983).

A. Boundary and Flagellar Zone All members of the algal order Volvocales have cell walls containing a crystalline layer overlying an amorphous inner layer (Goodenough and Heuser, 1985; Roberts et af., 1985). From studies of a large number of Volvocales species, Roberts and co-workers concluded that the cell walls of these algae fall into four major structural classes (Roberts, 1974; Roberts et al., 1982). Volvox and some other colonial Volvocales have a wall structure shared by Chlamydomonas reinhardtii (Roberts et al,, 1982; Roberts et al., 1985). In C. reinhardtii, the crystalline layer is composed of a number of hydroxyproline-rich glycoproteins that may be disassembled by chaotropic agents and recrystallized in vitro (Hills et al., 1975; Catt et al., 1976, 1978; Roberts, 1974, 1979; Roberts et al., 1980; Goodenough et af., 1986). As pointed out by Kirk et al. (1986), it is the boundary zone (tripartite layer, BZ2) corresponding to layers W2-W6 of Chlamydomonas that is closely related in both these organisms, whereas the inner zones of the cell walls/ECMs diverge and define species specificity. Previous work on the species-specific sensitivity of the inner wall structures to lytic enzymes provided an experimental basis for this generalization (Claes, 1971; Schlosser, 1976,1984;Matsuda etal., 1987).Proof for the evolutionary relationship between C. reinhardtii and V. carteri boundary zones was obtained by performing interspecific reconstitutions of cell walls. Adair et al. (1987) solubilized the crystalline layers of V. carteri and demonstrated that the insoluble layers of C. reinhardtii allowed nucleated assembly of the solubilized BZ2 components to yield hybrid walls. Vice versa, V . carteri can nucleate the assembly of C. reinhardtii crystalline layer, but not that from Chlamydomonas eugametos. Therefore, it is plausible to expect homologies among the glycoproteins of the boundary zones. Morphological and some biochemical data are available for the corresponding components of the crystalline layer of C. reinhardtii. Four glycoproteins are extractable; three are HRGPs (GP1, GP2, and GP3), and one is glycine-rich (GP 1.5) (Good-

58

MANFRED SUMPER AND ARMlN HALLMANN

enough et al., 1986; Goodenough and Heuser, 1988). Partial sequences deduced from cDNA clones have been published for GP1 and GP2 (Adair and Apt, 1990; data library accession nos. M58496 and M58597). Strangely, the reading frame used for prediction of the amino acid sequence of GP2 contains two TAG stop codons and therefore requires reexamination. Peptide-mapping studies, immunological cross-reactivity, and electronmicroscopic data support the presence of GP2 homologues in V. curteri (Adair and Appel, 1989; Goodenough and Heuser, 1988). As yet, only a single gene encoding a boundary zone glycoprotein from Volvox (ISG, see Section IV.A.2) has been cloned and characterized.

6. Cellular Zone As mentioned above, extraction of asexually growing Volvox spheroids with 1%SDS containing 0.5 M NaCl at 95°C solubilizes all cytoplasmic proteins and produces colorless Volvox “ghosts” that still exhibit features characteristic of the ECM morphology. In particular, the honeycomb-like cellular compartments (CZ3) remain intact, indicating the existence of covalent crosslinks in the coherent network constituting this ECM subzone. Such an insoluble polymer poses particular problems for a biochemical characterization of the building blocks involved. Therefore, it was important to realize that insoluble “ghosts” are nearly quantitatively converted to soluble components if treated with anhydrous hydrogen fluoride (HF). This procedure is known to cleave 0-glycosidic linkages without affecting peptide bonds (Mort and Lamport, 1977). Thus, HF cleavage enables the characterization of all the polypeptides present in the insoluble fraction of the Volvox ECM. SDS-PAGE analysis of HF-solubilized “ghosts” exhibits two main polypeptides with molecular masses around 60 and 70 kDa that were identified as being derived from the (deglycosylated) glycoproteins SSG 185 and pherophorin I (Fig. 4). 1. The ECM Glycoprotein SSG 185

The sulfated glycoprotein SSG 185has been characterized as the monomeric precursor of the CZ3 substructure using immunological techniques (Wenzl el al., 1984; Ertl et al., 1989).The primary structure of the SSG 185 polypeptide chain has been derived from cDNA and genomic DNA. A central domain of the protein, 80 amino acid residues long, consists almost exclusively of hydroxyproline residues. Most of these hydroxyproline residues are glycosylated with 1,Zlinked di- and tri-arabinosides. Proteolysis of SSG 185 results in a large, completely resistant 145-kDa fragment with a high content of hydroxyproline that represents this central domain of SSG 185.

59

BIOCHEMISTRY OF THE Volvox ECM

Coomassie stain

,SSG 185 antibody

FIG.4 Main components of the SDS-insoluble Volvox ECM. Volvox “ghosts,” representing the SDS-insoluble ECM, were solubilized by treatment with anhydrous hydrogen fluoride at o”C, loaded onto a SDS-polyacrylamide gel, and transferred by Western blotting onto a membrane. Proteins (deglycosylated) were stained with Coomassie blue (“Coomassie stuin”). Immune detection was performed with a monoclonal antibody directed against pherophorin I (“Pherophorin 1 antibody”) and a polyclonal antibody directed agains SSG 185 (“SSG 185 antibody”). Both times the second antibody was an anti-IgG alkaline phosphatase conjugate.

Most probably, the secondary structure of such a domain is the polyproline I1 helix conformation. This is the most extended helix formed by polypeptides with three residues per turn and a pitch of 0.94 nm. As revealed by EM, the central domain of SSG 185 indeed shows a rod-shaped morphology (Ertl et aZ., 1989).Attached to this central rod is a 21-nm-longpolysaccharide with highly acidic properties. This polysaccharide consists of a 1,3-linked polymannan backbone and each mannose unit carries at position 6 a 1,2linked di-arabinoside side chain (Fig. 5). The striking feature of this saccharide is its extreme degree of sulfation. Each arabinose carries two sulfate groups and the mannoses of the backbone are sulfated at position 4. Remarkably, the degree of sulfation in this SSG 185 polysaccharide is found to change under developmental control (see Section IV.B.l). The high density of negative charges should exert a strong influence on the overall physicochemical properties of the C Z . Cations and positively charged ECM proteins should bind to this strong cation exchanger. Incorporation studies with radioactive phosphate led to the detection of another unique structural element within the central rod-shaped domain of SSG 185. A phosphodiester bridging two arabinose residues via their 5positions was isolated from hydrolysates of polymeric SSG 185 (Holst et aZ., 1989).The possible function of this phosphodiester as a crosslink within the ECM is discussed in Section 1V.D.

60

MANFRED SUMPER AND ARMlN HALLMANN

1

I-

O$O-

5

FIG. 5 Proposed chemical structure of the sulfated saccharide covalently linked to SSG 185.

The N-terminal (about 230 amino acid residues) as well as the C-terminal (about 165 amino acid residues) extensions of the central hydroxyprolinerich domain exhibit no unusual amino acid preference except for cysteine. A sequence comparison of the N- and C-terminal domains of SSG 185 remarkably reveals a significant degree of homology (24% identity and 54% similarity over 206 residues). SSG 185 oligomers obtained by limited proteolytic degradation of polymeric SSG 185 were studied by electron microscopy. From the corresponding EM pictures, an overlapping staggered assembly of the monomeric units has been deduced. Monoclonal antibodies raised against the protease-resistant 145-kDa glycopeptide were shown to prevent in vivo expansion of young Volvox spheroids, presumably by inhibiting the insertion and polymerization of SSG 185 monomers into the CZ3 zone (Ertl et al., 1989). The in viva kinetics of SSG 185 polymerization could be followed by pulse-chase labeling experiments with radioactive sulfate (Wenzl et al., 1984). 2. Pherophorin I

The sex-inducing pheromone of Volvox is a glycoprotein. The pherophorins, a newly discovered ECM protein family, are multidomain glycoproteins with homology to this sex-inducing pheromone in their C-terminal domain (Sumper et al., 1993). Recent evidence suggests that pherophorins represent a major family of ECM glycoproteins with amazingly different properties with respect of localization within the ECM. Pherophorins I and I l l are constitutively expressed in asexually growing Volvox spheroids (God1 et al., 1995). Besides SSG 185, pherophorin I is a main constituent of the C Z

BIOCHEMISTRY OF THE Volvox ECM

61

(Fig. 4). In contrast to SSG 185, pherophorins I and I11 can be selectively extracted from Volvox “ghosts” by treatment with EDTA, indicating a noncovalent fixation of pherophorins within the CZ. Mg2+or Ca2+bridges may be involved in this interaction. The primary structure of pherophorin I was deduced from cDNA sequence analysis. A proline-rich (most probably hydroxyproline-rich) stretch of about 23 amino acid residues around position 170 indicates the existence of two well-separated domains in this polypeptide. Both these domains exhibit no unusual amino acid preference. Surprisingly, the N-terminal domain shares 21% identity and 56% similarity over 220 residues with the corresponding N-terminal domain of SSG 185. The biochemistry and ECM localizations of other members of the pherophorin family that are expressed under the control of the sex-inducing pheromone are described below (Section IV.B.2).

C. Deep Zone Although the constituents of the deep zone can easily be obtained, until recently very little was known about the biochemistry of this compartment. An early paper (Gilles et al., 1983) described an extracellular glycoprotein (290 kDa) of the deep zone that was reported to be phosphorylated on serine residues (“pp290”). Upon application of sex-inducing pheromone, pp290 was found to be replaced by two other phosphorylated proteins (“pp240” and “pp120”). A recently initiated biochemical analysis could not confirm the originally published data. These glycoproteins are not phosphoproteins, rather they bear their phosphate residues on saccharide chains. Again, the phosphodiester arabinose-5-phospho-5’-arabinose originally characterized in SSG 185 could be identified as a main structural element of pp290 and pp240 (Wenzl and Sumper, unpublished results). Therefore, we replace the term pp290 with HRGP-290 for the following reasons: The deglycosylated polypeptide turned out to be nearly resistant to proteases like pronase or subtilisin. Amino acid sequence analysis by automated Edman degradation yielded only hydroxyproline signals over 40 cycles. Amino acid analysis exhibited nearly exclusively hydroxyproline (Gorlach, 1994). Unfortunately, this strange composition excludes the cloning of the corresponding gene on the basis of amino acid sequence data. HRGP-290 contains the neutral sugars arabinose and galactose in a 1: 1 ratio, and permethylation analysis exhibited a terminally bound furanosidic galactose and 1,2,5-substituted arabinose as main products (Wenzl, Riegel, and Sumper, unpublished results). Figure 6 shows the composition of a deep zone extract after pulse labeling with [14C]hydrogencarbonate and [33P]phosphate,respectively. Relatively few labeled components are seen

62

MANFRED SUMPER AND ARMlN HALLMANN 14c 33p

I

I

kDa 290 b

HRGP-290

240 b

120 w

FIG. 6 Composition of a deep zone (DZ) extract after pulse labeling with [‘“Clhydrogen carbonate (‘“‘C”) and [33P]phosphate (“33P’).Fluorograrn of an SDS-polyacrylarnide gel. HRGP-290 represents a main constituent of the DZ.

and HRGP-290 represents a main constituent of the deep zone. Upon a chase, HRGP-290 turns out to be the monomeric precursor of an insoluble polymer. HRGP-290 is a likely precursor for the fibrous polymer found in the deep zone and defined as subzone DZ1. An early report by Mitchell (1980) described the existence of a polyhydroxyproline in Volvox. Possibly, these early data were derived from HRGP-290. Wounding of Volvox spheroids (see Section IV.C.2) induces biosynthesis of a multidomain protease and a lysozymekhitinase and both these enzymes were identified as components of the deep zone. Another newly identified constituent of the deep zone is pherophorin-S (see Section IV.B.2.b).

IV. ECM Biogenesis and Remodeling Scanning electron microscopy of cleaving embryos gave no evidence of any extracellular matrix in the vicinity of embryonic cells (Green and Kirk, 1981). Therefore, it was concluded that embryogenesis is the only stage of the life cycle during which Volvox cells appear to be totally devoid of any extracellular matrix (Kirk and Harper, 1986). At the end of embryogenesis, shortly after inversion, the close contacts between blastomeres are broken, permitting cells to draw away from one another as the young spheroid

BIOCHEMISTRY OF THE Volvox ECM

63

rapidly expands by deposition of ECM material. It is this stage of development at which massive production of ECM molecules and their assembly are initiated. Uncleaved gonidia as well as early embryos can easily be removed from the mother spheroid without affecting their developmental program (Starr, 1969,1970). This fact is an ideal prerequisite to resolve by pulse-chase labeling experiments the stage dependence of production and kinetics of assembly for individual ECM proteins.

A. Embryogenesis

1. Algal Cell Adhesion Molecule (Algal CAM) Proof that plants possess homologues of animal adhesion proteins has been lacking for a long time. The blastomeres of a developing Volvox embryo establish close contacts with their neighbors in a precisely predictable pattern. But even for Volvox embryos, it was concluded from electronmicroscopic investigations that embryonic cells of Volvox are exclusively linked by cytoplasmic bridges (Green and Kirk, 1981; Kirk and Harper, 1986) and do not possess cell-cell contacts mediated by cell adhesion molecules or an ECM. Considering the known importance of animal cell adhesion molecules in developmental processes, the nature of cell-cell contacts of Volvox embryos was reinvestigated using a biochemical approach (Huber and Sumper, 1994). Monoclonal antibodies were raised against a crude membrane fraction from Volvox embryos. The resulting monoclonals were then screened for their capability to interfere in vivo with cell-cell contact formation during embryogenesis. Several monoclonals were found that were able to disrupt cell-cell contacts of the 4-cell embryo. Disruption of these cell-cell contacts results in a big hole in the center of a 4-cell embryo. Confocal laser scanning immunofluorescence microscopy localized the corresponding antigen at the cell-cell contacts of the developing embryo, as expected for a potential CAM. Therefore, this antigen was denoted Algal CAM. Algal CAM was purified, and amino acid sequence data allowed the cloning of its cDNA and genomic DNA. The deduced amino acid sequence reveals a multidomain structure of Algal CAM with unexpected homologies to known plant as well as animal protein families. N-terminally located is a proline-rich domain with Ser-Pro-Pro-Pro-Pro repeats diagnostic of the extensins from higher plant cell walls. This plant-specific domain is followed by two repeats with homology to fasciclin I, a cell adhesion molecule involved in the neuronal development of Drosophilu. The fasciclin I protein in Drosophilu is a homophilic cell adhesion molecule (Elkins et ul., 1990), expressed on the surface of a subset of fasciculating axons, and seems to be involved in growth cone extension and/or guidance (Zinn el

64

MANFRED SUMPER AND ARMlN HALLMANN

al., 1988). Fasciclin I is composed of four homologous repeats of about 150 amino acids each (Zinn et al., 1988). Algal CAM contains two copies of related repeats and, therefore, represents the first plant homologue of animal cell adhesion molecules. Different variants of Algal CAM are produced under developmental control by alternative splicing. The C-terminal part of Algal CAM can be replaced by a different amino acid sequence that is encoded by an additional exon. This alternative sequence may represent a glycosyl-phosphatidylinositol (GPI) anchor addition signal (reviewed by Englund, 1993). The potential GPI-anchored variant of Algal CAM is absent in early embryos and becomes detectable at or immediately after the differentiating cleavage (i.e., the transition from the 32- to the 64-cell embryo). Four-cell embryos treated with Algal CAM antibody undergo a lower number of cleavages and produce only a few reproductive cells. These observations suggest an important function of Algal CAM variants during Volvox embryogenesis and support an early model (Sumper, 1979) using properties of cell adhesion molecules to explain the main features of Volvox embryogenesis: in a slightly modified form, this model is able to offer a mechanism by which the embryo could realize arrival at the 32-cell stage. At this stage, the differentiating cell cleavage takes place in exactly 16 blastomeres. Algal CAM antibody given at late stages of embryogenesis does no longer disrupt cell-cell contacts, but causes a quite different phenotype: the antibody selectively inhibits the process of embryonic inversion. 2. The ECM Glycoprotein ISG An ECM protein (denoted as ISG) with remarkable properties was discovered by pulse-labeling studies with radioactive sulfate over the period of embryonic inversion (Wenzl and Sumper, 1982; Schlipfenbacher et al., 1986). ISG is a sulfated, extracellular glycoprotein synthesized for less than 10 min in inverting Volvox embryos as well as in inverting sperm cell packets. Subsequently, ISG has been characterized by studies of protein chemistry and electron microscopy. The primary structure of ISG has been derived from genomic DNA and cDNA (Ertl et al., 1992). ISG is composed of a globular and a rod-shaped domain. The rod-shaped domain is again related to the extensin family with numerous repeats of Ser-(Hyp),-6 motifs. Virtually all of the hydroxy amino acids in this region appear to be glycosylated, with the dominant sugars being arabinose and galactose. Deglycosylation reduces the apparent molecular mass of ISG from about 200 kDa to about 60 kDa. The N-terminally located globular domain exhibits no unusual amino acid composition. The length of the rod-shaped domain was determined by electron microscopy to be 57 nm. Purified ISG oligomerizes to star-like particles with a central knob and a variable number of the 57-nm arms.

BIOCHEMISTRY OF THE Vo/vox ECM

65

Although ISG is synthesized only during an extremely short period in the 48-h life cycle of Volvox, the mature glycoprotein remains stable for at least 24 h, as revealed by pulse-chase experiments with radioactive sulfate. Immunofluorescencemicroscopy, using polyclonal antibodies raised against the globular domain of ISG produced in Escherichia coli by recombinant DNA technology, localizes ISG in the flagellar tunnel region and in the boundary zone of the ECM (Ertl et al., 1992). A distinct feature at the Cterminal end of ISG is the occurrence of a cluster of five positively charged amino acid residues. A synthetic decapeptide matching this sequence disturbed the early stages of ECM biogenesis and assembly at concentrations M without affecting the viability of Volvox cells. This as low as 5 X observation suggests that ISG may participate in early processes that organize ECM assembly. Based on these results and on the remarkable phenotype exhibited by a temperature-sensitive flagellaless mutant (flgCII) studied by Huskey (1979), an interesting hypothesis for the function of ISG was articulated by David Kirk (personal communication). When the mutant f?gCll was held at the restrictive temperature during embryonic inversion and early postembryonic development and then shifted to the permissive temperature, spheroids developed flagella that appeared to beat normally, but the spheroids were incapable of any coordinated swimming behavior. It turned out that the cells of the spheroid were randomly oriented with respect to the anterior-posterior axis of the spheroid and thus flagella of different cells were working antagonistically rather than in a coordinated manner. Therefore, it is concluded that flagella must be present at the time when the ECM is first laid down in order for the cells to be locked into the correct position within the spheroid. A very important early stage in ECM construction, prior to the breakdown of embryonic cell-cell interactions (cytoplasmic bridges and/or cell-cell contacts), should therefore involve deposition of boundary-zone matrix material around the bases of the flagella, and also out over the rest of the surface of the inverted embryo. This would permanently trap all flagella and hence the cells, thereby fixing the cellular orientations that had been established during embryonic cleavages. ISG, the first ECM component of the postinversion embryo to be synthesized, is a likely candidate to serve this function. If so, ISG would also assist in nucleation and assembly of other ECM components. This proposed scenario would explain the observation that a peptide analogue of ISG applied at the early stages of ECM biogenesis has such a drastic effect on the organization of all parts of the ECM. The striking developmental control of ISG synthesis operates at the level of transcription. ISG mRNA is virtually absent at the very beginning of the inversion process. A high level is observed during inversion and, toward

66

MANFRED SUMPER AND ARMlN HALLMANN

the end of inversion, the level of ISG mRNA again significantly decreases (Ertl et a[., 1992). The characterization of Algal CAM and ISG proves the existence of ECM glycoproteins during both the early and the late phase of embryogenesis. Possibly, these “embryonic” ECM molecules are not yet organized in supramolecular networks and may therefore escape from microscopic detection during embryogenesis. A putative Chlamydomonas cell wall glycoprotein identified by its cDNA clone is related to the globular domain of ISG (Woessner et al., 1994). Remarkably, the rod-shaped domain with the typical Ser-(Pro)4-6repeats in ISG is replaced by a different rod-shaped element with (Ser-Pro), repeats in the Chlamydomonas homologue. B. Modifications under Developmental Control

1. Asexual Life Cycle: SSG 185 Pulse-labeling studies with radioactive sulfate at different developmental stages revealed the existence of variants of the ECM glycoprotein SSG 185 that are defined by different electrophoretic mobilities in SDS-PAGE. A given variant is reproducibly synthesized at a defined stage of development. For instance, at the time of early embryogenesis, the somatic cells of the mother spheroid synthesize a SSG 185 with a significantly larger apparent molecular mass compared to the variant produced at later stages of embryogenesis (Wenzl et al., 1984). The molecular basis for this difference resides in the sulfated polysaccharide attached to SSG 185. In particular, SSG 185 variants with a slower electrophoretic mobility lack the sulfate group at the C 4-positions in the polymannan backbone (Mengele, 1988). These modifications of the SSG 185 monomer remain conserved in the polymer, indicating structural modifications of the ECM at defined developmental stages. At present, the biological reason for this ECM modulation is unknown. An exciting recent discovery in plant ECM biology has been the demonstration that ECM molecules may influence cell fate in plants (Brownlee and Berger, 1995). It appears that information may be stored in the structure of ECM. Somatic cells of Volvox exhibit programmed cell death (Pommerville and Kochert, 1982) and this event is influenced by the development program: In sexual (egg-containing) organisms, which do not enter embryonic cleavages, programmed cell death is delayed by 96 h. It is tempting to speculate that ECM modifications might be involved in this cross-talk between asexual embryos (or eggs) and somatic cells. 2. Sexual Development

Although V. carteri reproduces asexually much of the time, in nature it reproduces sexually at least once each year. V. carteri lives in temporary

BIOCHEMISTRY OF THE Vo/vox ECM

67

ponds that dry out in the heat of late summer. Asexual Volvox algae would die in minutes once the pond dried out, but V. carteri is able to escape this catastrophe by switching to the sexual life cycle shortly before the pond dries up, producing dormant zygotes that survive the drought of late summer and the cold of winter. When rain fills the ponds in spring, the zygotes hatch out to establish a new generation of asexually reproducing individuals. The stimulus for switching from the asexual to the sexual mode of reproduction in V. carteri is known to be a sex-inducing pheromone, a 32-kDa glycoprotein (Starr, 1970; Tschochner et al., 1987; Mages et al., 1988). This pheromone is one of the most potent biological effector molecules known. It triggers sexual development of gonidia at concentrations as low as M and initiates a modified developmental program that results in the production of eggs or sperm packets, depending on the genetic sex of the individual. What is the source of this sex-inducing pheromone and how do these simple organisms predict the coming of unfavorable conditions to produce a sexual generation just in time? Kirk and Kirk (1986) found a simple explanation: the sexual cycle is initiated by a heat shock that causes the somatic cells of the asexual Volvox spheroid to produce the sex-inducing pheromone. The level of pheromone is then further amplified by the ability of sperm cells to produce more sex-inducingpheromone (Starr, 1970;Gilles et al., 1984). By this strategy, all members of the population within a pond or lake become synchronously converted to the sexual pathway. A particularly fascinating problem is the molecular mechanism which enables the sex-inducing pheromone to act at a concentration as low as l O - I 7 M. A final answer is not yet available despite the efforts of several groups. However, it is established that the first biochemical responses to the sex-inducing pheromone are structural modifications within the ECM (Wenzl and Sumper, 1982, 1986; Gilles et al., 1983; Sumper et al., 1993). In particular, the earliest response, detectable a few minutes after the application of the pheromone, is the synthesis of a sulfated ECM glycoprotein, pherophorin 11, with an apparent molecular mass of 70 kDa. a. Pherophorin ZZ After application of the sex-inducing pheromone, the somatic cells (which are not the ultimate target of the pheromone’s message!) of an asexually growing Volvox spheroid respond with a strong induction of pherophorin I1 synthesis. As in the case of constitutively expressed pherophorin I, pherophorin I1 is again found to be localized in the insoluble CZ of the ECM. The primary structure of pherophorin I1 was deduced from cDNA sequence analysis (Sumper et al., 1993). A short proline-rich (most probably hydroxyproline-rich) stretch of about 12 amino acid residues around position 170 separates two domains that share sequence identities with the corresponding domains of pherophorin I (Fig. 7). The C-terminal domain exhibits 28% identity and 68% similarity with the sex-inducing pheromone. About 10 copies of pherophorin 11-related

BIOCHEMISTRY OF THE Volvox ECM

69

genes are found to exist, at least 3 of them in tandem arrangement, in the Volvox genome (Godl et al., 1995). This high gene dose may explain the massive production of pherophorin I1 in response to pheromone application. Even after its deposition within the ECM, pherophorin I1 (in contrast to pherophorin I ) turned out to be an unstable protein being proteolytically processed in a highly specific manner. With a half-life of about 6 h, pherophorin I1 is cleaved into a 42-kDa and a 30-kDa fragment. The cleavage site is exactly adjacent to the polyproline spacer. Interestingly, pherophorin I exhibits an insertion of seven amino acid residues at this cleavage site (Fig. 7). This may explain the resistance of pherophorin I to this specific processing. Pherophorin I1 processing causing the liberation of the pheromone-homologous domain was discussed as part of a signal amplification mechanism that would explain the exquisite sensitivity of the sexinducing pheromone. Inhibition of pherophorin I1 processing indeed correlates with a suppression of sexual induction (Godl et al., 1995). However, direct proof that the C-terminal domain of pherophorin I1 has sex-inducing activity is still lacking.

b. Pherophorin S Application of the sex-inducing pheromone not only induces a modulation of the C Z of the ECM but also leads to substantial changes in the chemical composition of the deep zone. At least two novel ECM glycoproteins are synthesized by somatic cells and targeted to the deep zone in response to the pheromone. Very recently, one of these pheromone-dependent glycoproteins with an apparent molecular mass of 110 kDa was biochemically characterized in detail (Godl et al., 1997). Unexpectedly, this protein of the deep zone turned out to be a true member of the pherophorin family (Fig. 7) (now denoted as pherophorin S) although it exhibits a completely novel set of properties. Besides its different location within the ECM, pherophorin S remains a highly soluble component of the deep zone. Main structural differences from other members of the family include the presence of a poly-hydroxyproline spacer in pherophorin S, 88 amino acid residues in length, that separates the N- and C-terminal domains and, in addition, the presence of incorporated phosphate. This phosphate-containing element that is part of a saccharide attached to the

FIG. 7 Alignment of the amino acid sequences of pherophorin S and pherophorin 1-111. The polyproline stretches are given on gray background. Gaps (-) are introduced to allow maximal alignment. The C-terminal part of the pherophorins found to be homologous to the sexinducing pheromone is boxed. The site of pherophorin I1 and 111 cleavage is marked by an arrowhead. Mature polypeptides are given; for pherophorin S, the N terminus of the mature protein has not been experimentally determined.

70

MANFRED SUMPER AND ARMlN HALLMANN

poly-hydroxyproline spacer again turned out to be identical to the phosphodiester originally discovered in SSG 185 (see Sections III.B.l and 1V.D) which also contains an extended stretch of hydroxyprolines as a spacer element. Since SSG 185 and pherophorin S are located in completely different zones of the ECM, it is unlikely that this very special spacer element provides the signal for targeting pherophorin S to the deep zone. There are a few additional changes in the ECM composition that have been reported to be caused by the sex-inducing pheromone. A corresponding ECM protein, denoted as FSG, will be mentioned only briefly, because as yet only limited biochemical data are available. Synthesis of FSG (-280 kDa) is initiated within 20-30 min after the addition of the pheromone (Wenzl and Sumper, 1982). FSG is a sulfated glycoprotein: Arabinose, xylose, mannose, and galactose (-4.5:0.2:0.2:1.0) are the predominant neutral sugars. FSG is continued to be synthesized by somatic cells until the early stages of embryogenesis.

C. ECM Modifications Induced by Stress 1. Arylsulfatase (Sulfur Deprivation)

Arylsulfatase activity is found in many organisms and plays an important role in mineralization of sulfate (Speir and Ross, 1978). Volvox synthesizes arylsulfatase in response to sulfur deprivation. Enzyme activity remains associated with the spheroid and is not secreted into the culture medium. However, in a Volvox mutant strain called “dissociator” (Dauwalder et al., 1980), which is unable to produce a structurally intact ECM and consequently dissociates into single cells shortly after the end of embryogenesis, large amounts of enzyme activity are detectable in the culture medium. These facts indicate deposition of the arylsulfatase within the ECM of the wild-type strain, but no information is available about the subzone involved. Properties of the Volvox arylsulfatase (like its inducible synthesis and its extracellular location) indicate that mineralization of sulfate in response to sulfur deprivation seems to be the function of this enzyme. The inducible arylsulfatase of Volvox has been purified to homogeneity and the corresponding gene and cDNA have been cloned (Hallmann and Sumper, 1994a). The gene is composed of 16 introns and 17 exons that encode a 649-aminoacid polypeptide chain. The presence of a hydrophobic leader sequence together with the existence of seven potential N-glycosylation sites suggests glycosylation of the polypeptide. A novel posttranslational protein modification has recently been described in two human sulfatases, by which a cysteine is replaced by a serinesemialdehyde (2-amino-3-oxopropionic acid) residue (Schmidt et al.,

BIOCHEMISTRY OF THE Volvox ECM

71

1995). Recently, the presence of this modification was also demonstrated in Volvox arylsulfatase (Selmer et al., 1996). The evolutionary conservation of this novel protein modification between sulfatases of Volvox and man supports the assumption that this modification is required for the catalytic activity of sulfatases and may be present in all sulfatases of eukaryotic origin (Selmer et al., 1996). Because the arylsulfatase enzyme is readily assayed using chromogenic substrates, but is not detectable in cells grown in sulfate-containing medium, the genes encoding arylsulfatase are useful reporter genes both in Chlamydomonas (Davies et al., 1992; Davies and Grossman, 1994; Quinn and Merchant, 1995) and in V. carteri (Hallmann and Sumper, 1994b; God1 et al., 1997). A remarkable property of the arylsulfatase from Volvox is its insensitivity toward detergents like dodecyl sulfate. Concentrations as high as 1%SDS do not affect enzyme activity. This property enables easy detection of the enzyme even in crude cell lysates (Hallmann and Sumper, 1994a). In addition, the highly regulated promoter of the arylsulfatase gene is a tool for producing inducible expression vectors for cloned genes.

2. Responses to Wounding By subtractive cDNA technology, a number of genes were recently identified that are induced in Volvox by wounding (Haas, Amon, and Sumper, unpublished results). Two of these cDNA clones have been characterized in detail (Amon et al., 1997) and turned out to encode a member of the chitin-binding proteins and a chitinaseAysozyme. In higher plants, these proteins have been implicated as part of a defense mechanism against fungi (Boller, 1988; Bowles, 1990; Linthorst, 1991). The first clone encodes a polypeptide with 309 amino acid residues including a typical signal sequence. The N-terminal half is composed of two nearly perfect repeats of a 48-amino-acid sequence motif. These repeats are separated by a typical spacer element (SGGGGSTPTSTAPPAR). Similar repeats located in phage lysozymes (Paces et al., 1986; Garvey et al., 1986), in a muramidase (Chu et al., 1992), and in an autolysin from Streptococcus faecalis (Beliveau et al., 1991) were identified as top-scoring hits in a BLASTP search (Altschul et al., 1990) of the Swiss-Prot Protein Sequence Database. The region of identities covers nearly the total length of the 48-amino-acid repeat. This repeat is diagnostic of enzymes of the lysozyme family (Joris et aZ., 1992) that also includes vacuolar and secreted chitinases from plants (Holm and Sander, 1994). A repeating unit structure is also a striking feature of the deduced amino acid sequence of the second clone. Three repeats of a domain, 48 amino acid residues in length, constitute the C-terminal part. Proline-rich se-

72

MANFRED SUMPER AND ARMlN HALLMANN

quences with Ser-(Pro), motifs typical of extensins separate these domains from each other. Again, a homology search revealed a close relation of the repeating unit to a well-known protein family, namely, to chitin-binding proteins (Raikhel et al., 1993). The chitin-binding domains of this Volvox polypeptide show extensive identity to the corresponding domains of other proteins like the class I chitinases (Linthorst et al., 1990), hevein (Broekaert et al., 1990), and the wound-induced WIN proteins from potato (Stanford et al., 1989). The N-terminal sequence of the Volvox chitin-binding protein shares high homology with a human cysteine protease (31% identity and 69% similarity over 241 amino acid residues) (Fuchs and Gassen, 1989). This remarkable combination of a protease domain, extensin-like spacer elements, and several chitin-binding domains suggests that this polypeptide is specialized for the degradation of chitin-linked proteins. A similar domain combination, however, with different types of spacer elements, has been described for a prokaryotic enzyme produced by Streptomyces griseus (Sidhu et al., 1994). In Section VI the domain organization of these Volvox ECM proteins induced by wounding is schematically illustrated. Both of these proteins were detected by Western blot experiments as components of the deep zone of the ECM.

D. Crosslinking The sulfated glycoprotein SSG 185 is the monomeric precursor of a highly insoluble polymer of Volvox ECM (CZ3). The phosphodiester found in SSG 185 has been investigated by methylation analysis, I3C-NMR, enzymatic assays, and mass spectrometry and identified as ~-Araf-5-phospho5’-~-Araf(Fig. 8). This phosphodiester bridge most probably crosslinks

/0

’

‘

P

O

H

HO-P=O

I I

OH

0

OH FIG. 8 Structure of the phosphodiester of arabinose isolated from the ECM glycoproteins SSG 185, pherophorin S, and HRGP-290 of Volvox carteri.

BIOCHEMISTRY OF THE !&ox

73

ECM

different carbohydrate chains either intra- or intermolecular in SSG 185 (Holst et af., 1989). There is indirect evidence for an intermolecular crosslink. Polymeric SSG 185 can easily be degraded to monomeric polypeptide chains by the action of anhydrous hydrogen fluoride at O"C, a procedure that selectively cleaves 0-glycosidic linkages (Mort and Lamport, 1977). This fact indicates the existence of intermolecular crosslinks between saccharide chains and excludes intermolecular crosslinks of the exceedingly stable diphenyl ether linkage of isodityrosine (Fry, 1982). Originally, this type of linkage was favored as an intermolecular crosslink in extensins from higher plants, but more recent evidence favors an intramolecular crosslink rigidifying the polypeptide backbone (Kieliszewski and Lamport, 1994). Isodityrosine was also detected in hydrolysates from Chlamydomonas W2 cell wall glycoprotein (Waffenschmidt et at., 1993), but again, proof for the existence of intermolecular crosslinks is lacking. Recently, the HRGPs present in the deep zone of Volvox ECM were found to contain many copies of the same phosphodiester of arabinose. Both of these novel glycoproteins are converted to polymeric structures, as is the case for SSG 185. A remarkable fact is the presence of the diester even in the monomeric precursors, which stimulates speculation about the chemical mechanism of crosslink formation. Without any energy requirement, an intramolecular phosphodiester bridge could be converted into an intermolecular bridge simply by a transesterification reaction (Fig. 9). This reaction would act in both directions. In principle, this chemistry is precisely what is required to remodel or rearrange a glycoprotein network in the

Ho-Ara

-1

A ra

1 A r a - O "

HO-Ara

Ara -@-

I

HO-Ara

I

Ara

Ara

A

+

Ara

FIG. 9 A speculative model for crosslink formation. Without any energy requirement, an intramolecular phosphodiester bridge could be converted into an intermolecular bridge simply by a transesterification reaction. This would be a reversible process.

74

MANFRED SUMPER AND ARMlN HALLMANN

wall to accommodate the insertion of new monomers and therefore permit extension. A comparison with the phosphodiesters of riboses in self-splicing RNAs drives speculations even further: Perhaps this transesterification works autocatalytically. Experiments are currently under way to check this possibility. E. ECM Lysis The liberation of Volvox daughters from the mother spheroid is effected by an enzymatic process, by which the sheath of the parental spheroid is lysed locally so that each daughter spheroid leaves through a hole. In Chlamydomonas, enzymes have been known for some time that are involved in the process of wall degradation (Claes, 1971; Schlosser, 1976, 1981). In C. reinhardtii there are at least two wall-degrading enzymes that function at specific developmental stages: a gamete lytic enzyme (GLE) and a vegetative lytic enzyme (VLE). GLE is a Zn2+-containingmetalloprotease with a molecular mass of 62 kDa (Matsuda et al., 1984, 1985). Its primary structure is homologous to those of mammalian collagenases (Kinoshita et al., 1992). VLE was partially purified and reported to be a 34-kDa glycoprotein (Jaenicke and Waffenschmidt, 1981; Jaenicke et al., 1987; Spessert and Waffenschmidt, 1990). However, the enzyme was recently purified to homogeneity and shown to be a serine protease with a molecular mass of 130 kDa (Matsuda et al., 1995), whereas the 34-kDa protein turned out to be a main impurity of the crude extract. Possibly, homologous proteases have also been described from V. carteri (Jaenicke and Waffenschmidt, 1979, 1981; Waffenschmidt et al., 1990). The lysins of Chlamydomonas and of Volvox degrade the cell wall/ECM in a species-specific manner. From the subtractive cDNA Volvox library mentioned above (Section IV.C.2), the existence of a Volvox homologue of Chlamydomonas GLE could recently be deduced exhibiting a remarkable mosaic structure (schematically illustrated in Section VI) (Haas, Godl, and Sumper, unpublished results). Like in SSG 185 and pherophorin S, a long stretch of prolines (hydroxyprolines?), -120 residues in length, is a central part of the polypeptide. N-terminally located is the sequence homologous to the metalloprotease GLE (38% identity and 74% similarity over 92 amino acid residues, including all active site residues), whereas the C-terminal part of this polypeptide consists of two modules, each repeated twice, of unknown function. V. Relationship to Higher Plant ECMs HRGPs found in the ECM of higher plants include extensins, repetitive proline-rich proteins (RPRPs), some nodulins, gum arabic glycoprotein

BIOCHEMISTRY OF THE Vdvox ECM

75

(GAGP), arabinogalactanproteins (AGPs), and chimeric proteins such as potato lectin which contain an extensin module fused to a lectin. The extensins, a large multigene family of higher plant HRGPs (Showalter and Varner, 1989), are the best characterized and probably the most abundant structural proteins of dicot cell walls (Cooper et al., 1984; Varner and Lin, 1989). Higher plant HRGPs are assumed to play key functions in cell wall self-assembly and cell extension. Their repetitive peptide motifs and the site-specific posttranslational modifications singly or in combination are believed to constitute functional sites involved in various aspects of cell wall biogenesis, as, for instance, self-assembly, adhesion, and crosslinking (Kieliszewski and Lamport, 1994). Based on their similar chemistry, it is reasonable to hypothesize that higher plant and VoZvox/Ch/arnydornonas HRGPs share similar functions in ECM assembly and architecture. Recent reviews concerning different aspects of higher plant ECM include Knox (1995), Brownlee and Berger (1995), Joseleau et al. (1994), Roberts (1994), Albersheim et a/. (1994), Lane (1994), Varner and Ye (1994), Gibeaut and Carpita (1994), Showalter (1993), Fry et al. (1993) and Levy and Staehelin (1992).

VI. Conclusions In this review, the structures of more than 10 ECM proteins from a single species of the multicellular alga Volvox are compared. For 10 ECM proteins, the complete primary structures have been deduced from their genes and the corresponding structures are summarized in the stylized drawing of Fig. 10. Together with structural data from cell wall glycoproteins of the unicellular relative C. reinhardtii, this pool of characterized proteins should allow recognition of common biochemical and structural features. Due to the high content of hydroxyproline, these proteins are usually denoted as HRGPs and are regarded as representing products of one giant supergene family. However, the sequence data do not support such a view. Rather, a typical feature of all algal ECM proteins is a striking modular composition, where hydroxyproline-rich sequences are strictly confined to the rod-shaped domains. Many other modules found in these ECM proteins are completely devoid of hydroxyproline residues. The multiple functions of a complex ECM can hardly be met on the basis of repetitive hydroxyproline-rich sequences. Instead of defining a HRGP family, it appears more appropriate to define a H R module family that can be combined with other modules to yield chimeric and multifunctional polypeptides. A particularly striking example for the confusion introduced by the term “HRGP” is found among the Vdv ox pherophorin protein family. By se-

Volvox ECM-protein

modular composition

localization I property

reference

SSG 185

CZ Imain component of the CZ; contains phosphodiester bridges

Wenzl and Sumper, Iga2; Wenzl et a/., 984; Ertl eta/., 1989

Pherophorin I

CZ Imain component of the CZ

~

Pherophorin II

CZ I C-terminal domain is liberated under the influence of the sex pheromone

sumper a/., 1993;

Pherophorin 111

cz

Godl et a/., 1995

PherophorinS

DZ Ithe only member of pherophorins Godl eta/., 1997 targeted lo DZ; contains phosphcdiester bridges

Algal-CAM

cell-cell contacts I cell adhesion molecule; homology to fasciclin I

IS0

BZ I synthesized for less than

chitin-binding protein

DZ I stress-induced; synthesized in response to wounding

Amon eta/., 1997

chitiflase/ lysozyme

DZ Istress-induced; synthesized in response to wounding

Amon eta/.,1997

~

p

~

~

~

~

Godl et af., 1995

,

= A-type domain

~

;

hydroxyproline-rich ~ = ~(HR)-module Q 3 ~

+rn -

= globular ISG domain = cysteine proteinase

Huber and Sumper' I

GLE4ike protein

10 min in invertingembryos

/homology to Chlamydomonas GLE

fascrclin I-like domain

Wenzl and Sumper, 1982; Schlipfenbacher eta/., 1986; Ertl ef a/. , 1992

chitin binding domain

= lysozvme domain

= OLE-like domain

3

(unpublished)

FIG.10 Domain organization of known Vofvox ECM glycoproteins.

@

= :Z'i?ikperties

-

domain with unknown properties

BIOCHEMISTRY

OF THE Vo/vox ECM

77

quence alignment (Fig. 7), all members are closely related; however, pherophorin I, 11, and 111 only contain a very short hydroxyproline-rich spacer that does not allow classification as a HRGP. In contrast, the amino acid composition of pherophorin S clearly qualifies this protein for membership in the HRGP family. But it is only the insertion of a long hydroxyprolinerich module between the N- and the C-terminal domains that converts this member of the pherophorins to the HRGP family. Most probably, the rodshaped HR modules have a structural function and serve as building blocks to create the defined framework of the ECM. This is supported by the unusually high variability of the HR modules found even among closely related ECM proteins. For instance, the Chlamydomonas homologue of the Volvox ECM protein ISG, denoted as VSP-3, replaces the typical Volvox H R module with numerous Ser-(Hyp)4-6 repeats by a different module with (Ser-Hyp), repeats. An even more striking example has been detected in cell wall proteins (denoted as frustulins) from diatoms which also exhibit a conspicuous modular composition (Kroger et al., 1994, 1996). Frustulins are composed of several repeats of a highly conserved module (ACR domains) which are separated by HR modules if isolated from the diatom Cylindrotheca fusiformis. In a highly homologous frustulin isolated from Navicula pelliculosa these ACR domains are separated by polyglycine modules. Probably, the purely structural task of the HR modules can be met by a large spectrum of rapidly evolving sequence variants, inserted in otherwise highly conserved ECM proteins. It has repeatedly been proposed that HRGPs should make excellent phylogenetic markers for all plants, because of the central role these proteins play in organizing cell and plant morphology. The HR modules within these proteins, however, do not appear to be suitable markers for assessing long-term evolution. In Table I the HR modules found so far in Volvox and Chlamydomonas cell wall/ECM proteins are compared. Where analyzed in more detail, these modules are found to be targets for extensive posttranslational modifications. Among the modifications found are 0-glycosylations with oligoarabinosides, introduction of phosphodiester bridges between arabinose residues, and, in a single case, the attachment of a highly sulfated polysaccharide. The mosaic structures of volvocine ECM proteins known so far appear to provide another example of the combinatorial advantage of shuffling modules, as it is so evident in the evolution of the metazoan ECM proteins (Doolittle, 1995). A high combinatorial potential may not only be a prerequisite for establishing the transition from a cell wall to the complex ECM of a multicellular organism, but also to achieve subsequent species diversification. Remarkably, the transition from unicellularity to multicellularity independently happened several times within the Volvocales and is, accord-

TABLE I HR Modules Known from Volvox and Chlamydomonas Cell WalllECM Proteins (Hydroxy)Proline-rich module Organism

ECM protein

Characteristic repeat

Volvox carteri Vo1vo.x carteri

SSG 185 Pherophorin I

Ser-(Pro)2.17 and (Pr0)2-4 -

Volvox carteri Volvox carteri Volvox carteri Volvox carteri Volvox carteri Chlamydomonas reinhardtii Chlarnydomonas reinhardtii Chlnmydomonas eugametos

Pherophorin S Algal CAM ISG Chitin-binding protein GLE-like protein

Ser-(Pro), Ser-(Pro)3-6 Ser-(Pr~)~_~ Ser-(Pro)z-4 Ser-(Pro)5

Zygote wall protein VSP-3 WP6

(Pro)j and (SerPro) (SerPro) (SerPro),

Maximum length of poly-(hydroxy)proline stretch (Pro) I X (Pro) 10 (pro)26 (Pro)6 (Pro17 (Pro17 (Proh

Data library accession no. X51616 X69801 YO7752 X80416 X65165

S44199 L29029 L29028

BIOCHEMISTRY OF THE Vdvox ECM

79

ing to rRNA sequence data, a very recent evolutionary event in contrast to textbook statements about Volvox (Rausch er al., 1989; Larson er al., 1992). In a stimulating essay on the origins of eukaryotic sex, Goodenough (1985) stressed the possible role of cell wall molecules in the evolution of eukaryotic sex. Derivatives of these molecules designed for self-assembly could have been recruited for promoting cell fusion, and, due to their specificity, for limiting such fusions to genetically appropriate partners. The structural properties of the sexual agglutinins from Chlurnydornonas (Adair and Snell, 1990) support this idea. Sexual speciation requires molecules that are, in addition, designed for variation. Again, the modular organization of ECM proteins may provide this important property. The sexual reproduction system of Volvox adds a new and unexpected aspect. Before entering the sexual reproductive cycle, asexually growing organisms have to be triggered to develop male and female organisms. This signal is provided by the sex-inducing pheromone (a glycoprotein). It was a great surprise that main compartments of the Volvox ECM contain proteins (pherophorins) that bear modules with homology to the sex-inducing pheromone. Possibly, the highly potent sex-inducing pheromone is evolutionarily derived from a member of the pherophorin family that originally served a structural function within the ECM. Sexual speciation again requires modulation of pheromone molecules. It is interesting to note that the pherophorins represent a large protein family, and, in particular, pherophorin 11-like proteins are represented by about 10 genes, in tandem arrangement, that encode similar but not identical sequences. Homologous recombination events at this repetitively organized DNA locus would create variability. Pherophorins are located in the CZ, and as mentioned earlier it is exactly this zone that exhibits species-specificity, whereas the BZ is highly conserved among different Volvocales. Volvox,the simplest multicellular organism, responds to environmental stimuli and wounding in much the same way as that observed in higher plants. These developmental processes depend on changes that take place in the structure of the ECM. Recent progress in the application of powerful molecular genetic techniques like transformation and gene replacement should support further advances in understanding the function of ECM proteins in these important mechanisms of plant biology.

Acknowledgment We are indebted to Dr. David L. Kirk for critical reading of the manuscript.

References Adair, W. S., and Appel, H. (1989). Identification of a highly conserved hydroxyproline-rich glycoprotein in the cell walls of Chlumydomonus reinhurdtii and two other Volvocales. Pluntu 179,381-386.

80

MANFRED SUMPER AND ARMlN HALLMANN

Adair, W. S., and Apt, K. E. (1990). Cell wall regeneration in Chlamydomonas: Accumulation of mRNAs encoding cell wall hydroxyproline-rich glycoproteins. Proc. Natl. Acad. Sci. USA 87,7355-7359. Adair, W. S., and Snell, W. J. (1990). The Chlamydomonas reinhardtii cell wall: Structure, biochemistry, and molecular biology. In “Organization and Assembly of Plant and Animal Extracellular Matrix” (W. S. Adair and R. P. Mecham, eds.), pp. 15-84. Academic Press, San Diego. Adair, W. S., Steinmetz, S. A., Mattson, D. M., Goodenough, U. W., and Heuser, J. E. (1987). Nucleated assembly of Chlamydomonas and Volvox cell walls. J. Cell Biol. 105,2373-2382. Albersheim, P., An, J., Freshour, G., Fuller, M. S., Guillen, R., Ham, K.-S., Hahn, M. G., Huang, J., O’Neill, M., Whitcombe, A., Williams, M. V., York, W. S., and Darvill, A. (1994). Structure and function studies of plant cell wall polysaccharides. Biochem. Soc. Trans. 22,374-378. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990). Basic local alignment search tool. J. Mol. Biol. 215, 403-410. Amon, P., Haas, E., and Sumper, M. (1997). The sexual pheromone and wounding triggers the same set of genes in the multicellular green alga Volvox. Submitted for publication. Beliveau, C., Potvin, C., Trudel, J., Asselin, A., and Bellemare, G. (1991). Cloning, sequencing, and expression in Escherichia coli of a Streptococcus faecab autolysin. J. Bacteriol. 173, 5619-5623. Boller, T. (1988). Ethylene and the regulation of antifungal hydrolases in plants. In “Surveys of Plant Molecular and Cell Biology” (B. J. Miflin, ed.), Vol. 5. pp. 145-174. Oxford Univ. Press, London. Bowles, D. (1990). Defense-related proteins in higher plants. Annu. Rev. Biochem. 59,873-907. Broekaert, I., Lee, H. I., Kush, A., Chua, N. H., and Raikhel, N. (1990). Wound-induced accumulation of mRNA containing a hevein sequence in laticifers of rubber tree (Hevea brasiliensis). Proc. Natl. Acad. Sci. USA 87, 7633-7637. Brownlee, C., and Berger, F. (1995). Extracellular matrix and pattern in plant embryos: On the lookout for developmental information. Trends Genet. 11,344-348. Catt, J. W., Hills, G. J., and Roberts, K. (1976). A structural glycoprotein, containing hydroxyproline, isolated from the cell wall of Chlamydomonas reinhardii. Planta 131, 165-171. Catt, J. W., Hills, G. J., and Roberts, K. (1978). Cell wall glycoproteins from Chlamydomonas reinhardii, and their self-assembly. Planta 138, 91-98. Chu, C. P., Kariyama, R., Daneo-Moore, L., and Shockman, G . D. (1992). Cloning and sequence analysis of the muramidase-2 gene from Enterococcus hirae. J . Bacteriol. 174,16191625. Claes, H. (1971). Autolyse der Zellwand bei den Gameten von Chlamydomonas reinhardii. Arch Mikrobiol. 108,221-229. Cooper, J. B., Chen, J. A., and Varner, J. E. (1984). The glycoprotein component of plant cell walls. In “Structure, Function, and Biosynthesis of Plant Cell Walls” (W. M. Dugger and S. Bartnicki-Garcia, eds.), pp. 75-88. Waverly Press, Baltimore. Dauwalder, M., Whaley, W. G., and Starr, R. C. (1980). Differentiation and secretion in Volvox. J. Ultrastruct. Res. 10, 318-335. Davies, J. P., and Grossman, A. R. (1994). Sequences controlling transcription of the Chlamydomonas reinhardtii /3 2-tubulin gene after detlagellation and during the cell cycle. Mol. Cell. Biol. 14, 5165-5174. Davies, J. P., Weeks, D. P., and Grossman, A. R. (1992). Expression of the arylsulfatase gene from the p 2-tubulin promoter in Chlarnydomonas reinhardfii. Nucleic Acids Res. 20,2959-2965. Debuchy, R., Purton, S., and Rochaix, J. D. (1989). The argininosuccinate lyase gene of Chlarnydomonas reinhardtii: An important tool for nuclear transformation and for correlating the genetic and molecular maps of the ARC7 locus. EMBO J. 8,2803-2809.

BIOCHEMISTRY OF THE V o h x ECM

81

Desnitski, A. G. (1995). A review on the evolution of development in Volvoxt Morphological and physiological aspects. Eur. J. Protist. 31, 241-247. Doolittle, R. F. (1995). The multiplicity of domains in proteins. Annu. Rev. Biochem. 64, 287-314. Elkins, T., Hortsch, M., Bieber, A. J., Snow, P. M., and Goodman, C. S. (1990). Drosophila fasciclin I is a novel homophilic adhesion molecule that along with fasciclin I11 can mediate cell sorting. J. Cell Biol. 110, 182551832, Englund, P. T. (1993). The structure and biosynthesis of glycosyl phosphatidylinositol protein anchors. Annu. Rev. Biochem. 62,121-138. Ertl, H., Mengele, R., Wenzl, S., Engel, J., and Sumper, M. (1989). The extracellular matrix of Volvox carteri; Molecular structure of the cellular compartment. J. Cell Biol. 109,3493-3501. Ertl, H., Hallmann, A., Wenzl, S., and Sumper, M. (1992). A novel extensin that may organize extracellular matrix biogenesis in Volvox carteri. EMBO J. 11,2055-2062. Fry, S. C. (1982). Isodityrosine, a new cross-linking amino acid from plant cell-wall glycoprotein. Biochem. J. 204, 449-455. Fry, S. C., Aldington, S., Hetherington, P. R., and Aitken, J. (1993). Oligosaccharides as signals and substrates in the plant cell wall. Pfant Physiot. 103, 1-5. Fuchs, R., and Gassen, H. G. (1989). Nucleotide sequence of human preprocathepsin H, a lysosomal cysteine proteinase. Nucleic Acids Res. 17, 9471. Garvey, K., Saedi, M. S., and Ito, J. (1986). Nucleotide sequence of Bacillus phage phi 29 genes 14 and 15: Homology of gene 15 with other phage lysozymes. Nucleic Acids Res. 14, 10001-10008. Gibeaut, D. M., and Carpita, N. C. (1994). Biosynthesis of plant cell wall polysaccharides. FASEB J. 8,904-915. Gilles, R., Gilles, C., and Jaenicke, L. (1983). Sexual differentiation of the green alga Volvox carteri: Involvement of extracellular phosphorylated proteins. Nafurwissenschaffen 70, 571-572. Gilles, R., Gilles, C., and Jaenicke, L. (1984). Pheromone-binding and matrix-mediated events in sexual induction of Volvox carteri. Z . Naturforsch. 39c, 5&1-592. Godl, K., Hallmann, A., Rappel, A., and Sumper, M. (1995). Pherophorins: A family of extracellular matrix glycoproteins from Volvox structurally related to the sex-inducing pheromone. Planfa 196,781-787. Godl, K., Hallmann, A,, Wenzl, S., and Sumper, M. (1997). Differential targeting of closely related ECM glycoproteins: The pherophorin family from Volvox. EMBO J. 16, 25-34. Goodenough, U. W. (1985). An essay on the origins and evolution of eukaryotic sex. In “The Origin and Evolution of Sex” (H. 0. Halvorson and S. Segal, eds.), pp. 123-140. A. R. Liss, New York. Goodenough, U. W., and Heuser, J. E. (1985). The Chlamydomonas cell wall and its constituent glycoproteins analyzed by the quick-freeze, deep-etch technique. J. Cell B i d . 101,1550-1568. Goodenough, U. W., and Heuser, J. E. (1988). Molecular organization of cell-wall crystals from Chlamydomonas reinhardtii and Volvox carferi.J. Cell Sci. 90, 717-734. Goodenough, U. W., Gebhart, B., Mecham, R. P., and Heuser, J. E. (1986). Crystals of the Chlamydomonas reinhardtii cell wall Polymerization, depolymerization, and purification of glycoprotein monomers. J. Cell Biol. 103,405-417. GOrlach, C. (1994). “Biochemische Charakterisierung einer Sexualpheromon-kontrollierten Komponente der Extrazellullren Matrix von Volvox carteri f: nagariensis.” Ph.D. thesis, Univ. Regensburg, Regensburg, Germany. Green, K. J., and Kirk, D. L. (1981). Cleavage patterns, cell lineages, and development of a cytoplasmic bridge system in Volvox embryos. J. Cell Biol. 91, 743-755. Gumpel, N. J., Rochaix, J. D., and Purton, S. (1994). Studies on homologous recombination in the green alga Chlamydornonas reinhardtii. Curr. Genet. 26, 438-442.

a2

MANFRED SUMPER AND ARMIN HALLMANN

Hallmann, A,, and Sumper, M. (1994a). A n inducible arylsulfatase of Volvox carteri with properties suitable for a reporter-gene system. Purification, characterization and molecular cloning. Eur. J. Biochem. 221, 143-150. Hallmann, A,, and Sumper, M. (1994b). Reporter genes and highly regulated promoters as tools for transformation experiments in Volvox carreri. Proc. Natl. Acad. Sci. USA 91, 11562-11566. Hallmann, A,, and Sumper, M. (1996). The Chlorella hexose/H+ symporter is a useful selectable marker and biochemical reagent when expressed in Volvox. Proc. Natl. Acad. Sci. USA 93,669-673. Hallmann, A , , Rappel, A,, and Sumper, M. (1997). Gene replacement by homologous recombination in the multicellular green alga Volvox carteri. Proc. Natl. Acad. Sci. USA 94,74697474. Hills. G. J., Philipps, J. M., Gay, M . R., and Roberts, K. (1975). Self-assembly of a plant cell wall in vitro. J. Mol. Biol. 96, 431-441. Holm, L., and Sander. C. (1994). Structural similarity of plant chitinase and lysozymes from animals and phage. An evolutionary connection. FEES Lett. 340, 129-132. Holst, O., Christoffel, V., Frund, R., Moll, H., and Sumper, M. (1989). A phosphodiester bridge between two arabinose residues as a structural element of an extracellular glycoprotein of Volvox carteri. Eur. J. Biochem. 181,345-350. Huber, O., and Sumper, M. (1994). Algal-CAMS: Isoforms of a cell adhesion molecule in embryos of the alga Volvox with homology to Drosophila fasciclin I. EMBO J . 13,4212-4222. Huskey, R. J. (1979). Mutants affecting vegetative cell orientation in Volvox carteri. Dev. Biol. 72, 236-243. Jaenicke, L., and Waffenschmidt, S. (1979). Matrix-lysis and release of daughter spheroids in Volvox carteri-A proteolytic process. FEBS Lett. 107, 250-253. Jaenicke, L., and Waffenschmidt, S. (1981). Liberation of reproductive units in Volvox and Chlamydomonas: Proteolytic processes. Ber. Dtsch. Bot. Ges. 94, 375-386. Jaenicke, L., Kuhne, W., Spessert, R., Wahle, U., and Waffenschmidt, S . (1987). Cell-wall lytic enzymes (autolysins) of Chlamydornonas reinhardtii are (hydroxy)proline-specific proteases. Eur. J. Biochem. 170,485-491. Joris, B., Englebert, S . , Chu, C. P., Kariyama, R., Daneo-Moore, L., Shockman, G. D., and Ghuysen, J. M. (1992). Modular design of the Enterococcus hirae muramidase-2 and Streptococcus faecalis autolysin. FEMS Microbiol. Lett. 70, 257-264. Joseleau, J.-P., Chambat, G., Cortelazzo, A , , Faik, A,,and Ruel, K. (1994). Putative biological action of oligosaccharides on enzymes involved in cell-wall development. Biochem. Soc. Trans. 22,403-407. Kieliszewski, M. J., and Lamport, D. T. A. (1994). Extensin: Repetitive motifs, functional sites, posttranslational codes, and phylogeny. Plant J. 5, 157-172. Kindle, K. L. (1990). High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc. Narl. Acad. Sci. USA 87, 1228-1232. Kindle, K. L., Schnell, R. A., Fernandez, E., and Lefebvre, P. A. (1989). Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase. J . Cell Biol. 109, 2589-2601. Kinoshita, T., Kukuzawa, H., Shimada, T., Saito, T., and Matsuda, Y. (1992). Primary structure and expression of a gamete lytic enzyme in Chlamydomonas reinhardtii; Similarity of functional domains to matrix metalloproteases. Proc. Natl. Acad. Sci. USA 89, 4693-4697. Kirk, D. L. (1988). The ontogeny and phylogeny of cellular differentiation in Volvox. Trends Genet. 4, 32-36. Kirk, D. L., and Harper, J. F. (1986). Genetic, biochemical and molecular approaches to Volvox development and evolution. Int. Rev. Cyrol. 99, 217-293. Kirk, D. L., and Kirk, M. M. (1986). Heat shock elicits production of sexual inducer in Volvox. Science 231, 51-54.

BIOCHEMISTRY OF THE Vo/vox ECM

a3

Kirk, D. L., Birchem, R., and King, N. (1986). The extracellular matrix of Volvox: A comparative study and proposed system of nomenclature. J. Cell Sci. 80, 207-231. Knox, P. (1995). Developmentally regulated proteoglycans and glycoproteins of the plant cell surface. FASEB J. 9, 1004-1012. Kroger, N.,Bergsdorf, C., and Sumper, M. (1994). A new calcium binding glycoprotein family constitutes a major diatom cell wall component. EMBO J . 13, 4676-4683. Kroger, N., Bergsdorf, C., and Sumper, M. (1996). Frustulins: Domain conservation in a protein family associated with diatom cell walls. Eur. J. Biochem. 239, 259-264. Lane, B. G. (1994). Oxalate, germine, and the extracellular matrix of higher plants. FASEB J. 8, 294-301. Larson, A,, Kirk, M. M., and Kirk, D. L. (1992). Molecular phylogeny of the volvocine flagellates. Mol. Biol. Evol. 9, 85-105. Levy, S., and Staehelin, L. A. (1992). Synthesis, assembly and function of plant cell wall macromolecules. Curr. Opin. Cell Biol. 4, 856-862. Linthorst, H. J. M. (1991). Pathogenesis-related proteins of plants. Crit. Rev. Plant Sci. 10, 123-150. Linthorst, H. J. M., Van Loon, L. C., Van Rossum, C. M. A., Mayer, A., Bol, J. F., Van Roekel, J. S. C., Meulenhoff, E. J. S., and Cornelissen, B. J. C. (1990). Analysis of acidic and basic chitinases from tobacco and petunia and their constitutive expression in transgenic tobacco. Mo1.-Plant Microb. Interact. 3,252-258. Mages, H.-W., Tschochner, H., and Sumper, M. (1988). The sexual inducer of Volvox carteri. Primary structure deduced from cDNA sequence. FEBS Lett. 234,407-410. Matsuda, Y., Yamasaki, A,, Saito, T., and Yamaguchi, T. (1984). Purification and characterization of cell wall lytic enzyme released by mating gametes of Chlamydomonas reinhardtii. FEBS Lett. 166,293-297. Matsuda, Y., Saito, T., Yamaguchi, T., and Kawase, H. (1985). Cell wall lytic enzyme released by mating gametes of Chlamydomonas reinhardrii is a metalloprotease and digests the sodium perchlorate-insoluble component of cell wall. J. Biol. Chem. 260, 6373-6377. Matsuda, Y., Saito, T., Yamaguchi, T., Koseki, M., and Hayashi, K. (1987). Topography of cell wall lytic enzyme in Chlamydomonas reinhardtii: Form and location of the stored enzyme in vegetative cell and gamete. J . Cell Biol. 104, 321-329. Matsuda, Y., Koseki, M., Shimada, T., and Saito, T. (1995). Purification and characterization of a vegetative lytic enzyme responsible for liberation of daughter cells during the proliferation of Chlamydomonas reinhardtii. Plant Cell Physiol. 36, 681-689. Mengele, R. (1988). “Versuche zur strukturellen Charakterisierung eines Glykoproteins aus der Extrazellularen Matrix von Volvox carteri.” Ph.D. thesis, Univ. Regensburg, Regensburg, Germany. Miller, D. H., Mellman, I. S., Lamport, D. T. A., and Miller, M. (1974). The chemical composition of the cell wall of Chtamydomonas gymnogama and the concept of a plant cell wall protein. J. Cell Biol. 63,420-429. Mitchell, B. A. (1980). “Evidence for Polyhydroxyproline in the Extracellular Matrix of Volvox.” M.S. thesis, Michigan State University, East Lansing, MI. Mort, A. J., and Lamport, D. T. A. (1977). Anhydrous hydrogen fluoride deglycosylates glycoproteins. Anal. Biochem. 82,289-309. Nelson, J. A,, and Lefebvre, P. A. (1995). Targeted disruption of the NIT8 gene in Chlamydomonas reinhardtii. Mol. Cell. Biol. 15, 5762-5769. Nozaki, H. (1996). Morphology and evolution of sexual reproduction in the Volvocaceae (Chlorophyta). J . Plant Res. 109,353-361. Paces, V., Vlcek, C., and Urbanek, P. (1986). Nucleotide sequence of the late region of Bacillus subtilis phage PZA, a close relative of phi 29. Gene 44, 107-114. Pommerville, J., and Kochert, G. (1982). Effects of senescence on somatic cell physiology in the green alga Volvox carteri. Exp. Cell Res. 140, 39-45.

84

MANFRED SUMPER AND ARMlN HALLMANN

Quinn, J. M., and Merchant, S. (1995). Two copper-responsive elements associated with the Chlamydomonas Cyc6 gene function as targets for transcriptional activators. Plant Cell 7 , 623-628. Raikhel, N. V., Lee, H. I., and Broekaert, W. F. (1993). Structure and function of chitinbinding proteins. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44,591-615. Rausch, H., Larsen, N., and Schmitt. R. (1989). Phylogenetic relationships of the green alga Volvox carteri deduced from small-subunit ribosomal RNA comparisons. J. Mol. Evol. 29,255-265. Roberts, K. (1974). Crystalline glycoprotein cell walls of algae: Their structure, composition and assembly. Philos. Trans. R. Soc. London, Ser. B 268, 129-146. Roberts, K. (1979). Hydroxyproline: Its asymmetric distribution in a cell wall glycoprotein. Planta 146,275-279. Roberts, K. (1994). The plant extracellular matrix: In a new expansive mood. Curr. Opin. Cell Biol. 6, 688-694. Roberts, K., Gay, M. R., and Hills, G. J. (1980). Cell wall glycoproteins from Chlamydomonas are sulphated. Physiol. Plant. 49, 421-424. Roberts, K., Hills, G. J., and Shaw, P. J. (1982). The structure of algal cell walls. In “Electron Microscopy of Proteins’’ (J. R. Harris, ed.), Vol. 3, pp. 1-40. Academic Press, New York. Roberts, K., Grief, C., Hills, G. J., and Shaw, P. J. (1985). Cell wall glycoproteins: Structure and function. J. Cell Sci. Suppl. 2, 105-127. Schiedlmeier, B., Schmitt, R., Muller, W., Kirk, M. M., Gruber, H., Mages, W., and Kirk, D. L. (1994). Nuclear transformation of Volvox carteri. Proc. Natl. Acad. Sci. USA 91,5080-5084. Schlipfenbacher, R., Wenzl, S., Lottspeich, F., and Sumper, M. (1986). An extremely hydroxyproline-rich glycoprotein is expressed in inverting Volvox embryos. FEBS Lett. 209,57-62. Schlosser, U. G. (1976). Entwicklungsstadien- und sippenspezifische Zellwandautolysine bei der Freisetzung von Fortpflanzungszellen in der Gattung Chlamydomonas. Ber. Dtsch. Bot. Ges. 89, 1-56. Schlosser, U. G. (1981). Algal wall-degrading enzymes-Autolysins. In “Encyclopedia of Plant Physiology New Series” (W. Tanner and F. A. Loewus, eds.), pp. 333-351. SpringerVerlag, Berlin. Schlosser, U. G. (1984). Species-specific sporangium autolysins (cell-wall dissolving enzymes) in the genus Chlamydomonas. In “The Systematics of Green Algae” (D. E. G. Irvine and D. Johns, eds.), pp. 409-418. Academic Press, London. Schmidt, B., Selmer, T., Ingendoh, A., and von Figura, K. (1995). A novel amino acid modification in sulfatases that is defective in multiple sulfatase deficiency. Cell 82, 271-278. Schmitt, R., Fabry, S., and Kirk, D. L. (1992). In search of molecular origins of cellular differentiation in Volvox and its relatives. Int. Rev. Cytol. 139, 189-265. Selmer, T., Hallmann, A., Schmidt, B., Sumper, M., andvon Figura, K. (1996).The evolutionary conservation of a novel protein modification, the conversion of cysteine to serinesemialdehyde in arylsulfatase from Volvox carteri. Eur. J. Biochem. 238, 341-345. Showalter, A. M. (1993). Structure and function of plant cell wall proteins. Plant Cell 5,9-23. Showalter, A. M., and Varner, J. E. (1989). Plant hydroxyproline-rich glycoproteins. In “The Biochemistry of Plants” (P. K. Stumpf and E. E. Conn, eds.), Vol. 15, pp. 485-520. Academic Press, New York. Sidhu, S. S., Kalmar, G. B., Willis, L. G., and Borgford, T. J. (1994). Streptomyces griseus protease C. A novel enzyme of the chymotrypsin superfamily. J. Biol. Chem. 269,2016720171. Snell, W. J. (1983). Characterization of the flagellar collar. Cell Motil. 3, 273-280. Sodeinde, 0.A., and Kindle, K. L. (1993). Homologous recombination in the nuclear genome of Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA 90, 9199-9203. Speir, T. W., and Ross, D. J. (1978). Soil phosphatase and sulfatase. In “Soil enzymes” (R. G. Burns, ed.), pp. 197-250. Academic Press, London.

BIOCHEMISTRY OF

THE Vo/vox ECM

85

Spessert, R., and Waffenschmidt, S. (1990). Studies on the vegetative autolysin during the vegetative life cycle in Chlamydvmvnas. Eur. J. Cell Biol. 51, 17-22. Stanford, A,, Bevan, M., and Northcote, D. (1989). Differential expression within a family of novel wound-induced genes in potato. Mvf. Gen. Genet. 215,200-208. Starr, R. C. (1969). Structure, reproduction, and differentiation in Volvvx carterif: nagariensis Iyengar, strains HK9 & 10. Arch. Prvtistenk. 111,204-222. Starr, R. C . (1970). Control of differentiation in Vvlvvx. Dev. Biof. Suppl. 4, 59-100. Stevens, D. R., Rochaix, J. D., and Purton, S . (1996). The bacterial phleomycin resistance gene ble as a dominant selectable marker in Chlurnydvrnvnas. Mol. Gen. Genet. 251,23-30. Sumper, M. (1979). Control of differentiation in Vvlvvx carteri: A model explaining pattern formation during embryogenesis. FEES Lett. 107, 241-246. Sumper, M., Berg, E., Wenzl, S., and Godl, K. (1993). How a sex pheromone might act at a M.EMBO J. 12, 831-836. concentration below Tschochner, H., Lottspeich, F., and Sumper, M. (1987). The sexual inducer of Vvlvvx carferi: Purification, chemical characterization and identification of its gene. EMBO J. 6,2203-2207. Varner, J. E., and Lin, L. (1989). Plant cell wall architecture. Cell 56, 231-239. Varner, J. E., and Ye, Z . (1994). Tissue printing. FASEB J . 8, 378-384. Waffenschmidt, S., Knittler, M., and Jaenicke, L. (1990). Characterization of a sperm lysin of Vvlvvx carteri. Sex. Plant Reprvd. 3, 1-6. Waffenschmidt, S., Woessner, J. P., Beer, K., and Goodenough, U. W. (1993). Isodityrosine crosslinking mediates insolubilization of cell walls in Chlarnydomvnas. Plant Cell 5,809-820. Wenzl, S., and Sumper, M. (1982). The occurrence of different sulphated cell surface glycoproteins correlates with defined developmental events in Vvlvvx. FEES Letr. 143,311-315. Wenzl, S., and Sumper, M. (1986). Early event of sexual induction in Vvlvox: Chemical modification of the extracellular matrix. Dev. B i d . 115, 119-128. Wenzl, S., Thym, D., and Sumper, M. (1984). Development-dependent modification of the extracellular matrix by a sulphated glycoprotein in Volvox carferi. EMBO J. 3,739-744. Woessner, J. P., Molendijk, A. J., van Egmond, P., Klis, F. M., Goodenough, U. W., and Haring, M. A. (1994). Domain conservation in several volvocacean cell wall proteins. Plant Mol. B i d . 26,947-960. Zinn, K., McAllister, L., and Goodman, C. S. (1988). Sequence analysis and neuronal expression of fasciclin I in grasshopper and Drvsophila. Cell 53, 577-587.

This Page Intentionally Left Blank

Cell Biology of the Basophil Ann M. Dvorak Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215

The cell biology of basophils, based on published studies spanning 1990-1997, is reviewed. These rarest cells of granulocyte lineages are now available in sufficient numbers for such studies to be done, based on new methods for isolating and purifying the cells from peripheral blood and organ sources and for their derivation in growth factorcontaining cultures from their precursors de now. These studies are dependent on electron microscopy for the accurate identification of basophils, studies which have recently established the presence of basophils in two new species-mice and monkeys. Secretory, endocytotic and storage properties of basophils constitute their mechanistic role@)in human disease; their role@)in health is, however, obscure. Development of immunoaffinity and enzyme-affinity ultrastructural labeling techniques to image the Charcot-Leyden crystal protein and histamine in human basophils, coupled with ultrastructuralanalysis of kinetic samples of cells obtained after stimulation with diverse secretogogues, has provided insight into the role of vesicles in secretory transport mechanisms in human basophils as well as the definition of key ultrastructural phenotypes of secreting basophils. KEY WORDS: Basophil, Electron microscopy, Histamine, Charcot-Leyden crystal protein, Vesicle transport, Allergy, Cytokines, Chemokines, Interleukin-3, Histaminereleasing factor, Monocyte chemotactic protein-1, Inflammation.

1. Introduction Basophils, granulocytes produced in the bone marrow, are cells that circulate as mature cells and have the capacity to invade tissues. They are the rarest of all circulating cell lineages, a fact that has hampered their study following their identification in humans in 1879 (Ehrlich, 1879). The historical, morphological, and immunological characterization of human basophils Inrernorional Review of Cylology. Vol. 180

0074-7696198 $25.00

07

Copyright Q 1998 by Academic Press. All rights of reproduction in any form reserved.

88

ANN

M. DVORAK

from 1879 to 1985 was recently reviewed (Dvorak, 1988a). Highlights roughly spanned three eras of development. Between 1879 and the late 1960s, early structural and histochemical studies utilized more easily obtained populations of malignant human basophils rather than rare and difficult-to-obtain normal human basophils. The participation of basophils in inflammatory exudates, definition of two forms of immunity [cellmediated immunity (CMI) and immediate hypersensitivity), recognition of the histamine content and releasability of basophils, and identification of a new immunoglobulin class (IgE) responsible for reaginic activity in serum-mediated hypersensitivity were all elucidated in the early era (Dvorak, 1988a). In the middle era (1970-1979), advances in the structural and cytochemical definitions of human basophils, documentation of basophils in a variety of cell-mediated and inflammatory events, description of a slow release reaction [termed piecemeal degranulation (PMD)] in these in vivo events, and extensive studies of the biochemistry of immediate hypersensitivity release reactions [termed anaphylactic degranulation (AND)] involving human basophils were accomplished (Dvorak, 1988a). The years 1980-1985 showed a rapid progress in technical developments allowing purification of large numbers of normal peripheral blood basophils and stimulation of human basophil growth from precursors by growth factors. These new sources of normal basophils facilitated ultrastructural analyses of AND of human basophils and of human basophil development (Dvorak, 1988a). Additional reviews have covered many aspects of the biology of basophils of humans and guinea pigs (Dvorak and Dvorak, 1972, 1973, 1974, 1975, 1979,1993;Dvorak et al., 1974a, 1979a, 1980a,f, 1981a, 1983a,b;1986;Galli et al., 1984; Dvorak, l978,1986,1987,1988b, l989,1991,1992a, l993,1994a,b, 1995a, 1997; Dvorak and Ishizaka, 1995; Monahan and Dvorak, 1985; Orenstein et al., 1981; MacGlashan et al., 1982a). The topics of some of these reviews are as follows: (i) basophils of man and guinea pigs in CMI (Dvorak, 1992a; Dvorak and Dvorak, 1972, 1973; Dvorak et al., 1974a, 1980f, 1986; Galli et al., 1984); (ii) a special form of CMI, termed cutaneous basophil hypersensitivity (CBH), based on the extensive tissue infiltration of basophils (Dvorak, 1992a; Dvorak and Dvorak, 1974, 1993; Dvorak et al., 1979b;Galli et aL., 1984); (iii) descriptions of PMD by human and guinea pig basophils, of uptake and granule storage of exogenous proteins, and presentation of a general degranulation model for basophil degranulation (Dvorak, l978,1988a, 1991,1992a, 1993; Dvorak et al., 1980a; Dvorak and Dvorak, 1975; Galli et al., 1984); (iv) ultrastructure of AND by guinea pig basophils (Dvorak, 1978, 1991; Dvorak and Dvorak, 1979; Dvorak et al., 1983b; Galli et al., 1984); (v) plasminogen activator, a surface protease on guinea pig basophils (Dvorak et al., 1979a); (vi) identification and ultrastructure of malignant basophils in granulocytic leukemias (Dvorak et al., 1981a;

CELL BIOLOGY OF THE BASOPHIL

89

Monahan and Dvorak, 1985); (vii) proteoglycans in guinea pig basophils (Orenstein et af.,1981);(viii) purification of human and guinea pig basophils (MacGlashan et af., 1982a;Dvorak, 1988a, 1991);(ix) ultrastructural criteria for identification of basophils of multiple species (Dvorak, 1986, 1988b, 1989, 1991, 1994a,b; Dvorak et al., 1983a); (x) ultrastructural comparison of human granulocytic myelocytes (Dvorak, l987,1988a, 1994a); (xi) development of human basophils in vitro (Dvorak and Ishizaka, 1995); (xii) ultrastructure of AND by human basophils (Dvorak, 1988a,b, 1991,1993, 1994a, 1997; Dvorak et af., 1983b). In this review, progress in the cell biology of basophils, published in 1985-1997, will be emphasized. Human basophils continued to be a source for newer studies, and basophils in two new species (mouse and monkey) were identified (Dvorak et af., 1982a, 1989a, 1993a, 1994a; Seder et al., 1991a). Cytokines as growth factors, as cell activators, and as cell products were characterized, and the relevance of chemokines as potent secretogogues of basophils was established (Dvorak and Ishizaka, 1995; MacDonald, 1993; Grant et al., 1991; Kaplan et af., 1991; Ishizaka et al., 1989a,b). New sources of sufficient numbers of basophils for cell-biological studies were realized for human basophils in the development from cytokinestimulated precursors in vitro (Dvorak and Ishizaka, 1995; Ishizaka et al., 1989a,b),application of sophisticated cell-sorting methods for collection of mouse basophils from several organ sources (Seder et al., 1991a; Dvorak el al., 1993a, 1994a), and in vivo administration of interleukin (1L)-3 to obtain large numbers of circulating monkey basophils (Dvorak et af., 1989a). In-depth kinetic analyses, using a coordinated biochemical-morphological approach, of two different secretogogues, formyl methionyl leucyl phenylalanine (FMLP) and tetradecanoyl phorbol acetate (TPA), was accomplished (Dvorak et af., 1991a, 1992a; Warner et al., 1989; Schleimer et af., 1981, 1982), providing a solid anatomic foundation for subsequent studies of subcellular distributions of the Charcot-Leyden crystal (CLC) protein (Dvorak et al., 1996a, 1997a,b) and histamine (Dvorak, 1997; Dvorak et al., 1994b, 1995a, 1996f) in activated human basophils. Each of these new areas of study required the development of immunogold (Dvorak et al., 1988; Dvorak and Ackerman, 1989) and enzyme-affinity-gold (Dvorak, 1995a, 1997; Dvorak et al., 1993b, 1995b) ultrastructural methods for visualizing CLC protein and histamine. In aggregate, these new tools have facilitated better understanding of basophil secretion and recovery from secretion (Dvorak, 1991,1993,1994q 1995a, 1996,1997; Dvorak and Ishizaka, 1995; Dvorak et af., 1991a, 1992a, 1993a, 1995a, 1996a,b,f,1997a,b). Specifically,documentation of a degranulation continuum, originally proposed in 1975 as a general degranulation model for basophils (Dvorak and Dvorak, 1975), and of the key role for vesicular transport in effecting this process was accomplished (Dvorak,

90

ANN

M. DVORAK

1995b; Dvorak et al., 1994b, 1996a,e,f, 1997a,b). Also, extension of the principle for uptake and storage of soluble exogenous proteins in basophil secretory granules to other secretory cells reinforced the general importance of this biological mechanism (Dvorak, 1991; Dvorak and Monahan-Earley, 1995a,b; Dvorak et al., 1985a,b). Finally, the morphometric analysis of activated, labeled human basophils revealed distinctive morphologic phenotypes (Dvorak et al., 1997a), giving rise to the realization that asynchronous events stimulated in this extremely rare blood granulocyte provide an anatomic basis for the daunting task of interfering with the regulation of these events to understand more fully basophil function in health and disease.

II. Basophil Identification Basophils were first identified by Paul Ehrlich, based on the affinity of their cytoplasmic granules for basic dyes (Ehrlich, 1879), a property shared by a similar cellular lineage, mast cells, also identified by Ehrlich (1877). The standard way for identifying basophils and mast cells for many years was based on this metachromatic dye-binding capacity of basophils and mast cells. Ehrlich and other early anatomists (Ehrlich, 1879; Jolly, 1900; Maximow, 1913; Michaelis, 1902; Zimmermann, 1908) clearly distinguished basophils from mast cells in man based upon morphologic criteria and dyebinding, using light microscopy. Despite recurrent confusion regarding whether these are two distinct cell types or maturational phases of a single cell lineage, the balance of anatomic data favors the original view of these early anatomists, i.e., that basophils and mast cells are indeed two separate cell lineages (Dvorak, 1988a). The basic ultrastructure of basophils and mast cells elucidates their individual, unique morphologies and changes superimposed on them by maturational and functional programs (Dvorak, 1986, 1988a,b, 1989, 1991, 1994a; Dvorak and Ishizaka, 1995; Dvorak er al., 1983a, 1985b; Kepley et al., 1994; Hastie, 1990; Eguchi, 1991). Use of metachromatic stains to identify basophils by light microscopy has certain pitfalls that are not widely recognized when predominantly immature cells are assessed (Dvorak, 1988a). In humans, large basophilic myelocytes packed with metachromatically staining granules could easily be confused either with mast cells of similar size or with eosinophilic myelocytes in which many immature granules also bind metachromatic dyes. By electron microscopy, myelocytes of the basophil and eosinophil lineages are readily identified, based on specific anatomic and cytochemical criteria (Dvorak, l986,1987,1988a, 1991,1994a; Dvorak and Ishizaka, 1995;Dvorak et al., 1982a, 1993a, 1994a).

CELL BIOLOGY OF THE BASOPHIL

91

A. Electron Microscopy Mature human basophils (Fig. 1) typically contain a granulocyte nucleus, that is, a polylobed nucleus with extensive chromatin condensation. Cytoplasmic contents include mitochondria, cytoskeletal elements, glycogen, vesicles, and granules. Golgi structures are inconspicuous, and small amounts of free or membrane-bound ribosomes are present. Granule content is predominantly particulate but may be subcompartmentalized by multiple dense membranous arrays. Cell surface architecture is characterized by multiple broad surface processes that are irregularly spaced. A numerically minor small granule subset contains homogeneously dense material and resides near nuclear lobes (Dvorak, 1988a; Hastie, 1974,1990; Nichols and Bainton, 1973). Charcot-Leyden crystals form within the particulate matrix of the numerically predominant large secretory granules of

FIG. 1 Human basophil. Note the polylobed nucleus, irregular, broad surface processes, and secretory granules filled with particles. One small granule is also present. It does not contain particles (arrow). The cell surface is stained with cationized ferritin (with permission, from Dvorak, 1988b). Bar: 1.2 Fm.

92

ANN M. DVORAK

human basophils (Dvorak, 1988a, 1994a; Dvorak and Ackerman, 1989; Dvorak and Monahan, 1982).

1. Basophils in New Species a. Mouse Basophils The ultrastructural morphology of mature mouse basophils and immature mouse basophilic myelocytes was described and differentiated from the ultrastructural morphology of mature and immature mouse mast cells, mature eosinophils and neutrophils, and immature mouse eosinophilic myelocytes and neutrophilic myelocytes (Dvorak, 1986, 1991, 1994b, 1995b; Dvorak and Ishizaka, 1995; Dvorak et af., 1982a, 1983a,c, 1993a, 1994a; Seder et al., 1991a; Galli et al., 1983a, 1984). Mature mouse basophils have polylobed granulocytic nuclei with heavily condensed chromatin and small numbers of homogeneously dense secretory granules. Other cytoplasmic organelles include vesicles, mitochondria, cytoskeletal structures, small Golgi structures, glycogen, and free and membrane-bound ribosomes; the cell surface has irregular, broad processes (Dvorak et af., 1982a). Mouse basophils were discovered in large numbers, using these ultrastructural criteria, in sorted samples of mouse spleen and bone marrow non-B, non-T cells which expressed high-affinity Fc, receptors and produced interleukin-4 (IL-4) in response to crosslinkage of Fc, receptors, Fc, receptors, or to exposure to ionomycin (Seder et aL, 1991a). Thus, an association of IL-4 production with mouse basophils was first established (Seder et af., 1991a). Mouse basophils were identified ultrastructurally in non-B, non-T cells sorted from normal mouse bone marrow or spleen and were substantially increased when mice were previously injected with goat anti-mouse IgD antibodies (Dvorak et af.,1993a;Conrad et al., 1990). Of the granulated cells present in Fc,R+ non-T, non-B cells sorted from spleen or bone marrow of goat anti-mouse IgD-injected animals, -90% were in the basophil lineage (Dvorak et aL, 1993a). Of these, 97% were mature basophils in the spleen samples. By contrast, 31% of the cells in the basophil lineage in bone marrow samples were basophilic myelocytes (Dvorak et al., 1993a). Shortterm cultures of mouse bone marrow cells containing IL-3, with or without stem cell factor (SCF), were examined for mouse basophils (Dvorak et aZ., 1994a). Basophils did not develop increased granules and underwent apoptosis in cultures containing both factors, whereas mast cells thrived and did develop increased granule numbers. Basophils were Fc,R+ and ckit- when sorted after culture in IL-3 and SCF. Thus, mouse basophils, identified by electron microscopy, expressed little or no c-kit receptor on their cell surface, nor did they survive for long periods in SCF-supplemented cultures (Dvorak et af., 1994a).

b. Monkey Basophils Ultrastructural and cytochemical studies of monkey peripheral blood samples were performed after recombinant human

93

CELL BIOLOGY OF THE BASOPHIL

(rh) IL-3 was infused (Dvorak et al., 1989a). rhIL-3 stimulated a delayed granulocytosis (Donahue et af., 1988) primarily characterized by numerous mature basophils and fewer neutrophils and eosinophils (Dvorak et al., 1989a). Mature basophils were identified by electron microscopy (Dvorak et al., 1989a). They were found to be typical granulocytes with polylobed nuclei containing condensed chromatin. Numerous secretory granules were filled with homogeneously dense contents. Other cytoplasmic organelles included mitochondria, vesicles, cytoskeletal elements, glycogen, small Golgi structures, and minor amounts of free and membrane-bound ribosomes. Irregularly spaced broad surface processes were evident. Cytochemical and routine ultrastructural criteria aIIowed monkey basophils to be distinguished from other granulocytes and mast cells in this species (Dvorak et al., 1989a; Ts’ao et al., 1976; Patterson et al., 1980). Confirmation that IL-3 is a basophilopoietin in monkeys has appeared from the work of numerous laboratories (Mayer et al., 1989; Wagemaker et al., 1990; VolcPlatzer et al., 1991; Wognum et al., 1995; van Gils et al., 1995). 2. Basophils in New Circumstances-Human in Vitro

Basophils

We recently reviewed our experience in the ultrastructural analysis of a variety of culture systems of human cord blood mononuclear cells, spanning a 10-year period (Dvorak and Ishizaka, 1995). Suspension cultures of human cord blood mononuclear cells reliably gave rise to selective growth of human basophils (Ishizaka et al., 1985a) when supplemented with a fraction of culture supernatant of phytohemagglutinin-stimulated human T cells which lacked IL-2 (Ishizaka et al., 1985a; Ogawa et aL, 1983). These cells contained amounts of histamine similar to those in normal human peripheral blood basophils and had functional Fc, receptors of high affinity (Ishizaka et al., 1985a). Sequentially prepared cultures demonstrated the development of large, immature basophilic myelocytes that matured into small, mature basophils (Dvorak et al., 1985b), a process generally completed in -3 weeks. Similar cultures supplemented with either rhI1-3 or IL-5 (but not IL-4) gave rise primarily to eosinophils and basophils (IL-3) or eosinophils (IL-5) but not to mast cells (Ishizaka et al., 1989a,b; Saito et al., 1988; Dvorak et aZ., 1989b). Small numbers of mature human basophils were present in similar suspension cultures supplemented with the c-kit ligand, i.e., SCF, in its recombinant or naturally occurring form (Dvorak et al., 1993~).The basophils contained immunoreactive gold label for the CLC protein, whereas the more numerous mast cells developing therein did not (Dvorak et al., 1994~).Additional reports emphasize the utilization of electron-microscopic analysis to identify developing human basophils in a variety of culture systems (Eguchi, 1991; Eguchi et al., 1985,1989; Tanno et al., 1987;Fishman

94

ANN M. DVORAK

et al., 1985; Suda et al., 1985; Rimmer and Horton, 1986; Hermine et al., 1992; Boyce et al., 1995; Sorensen et al., 1988; Seldin et al., 1986); some of these reports concern primarily development of eosinophils (Saito et al., 1988; Eguchi et al., 1985; Rimmer and Horton, 1986; Hermine et al., 1992; Boyce et al., 1995; Sorensen et al., 1988; Seldin et al., 1986). Earlier reports

(i,e., prior to 1985) of utilization of electron microscopy to identify human basophils developing in vitro are summarized in Dvorak (1988a).

6. Cell Markers Considerable progress in characterizing cell markers that are displayed by human basophils has revealed commonalities with other cell lineages as well as several unique markers. These studies, recently reviewed (Valent and Bettelheim, 1992; Valent et al., 1990a), provide additional tools with which basophils can be identified in mixed cellular populations. Basophils and mast cells bind IgE on their surfaces via high-affinity Fc, receptors (Ishizaka et al., 1970; Ishizaka and Ishizaka, 1975; Sullivan et al., 1971; Becker et al., 1973), whereas basophils lack significant low-affinity Fc,RII (CD23) molecules (Stain et al., 1987). Basophils also have low-affinity binding sites for IgG (Valent et al., 1990a; Stain et al., 1987; Ishizaka et al., 1979; Nakagawa and de Weck, 1983), receptors for complement (Stain et al., 1987; Valent et al., 1990a; Olsen et al., 1988; Glovsky et al., 1979; de Boer and Roos, 1986) for the cytokines JL-3 (Lopez et al., 1990; Valent et al., 1989), IL-4 (Valent et al., 1990b), IL-2 (Stain et al., 1987; Stockinger et al., 1990), GM-CSF (Lopez et al., 1990), NGF (Valent et al., 1990a), and for histamine (Lichtenstein and Gillesie, 1973). Cell surfaces of basophils express HLA class I antigen (de Boer and Roos, 1986; Reshef and MacGlashan, 1987; Bochner et al., 1989), intercellular adhesion molecule-1 (Bochner et al., 1989), leukocyte common antigen (CD45) (Stain et al., 1987; de Boer and Roos, 1986), and the Bsp-1 antigen, a specific marker for human basophils (Valent et al., 1990a;Bodger and Newton, 1987;Bodger etal., 1987). A unique marker for human basophils, consistent with a granule location, has been reported (Kepley et al., 1995). The expression of surface integrins on human basophils includes B1 and B2 integrins and differs from the integrin pattern expressed by human mast cells (Sperr et al., 1992). Quantitative comparison of myeloid antigens on individual mature peripheral blood lineages reveals that these lineages can be distinguished based on this approach to cell identification with surface markers (Terstappen et al., 1990).A panel of 60 monoclonal antibodies (used to distinguish circulating basophils from eosinophils and neutrophils) has produced an antigenic membrane profile useful for basophil identification (Stain et al., 1987).

CELL BIOLOGY OF THE BASOPHIL

95

Similarly, this approach has revealed antigenic membrane profiles useful for distinguishing basophils from mast cells (Valent and Bettelheim, 1992).

C. Cell Contents

In man, histamine is present in the granules of two cells-mast cells and basophils (Dvorak 1991; Dvorak et al., 1993b, 1994b; Riley and West, 1953; Lagunoff et al., 1961; Pruzansky and Patterson, 1967; Ehrich, 1953). Basophils are smaller cells, containing an average of 1.3 pg of histamine per cell (Schroeder and Hanrahan, 1990), whereas larger mast cells are a rich source of histamine, containing 3.74 pgkell (Dvorak et al., 1985~).Proteoglycans are glycoconjugates composed of covalently linked glycosaminoglycans and proteins. These materials are responsible for the metachromasia displayed by basophils. Human (Galli et al., 1979; Ishizaka et al., 1985b; Metcalfe et al., 1984), guinea pig (Orenstein et al., 1978), rabbit (Sue and Jaques, 1974), and rat (Metcalfe et al., 1980) basophils contain chondroitin sulfates as their proteoglycan. Ultrastructural autoradiographic localization of radiolabeled sulfur to basophil granules (Orenstein et al., 1978;Galli etal., 1984) indicates that sulfur-containing granule proteoglycans are responsible for the metachromasia displayed by granules in appropriately stained smears of basophils. Mast cells, in contrast, generally contain heparin as the major proteoglycan (Metcalfe et al., 1979). Tryptase and chymase have been used to differentiate human mast cells in various anatomic sites (Irani et al., 1989) and to rule out basophil participation in mixed populations of human cells (Furitsu et al., 1989; Mitsui et al., 1993). Basophils lack chymase but contain extremely small amounts of tryptase (0.05 pgkell) (Castells et al., 1987), whereas mast cells contain as much as 35 pgkell of tryptase (Schwartz et al., 1987). Several eosinophil granule proteins are also found in basophilic leukocytes. Among these are eosinophil-derived neurotoxin (Abu-Ghazaleh et al., 1992), major basic protein (MBP) (Abu-Ghazaleh et al., 1992;Ackerman et al., 1983), and the CLC protein (Dvorak, 1996; Dvorak and Ackerman, 1989; Dvorak et a[.,1994c, 1996a, 1997a,b;Abu-Ghazaleh et a[., 1992;Ackerman et al., 1982). In contrast, MBP (Leiferman et al., 1986) and CLC protein (Dvorak, A.M. et al., 1994c; Leiferman et al., 1986) are absent from mast cells. It has recently been recognized that basophils (and mast cells and eosinophils) produce cytokines (Seder et al., 1991a; Burd et al., 1989; Galli et al., 1991). Initial studies in mice revealed that splenic non-B, non-T cells were associated with IL-4 production (Conrad et al., 1990; Ben-Sasson et al., 1990; Le Gros et al., 1990; Seder et al., 1991b; Paul et al., 1993) and that these Fc,R-positive cells were basophils (Seder et al., 1991a; Dvorak et aL, 1993a, 1994a). Ample evidence now exists showing that human basophils

96

ANN M. DVORAK

also produce IL-4 (Brunner et al., 1993; Arock et al., 1993; Schroeder et al., 1994,1996;MacGlashan etal., 1994a;Mueller et al., 1994; Ochensberger et aZ., 1995). In one of these reports, immunoreactive IL-4 was localized to Fc,RI-positive, activated peripheral blood basophils derived from normal donors, providing direct evidence that IL-4 is produced by activated human basophils (Mueller et al., 1994). More recently, immunoreactive IL-13, a cytokine closely resembling IL-4, has been localized in human basophils by immunocytochemistry (Li et al., 1996). D. Secretogogues and Secreted Cellular Products Biochemical studies of human basophil secretogogues and identification of secreted products are extensive. The unique patterns of secretogogue releasability and secretory products induced from human basophils allow identification of responding cells by analysis of fluids that bathe inflammatory and immunologicprocesses. For example, in late antigen-induced rhinitis, the mixture of inflammatory mediators present does not include prostaglandin D2 which is a mast cell product, thus implicating basophils in this immunologically mediated inflammatory process (Naclerio et al., 1985). Failure to detect LTB4,a leukotriene, in inflammatory fluids also implicates basophils and not mast cells, since basophils do not generate LTB4 (Warner et al., 1987). The ability to analyze such complex in uiuo events is based on a large background of biochemical studies of release reactions and activation of purified populations of human basophils and mast cells (Warner et d., 1987; MacGlashan and Guo, 1991; MacGlashan and Warner, 1991; MacGlashan and Lichtenstein, 1980; MacGlashan et al., 1982b, 1983; Peters et al., 1984; Lichtenstein et af., 1983). In these studies, it is clear that histamine is released from both basophils and mast cells, albeit in different amounts and sometimes with different kinetics. Histamine-releasing secretogogues can differ between these two cellular lineages, and in man, secretogogues for the release of histamine from mast cells derived from different organ sources (e.g., lung, skin, synovium, heart, uterus, gut) can have a different activation profile. Lipid products of basophils and mast cells (and subsets of mast cells) may also differ (MacGlashan and Warner, 1991; MacGlashan etaZ., 1982b;Peters etal., 1984;Lichtenstein et al., 1983;Warner et al., 1987; Naclerio et al., 1985). This group of inflammatory mediators is not performed; rather, it is generated from cellular lipids after stimulation. Analysis of lipid products of either cyclooxygenase or lipoxygenase metabolism of arachidonic acid produced by purified cellular populations is helpful in understanding contributions of individual cells to inflammatory exudates in uivo. Some of the secretogogues useful for examining these events in human basophils include those operating via an IgE-mediated mechanism

CELL BIOLOGY OF THE BASOPHIL

97

(Warner ef al., 1989; Lichtenstein, 1971, 1975; Findlay et al., 1980; KageySobotka et af., 1981), the calcium ionophore A23187 (Findlay et al., 1980; Lichtenstein, 1975; Warner et al., 1989), complement (Warner et aL, 1989; Siraganian and Hook, 1976; Hook and Siraganian, 1977; Hook et al., 1975; Grant et al., 1975, 1976, 1977; Farnam et al., 1985; Findlay et af., 1980), tumor-promoting phorbol esters (Schleimer et al., 1981, 1982; Warner et al., 1989), concanavalin A (Siraganian and Siraganian, 1974, 1975), or the bacterial peptide FMLP (Hook et al., 1976; Siraganian and Hook, 1977; Warner et al., 1989). Profiles of released mediators and kinetics of mediator release differ when purified human basophils are stimulated with different secretogogues. For example, anti-IgE elicited release of both histamine and LTC4 from human basophils within 15-30 min; FMLP released histamine and LTC4 in 2-5 min; C5a rapidly released histamine only; A23187 caused extensive release of histamine and LTC,, but TPA released only histamine and did so with slow kinetics-45-60 min (Warner et al., 1989). Thus, complex characteristics of these regulated secretov events in human basophils can be expected to impact differently on the pathogenesis of disease, where basophils prevail.

111. Basophils in Disease In 1970, we demonstrated in guinea pigs extensive infiltration of mature basophils from blood into tissues during cellular immunity of delayed onset mediated by lymphocytes (Dvorak et af., 1970). We named such reactions cutaneous basophil hypersensitivity (CBH) to distinguish them from classical delayed hypersensitivity (DH). Subsequently, invasion of tissues by basophils in a variety of circumstances and in numerous species was identified (Dvorak, 1988a; Dvorak and Dvorak, 1972,1973,1974,1975; Dvorak et af., 1973, 1974a,b, 1976a,b; 1979b,c, 1980f; Galli and Dvorak, 1995; Galli et al., 1984). Two recent reviews summarize the current status of knowledge regarding the tissue migration of circulating basophils in disease (Dvorak and Dvorak, 1993; Dvorak, 1992a). Thus, extensive circumstantial evidence exists implicating basophils in delayed-onset, cell-mediated disease and reactions such as chronic inflammation and graft rejection. Basophils have the capacity to produce fatal disease when systemic anaphylaxis is produced secondary to basophil degranulation (Bochner and Lichtenstein, 1991). These massive, often fatal events are examples of antibody-mediated immediate hypersensitivity (IH). Other IH disorders with a basophil-mediated component include hay fever (Creticos et aL, 1985), rhinitis (Naclerio et al., 1985; Bascom et al., 1988; Hastie et al., 1979;

98

ANN M. DVORAK

Iliopoulos et al., 1992), eczema (deShazo et al., 1979; Mitchell et al., 1982), and asthma (Lichtenstein and Bochner, 1991). A role for basophils in inflammation has been suggested (Lichtenstein et al., 1978). In order for basophils to participate in inflammatory processes, they need to migrate from the blood into tissues, as they must do in D H or CBH reactions. Cell migration is facilitated by complex adhesions between circulating cells and endothelia. Recently, endothelial and basophil adhesion molecules have been the subject of numerous studies (Bochner and Schleimer, 1994). That basophils participate in inflammatory responses to bacterial and viral pathogens in infectious diseases is suggested by the large number of studies showing that these pathogens can stimulate histamine secretion from basophils. Among the pathogens associated with secretion from basophils are staphylococci (Martin and White, 1969; Marone et al., 1982; Espersen et al., 1984), Escherichia coli (Norn et al., 1986),Salmonella (Norn et al., 1986), Candida albicans (Pedersen et al., 1987), Herpes simplex (Pedersen et al., 1987), influenza A (Clementsen et al., 1988a,b; Busse et al., 1983), and Vibrio cholera (Clementsen et al., 1988a).

IV. Basophils as Secretory Cells A. Analogies of Basophil Secretary Granules to Secretory Granules of Endocrine, Exocrine, and Mast Cells Basophils, like endocrine, exocrine, and mast cells, store their specific secretory products in membrane-bound containers in the cytoplasm. These storage organelles participate in secretion by exocytosis-i.e., fusion of granule and plasma membranes with extrusion of their contents to the extracellular space (Dvorak, 1988a,b, 1989, 1991, 1992b; Dvorak and Monahan-Earley, 1992, 1995c,d). Secretory granules, by routine ultrastructural examination, are filled with electron-dense contents and vary in size, shape, number, and substructural intragranular patterns. Some of these variations are typical for individual cells or for the same cells in different species. Yet all mature granules termed secretory granules (in the absence of PMD) are filled with secretory materials. Secretory granules are often referred to in the literature as “vesicles,” but we choose not to use this term for bona fide granules (see Vesicle Transport and PMD sections) in order to avoid confusion with processes that make use of vesicles to transport materials in and out of cells. The substructural patterns of electron-dense secretory granules are known for mature basophils in a number of species (Dvorak, 1992a). Since mast cells are the cells most often confused with basophils, we list their substructural patterns for the same species in Table I. Thus, mature basophil

99

CELL BIOLOGY OF THE BASOPHIL TABLE I SubstructuralArchitecture of Human, Guinea Pig, Mouse, Rat, Monkey, and Rabbit Basophil and Mast Cell Granules Species Human (Dvorak, 1988a,b, 1989, 1991; Dvorak and Ackerman, 1989; Dvorak and Kissell, 1991; Dvorak and Monahan, 1982; Dvorak ef al., 1976a, 1980b, 1984a, 1989b, 1993c, 1994c; Eguchi, 1991; Eguchi et al., 1985, 1989; Fedorko and Hirsch, 1965; Fox et a1.,1981, 1984; Hastie, 1974,1990; Ishizaka etal., 1985a; Kobayasi eral., 1968; Orr, 1977; Parmley er al., 1976; Ts'ao et al., 1977; Wetzel, 1970; ZuckerFranklin, 1967)

Mature basophil granule patterns 1. Particles 2. Multiple concentric membrane arrays 3. CLCS 4. Homogeneously dense

Mature mast cell granule patterns

Scrolls Crystals Particles Concentric and tangled thick threads 5. Homogeneously dense

1. 2. 3. 4.

1. Crystals Guinea pig (Chan and Yoffey, 1960; Dvorak 1978,1986,1991; Dvoraketnl., 1981b;Fedorko 2. Finely granular and Hirsch, 1965; Hebert and Lindberg, 1982; 3. Homogeneously dense Murata and Spicer, 1974; Pearce et al., 1977; Stock et a!., 1989; Taichman, 1970,1971; Terry et al., 1969; Watanabe, 1954; Wetzel, 1970; Winqvist, 1960, 1963)

1. Crystals 2. Particles 3. Homogeneously dense 4. Irregular thick threads 5. Regular arrays of 12-nm tubules

1. Homogeneously dense

1. Homogeneously dense

Rat (Bentfeld et al., 1977; Bentfeld-Barker and 1. Homogeneously Bainton, 1980; Combs, 1966; Hoenig and dense Levine, 1974; Login et al., 1987; Wetzel, 1970)

1. Homogeneously dense

Monkey (Barrett and Metcalfe, 1985; Dvorak et al., 1989a; Patterson et al., 1980; Ts'ao et al., 1976; Wetzel, 1970)

1. Homogeneously dense

1. Particles 2. Concentric and irregular threads 3. Homogeneously dense

1. Particles

1. Homogeneously dense

Mouse (Combs, 1971; Dvorak, 1991; Dvorak et al., 1982a; Galli er al., 1987; Hammel et aL, 1987; Wetzel, 1970)

Rabbit (Benveniste ef a1.,1972; Dvorak, 1992a; Hardin and Spicer, 1971; Horn and Spicer, 1964; Komiyama and Spicer, 1974 Wetzel et al., 1967)

2. Concentric and

tangled thick threads 3. Finely granular 4. Homogeneously dense

Note. Modified (with permission) from Dvorak (1992a).

100

ANN M. DVORAK

granules in humans contain particles, multiple concentric membranous arrays, or are homogeneously dense (Dvorak, 1988a, 1991; Dvorak and Ackerman, 1989; Dvorak and Monahan, 1982; Dvorak et al., 1976a, 1980b, 1985b, 1989b, 1991a, 1992a, 1993c, 1994b,c, 1995a, 1996a,b,e, 1997a,b; Ishizaka et al., 1985a; Hastie, 1974,1990; Eguchi, 1991; Fox et al., 1984; ZuckerFranklin, 1967; Parmley et al., 1976; Wetzel, 1970). In guinea pigs, mature basophil granules display crystals and are finely granular or homogeneously dense (Wetzel, 1970; Fedorko and Hirsch, 1965; Dvorak, 1991; Dvorak and Monahan, 1985; Dvorak et al., 1970, 1981b, 1987; Winqvist, 1960, 1963; Terry et al., 1969; Murata and Spicer, 1974; Chan and Yoffey, 1960; Watanabe, 1954; Hebert and Lindberg, 1982). The electron-dense material is homogeneous without substructural patterns in mature basophils of mice (Wetzel, 1970; Dvorak, 1991; Dvorak et al., 1982a, 1983c, 1993a, 1994a; Seder et al., 1991a; Galli et al., 1983a), rats (Wetzel, 1970; Hoenig and Levine, 1974; Palade, 1955; Bentfeld-Barker and Bainton, 1980), and monkeys (Wetzel, 1970; Dvorak ef al., 1989a). In rabbits, mature basophil granules contain particles, concentric and tangled thick threads, and are finely granular or homogeneously dense (Wetzel et al., 1967; Benveniste et al., 1972; Hardin and Spicer, 1971; Komiyama and Spicer, 1974; Horn and Spicer, 1964; Dvorak, 1992a). 8 . Degranulation of Basophils

1. Anaphylactic Degranulation Anaphylactic d e g r a d a t i o n is the general term used to describe the rapid regulated secretory events of which basophils are capable (Fig. 2B). It corresponds to granule extrusion by exocytosis, a regulated secretory process common to secretory cells in general. AND in basophils is the coordinated secretion of granule mediators, accompanied by the visible extrusion, or solubilization within specially constructed intracytoplasmic degranula-

FIG.2 Purified human peripheral blood basophils, stimulated with FMLP for 20 s, show PMD (A) and AND (B). In A, a polymorphonuclear (N) basophil devoid of full granules shows extraordinary enlargement of discrete, empty granule containers (G) in the cytoplasm. In B, a polymorphonuclear (N) basophil shows extrusion of membrane-free granules (solid arrowheads) which are located in cul-de-sacs closely associated with the cell surface at multiple points around the cell. Markedly elongated, complex surface processes are noted. Focal collections of released, dense concentric membranes (open arrowheads) are associated with the complex cell surfaces of this basophil. The cytoplasm contains numerous aggregates of dense glycogen and elongated tubules of smooth endoplasmic reticulum (arrow) (with permission, from Dvorak et al., 1991a). Bars: (A) 1.4 pm; (B) 0.8 pm.

102

ANN M. DVORAK

tion chambers, of typical secretory granules, stimulated by IgE-mediated mechanisms (Dvorak, 1988a, 1991). Thus, this is an explosive and rapid secretory event in human basophils which is completed within minutes of stimulation. In contrast, slow emptying of cytoplasmic granules characterizes PMD of human basophils. AND, then, is a special type of regulated secretion, of which all granule-containing secretory cells, including basophils and mast cells, are capable. Important anatomical findings associated with AND in appropriately stimulated human basophils include extrusion of CLCs and dense concentric membranes in concert with granule particulate contents through multiple pores in the cell membrane; shedding of multiple membranes and portions of cell processes; surface amplification by externalization of granule containers; formation of intracytoplasmic degranulation chambers or sacs by fusion of multiple granule membranes; decreased granule numbers; decreased numbers of cytoplamic vesicles; and completely degranulated, viable basophils. AND has not been identified in human basophils in vivo for several reasons. These include the rarity of these cells, the sampling problem inherent in electron-microscopic evaluation of tissues into which basophils have migrated, and the rapidity of the reaction. The clinical condition caused by the massive release of histamine, known as anaphylaxis, would surely be accompanied by evidence of AND in human basophils and mast cells, if samples were available. However, ultrastructural studies have not been carried out in these life-threatening circumstances. A large number of studies have been performed, however, using isolated blood basophils (MacGlashan and Lichtenstein, 1980). Various secretogogues have been used to stimulate AND in isolated, partially purified human blood basophils from allergic and nonallergic donors. Our early ultrastructural studies are summarized in Dvorak (1988a). The secretogogues studied include antigen (Dvorak et al., 1980b), anti-IgE (Ishizaka et al., 1985a), complement (Dvorak et al., 1981c), histaminereleasing activity (HRA) from cultured mononuclear cell supernatants (Dvorak et al., 1984b), and mannitol (Findlay et al., 1981). Although many anatomical findings are similar in these studies, important differences have been established. Initially, we examined AND induced by antigen E in basophils obtained from allergic donors (Dvorak et al., 1980b). These studies showed that the main untrastructural event was the extrusion of membrane-free granules through multiple pores in the plasma membrane around the circumference of the cell. Rarely, several granules fused before extrusion. Maximum granule and histamine release occurred together over 15 min after antigen stimulation. Granule containers were also exteriorized, leading to an extensively amplified cell surface in cells with maximal release of granules. Mature basophils developing in growth factor-containing cultures of human cord

CELL BIOLOGY OF THE BASOPHIL

103

blood cells underwent similar AND when passively sensitized cells were stimulated with anti-IgE (Ishizaka et al., 1985a). Release of histamine by hyperosmolar materials was studied when peripheral blood basophils were exposed to mannitol (Findlay et al., 1981). In addition to AND, as described above, some of these basophils developed large, central, intracytoplasmic d e g r a d a t i o n sacs containing membranefree granules analogous to those that regularly develop during AND induced in guinea pig basophils (Dvorak et al., 1981b). The mononuclear cell product, HRA, also stimulates histamine release from human basophils (Thueson et al., 1979a,b). Several time points after stimulation with HRA were examined by electron microscopy (Dvorak et al., 1984b). Typical AND characterized the anatomy associated with histamine release. Some cells failed to undergo AND but formed prominent motile structures, or uropods, instead. Stimulation of human basophils with the complement factor C5a resulted in rapid histamine release, accompanied by the morphology of AND (Dvorak et al., 1981~).Additionally, we noted minute openings between the plasma membrane and underlying unaltered granules which readily admitted the electron-dense tracer, cationized ferritin. These were accompanied by plasma membrane invaginations. These events preceded the widening of the pores and extrusion of granules to cul-de-sacs among elongated surface processes. Extensive generation of intragranular concentric dense membranes did not occur in these C5a peptide-stimulated cells. 2. Piecemeal Degradation

Piecemeal d e g r a d a t i o n is a term introduced to explain the ultrastructural finding of partially and completely empty cytoplasmic granule containers, in the absence of intergranule fusions or granule fusions to the plasma membrane and subsequent extrusion of granule contents to the microenvironment (Fig. 2A). It occurs in human basophils participating in large numbers in experimentally induced and sequentially biopsied contact allergy lesions in human skin (Dvorak and Dvorak, 1975; Dvorak et al., 1974b, 1976a; Galli et al., 1984). These mature basophils are also characterized by large numbers of cytoplasmic vesicles, some of which are attached to granules. Visible particles like those in granules, homogeneously dense contents, or apparently empty (electron-lucent) interiors prevail among these smooth membrane-bound, small cytoplasmic vesicles. Therefore, PMD, simply stated, defines the release of granule materials, in the absence of typical granule extrusion, from basophils. It is a process mediated by vesicular transport that develops slowly over days in skin biopsies of evolving contact allergies (Dvorak et al., 1976a). This form of stimulated secretory activity is to be distinguished from AND.

104

ANN

M. DVORAK

PMD of basophils has been identified in a number of experimentally induced or naturally occurring circumstances (reviewed in Dvorak, 1992a). Initially, we performed a kinetic analysis of experimentally produced lesions of contact allergy (Dvorak et aL, 1974b, 1976a) and sequential biopsies taken during skin graft rejection in humans (Dvorak et al., 1979c, 1980g). Basophils migrated into involved tissues from blood vessels and over time developed piecemeal losses of their particulate granule content. Quantification of skin contact allergy lesions over 6 days revealed significant increases in basophils undergoing PMD. Subsequent to these experimental studies of human skin contact allergy, we examined participating basophils in the peripheral blood and tissues from patients with Crohn’s disease, an inflammatory bowel disease of uncertain etiology (Dvorak et al., 1980~).Again, we were able to identify PMD as the predominant morphological expression of these reactive basophils. More recently, we have identified PMD in human basophils in uninvolved tissues of the small intestine in patients with ulcerative colitis, another inflammatory bowel disease (Dvorak et al., 1992b). Basophils migrate into tissues and body fluid from the blood in several human diseases (Dvorak, 1992a). In many of these circumstances, we have identified PMD of these participating cells (Dvorak, 1988a, 1991; Dvorak et al., 1982b; Fox et al., 1984), and PMD is evident in published electron micrographs (Collin and Allansmith, 1977; Glasser et al., 1976).

3. A Degranulation Model Proposed in 1975 In 1975, we proposed a general model of basophil d e g r a d a tio n (Fig. 3), based on our studies of guinea pig and human basophils, to account for the varied rates of granule substance release occurring in a variety of physiological and pathological circumstances (Dvorak and Dvorak, 1975). The model holds that PMD occurs by means of exocytotic vesicles which bud from the granule membrane, carrying with them small quanta of intact or dissolved granule material which flow to the cell surface, where they fuse with the plasma membrane and discharge their contents into the extracellular space. Glycogen aggregates that are closely associated with granules at sites of vesicle attachment may afford an energy source for vesicle budding. Once separation of vesicle from the granule membrane is complete, transport of vesicles may proceed at random. A net flux of granule contents out of the cell will result from vesicle movement if, for reasons of chemical structure, vesicles are able to fuse only with each other and with the membranes of other granules and the plasma membrane but not with the membranes limiting other cellular organelles. It is useful to postulate a closely coupled transport of endocytotic vesicles migrating from the cell surface to the cytoplasmic granules associated with vesicular exocytosis. Coupled exocytosis is necessary to account for the

BASOPHIL DEGRANULATION FIG. 3 Schematic diagram of a degranulation model in basophils. (A) Anaphylactic degranulation of guinea pig basophils. (1) Increased cytoplasmic vesicles 1 min after triggering with concanavalin A. Vesicles have fused with some granules; one has formed a bridge joining adjacent granules. (2) At 5-20 min, membranes of granules and cytoplasmic vesicles have fused with each other and with the plasma membrane, forming a “degranulation sac” containing membrane-free granules that open to the cell exterior by a narrow pore. Only occasional granules have escaped outside the cell, and many of these remain adherent to the cell surface. (3) Filaments have become prominent in the cytoplasmic processes embracing the degranulation sac. Over a period of hours, these processes retract, as indicated by arrows, presumably powered by the filaments, opening the degranulation sac to the exterior and permitting granule escape. (B) Anaphylactic degranulation of human basophils. (1, 2) Fusion of membranes of individual or joined granules directly with plasma membrane, with resulting discharge of granule contents. (3) Empty granule (EG) membranes eventually become part of plasma membrane as in 4. Note that portions of granule matrix remain adherent to plasma membrane. (C) Piecemeal degranulation of human (or guinea pig) basophils as in certain cell-mediated immune reactions. (1) Endocytotic vesicles travel from plasma membrane through the cytoplasm to basophilic granules, with which they fuse. (2) Exocytotic vesicles, containing bits of granule matrix, bud from granule membrane, pass through the cytoplasm, and fuse with the plasma membrane, discharging their contents to the extracellular space. (3) Same as 2, but budding proceeds at a faster rate. (4) Still more rapid exocytosis, in which vesicles bud from granule at so fast a rate that they do not separate from each other and form a channel joining the granule with the extracellular space. This is equivalent to anaphylaxis. N, nucleus (with permission, from Orenstein et al., 1981).

106

ANN M. DVORAK

undiminished (often slightly increased) size of granules which are releasing their contents and, hence, continuously losing portions of their enveloping membranes. An imbalance in the amount of vesicular traffic would result in changes in granule size. For example, excess efflux would produce smaller granules, and excess influx would produce larger granules. The extracellular fluid transported to the granules by endocytotic vesicles may provide solvent for the parital dissolution of granules that is regularly observed, for example, in the basophils infiltrating the reactions of allergic contact dermatitis (Dvorak et al., 1976a). Conceivably, the extra membranes accumulating within these basophil granules may be derived from those of the endocytotic vesicles, also indicating an imbalance between vesicular influx and efflux. In our vesicular transport model, the release of granule contents is postulated to proceed at a rate governed by the frequency of discharge of microvesicles from the granule membrane. The frequency of exocytosis is possibly determined by the rate of endocytosis which, in turn, may be controlled and modified by agents (antibodies, and probably other materials, such as cytokines, lymphokines, interleukins, bacterial peptides, etc.) acting at the cell surface. Based on the occasional granule alterations observed in “normal” circulating basophils, it is likely that a slow release of granule substance occurs under physiological conditions. In delayed-type cell-mediated immunologic reactions, degranulation apparently proceeds at a substantially greater pace. At still faster rates of degranulation, a threshold would eventually be reached, above which there would be insufficient time between successive discharges to permit complete separation of individual vesicles from the plasma or granule membranes or from each other. Under these conditions, endocytotic and/or exocytotic vesicles budding from the plasma or granule membranes would not form discrete, spherical structures; rather, they would coalesce to form continuous channels. Better developed channels might link the cell surface with granules and/or interconnect neighboring granules, depending on random collisions with either the plasma or the granule membranes. Continuous channels of this sort would lead to fusion of granule membranes with each other and with the plasma membrane, characteristic features of AND. It is clear that basophils have a role in mediating immediate hypersensitivity reactions or allergic diseases. It also seems clear that PMD of basophils may provide materials important to the onset, sustenance, or healing of a wide variety of disorders collectively involved with cell-mediated immunity. It is not clear, however, what role(s) these cells have in normal physiology, ie., in health. Secretion biologists categorize release mechanics from the various secreting cells in the body (the normal functions for most of which are known) as regulated or constitutive (Lacy, 1975; Orci et al., 1973; Palade, 1975;

CELL BIOLOGY OF THE BASOPHIL

107

Jamieson and Palade, 1971; Kelly, 1985). Regulated secretion, put simply, means an episodic, triggered release of stored cellular synthetic products; constitutive secretion means a continuous flow of synthetic cellular products. Regulated secretion utilizes classic exocytosis of membrane-free granules; constitutive secretion occurs by vesicular transport. AND of basophils, then, is an example of regulated secretion; PMD of basophils is similar to, and perhaps an example of, “up-regulated” constitutive secretion.

C. Basophil Secretory Granules as Storage Organelles for Exogenous Proteins Classically, secretory granules were not considered to be storage repositories €orexogenous proteins. Rather, their contents were generally considered to derive from the synthetic capacities of individual cell lineages, such contents being held in reserve until appropriate secretogogues initiated their secretion by exocytosis of entire granules. So, too, were the granular contents of basophils considered. However, in 1972,we published our initial studies which showed that basophils stored an electron-dense exogenous tracer protein, horseradish peroxidase (Fig. 4A), in their secretory granules (Dvorak et al., 1972), thus setting the stage for release of such exogenous proteins when appropriately stimulated. In certain circumstances, internalization of the cellular products of another lineage, such as eosinophil peroxidase (EPO) (Fig. 4B), could render the cytochemical profile of basophils as mimicking that of eosinophils (Dvorak et aL, 1985a,b) in inflammatory reactions rich in EPO released from reactive eosinophils. Our initial studies of this general cell-biological mechanism in purified guinea pig basophils (Dvorak et al., 1974c, 1980d, 1985a), and later in human basophils arising in cultures replete with eosinophils undergoing EPO secretion (Dvorak et al., 1985b), have further documented this mechanism. Substantial studies of other cell lineages now provide greater generality €or this event (Handagama et al., 1987,1989, 1993; Harrison et al,, 1990; Hill et al., 1996; Wagner et al., 1996).

1. Uptake and Granule Storage of Exogenous Proteins by Basophils We noted a uniquely rich complement of small (100 to 150 nm) smooth vesicles in the cytoplasm of guinea pig bone marrow and peripheral blood basophils in situ (Dvorak et al., 1972; Dvorak and Dvorak, 1975). These structures suggested the possibility that basophils were engaged in endocytosis and perhaps secretion, using these vesicles as transport vehicles. Initially, we demonstrated the uptake function of these structures using an

FIG. 4 Guinea pig basophils prepared to demonstrate peroxidase activity by a cytochemical technique after exposure to HRP for 30 min at 37°C (A) or EPO for 15 min at 37°C (B). Note the large number of HRP-filled, dense, black cytoplasmic vesicles adjacent to a nonperoxidase-containing guinea pig basophil granule with a mixture of longitudinal and hexagonal arrays in A. The basophil in B contains EPO-loaded, dense, black vesicles and granules that are completely filled with dense. EPO, partially filled with EPO, or do not contain any EPO (arrow). N, nucleus (with permission, from Dvorak and Dvorak, 1993). Bars: (A) 0.6 pm; (B) 1.8 pm.

CELL BIOLOGY OF THE BASOPHIL

109

intravenous tracer, horseradish peroxidase, which is rendered electrondense by a cytochemical procedure (Dvorak et al., 1972). These studies clearly showed uptake and transport of this protein to granules in viva Subsequently, using purified guinea pig basophils in v i m and wash-out experiments, we were able to show transport of horseradish peroxidase in vesicles in the reverse direction, such as cellular secretion might utilize in certain circumstances (Dvorak et al., 1980d). These studies of vesicular transport were extended to include uptake and transport to granules of exogenous EPO by guinea pig basophils, mouse mast cells, and mouse granular lymphocytes displaying NK activity in vitro, thereby documenting a role for vesicular transport of a biologically important protein released in eosinophil-rich inflammatory reactions and extending this uptake mechanism to mast cells and NK cells (Dvorak, 1991; Dvorak et al., 1985a). In studies of human basophils arising in factor-supplemented, eosinophilrich cultures, we similarly demonstrated endocytosis, vesicle attachment, and discharge into secretory granules in basophils of EPO released by the eosinophils (Dvorak et al., 1985b).

2. Uptake and Granule Storage of Exogenous Proteins by Other Secretory Cell Lineages Handagama et al. (1987, 1989, 1993) confirmed our findings in basophils by demonstrating the ability of megakaryocytes (MK) and platelets to store horseradish peroxidase in their alpha granules. Additionally, they showed that intravenously injected albumin, IgG, and fibrinogen were stored in guinea pig MK and platelet alpha granules. Platelet alpha granule contents include those proteins synthesized by precursor MK as well as numerous exogenous proteins that are synthesized elsewhere and are taken up by platelets from the plasma. Thus, platelet alpha granule endogenous proteins include platelet factor 4 and P-thromboglobulin, whereas exogenous proteins resembling plasma factors include albumin, factor V, thrombospondin, fibronectin, fibrinogen, and vitronectin (Harrison et al., 1990). Patients with congenital afibrinogenemia do not have fibrinogen in their platelets, suggesting that all fibrinogen in normal platelets is obtained by internalization of plasma stores (Harrison et al., 1990). Similarly, the platelets of patients with Glanzmann’s thrombasthenia are devoid of the fibrinogen receptor, GPIIb/IILa, and also do not have fibrinogen stores in their platelets (Harrison et al., 1990). These two illustrations show that circulating fibrinogen is necessary in order for platelets to store fibrinogen in alpha granules (Harrison et al., 1990), and, if platelets are devoid of their fibrinogen receptor, they also cannot internalize and store fibrinogen taken up from the plasma (Harrison et al., 1990). More recently, a unique disintegrin, which is a specific antagonist of the fibrinogen receptor GPIIb-IIIa, was used to

110

ANN M. DVORAK

demonstrate that all endocytotic uptake of plasma fibrinogen by platelet and MK alpha granules was inhibited (Handagama et al., 1993). Thus, platelet fibrinogen stored in alpha granules is derived entirely by endocytosis and not by synthesis in platelet precursor MK (Handagama et al., 1993). Similarly, a human megakaryocytic cell line was used to show that vitronectin is not a synthetic product of MKs but is endocytosed from serumderived vitronectin and stored in secretory granules where it was colocalized with an MK secretory protein, type 1 plasminogen activator inhibitor (Hill et al., 1996). In another study, it was determined that haptoglobin was not synthesized de novo by neutrophils. Rather, this ubiquitous mammalian serine protein (Javid, 1978) was internalized and concentrated in cytoplasmic granules and, in turn, was secreted following neutrophil activation (Wagner et al., 1996). Thus, at least two additional secretory cells, neutrophils and megakaryocytes (and their progeny, platelets), as well as basophils, store exogenous proteins in secretory granules.

V. Development of Tools for Advanced Cell-Biological Studies of Basophils A. Early Tools For -90 years following Ehrlich’s discovery of human basophils (Ehrlich, 1879), studies were seriously impeded by the inability to obtain cells for study. Since only 0.5% of the peripheral blood cells are basophils, most studies utilized malignant basophils, which increase in myelogenous leukemia (Dvorak, 1988a; Dvorak et al., 1981a; Dvorak and Dvorak, 197.5; Galli et al., 1983b; Maloney and Lange, 19.54) and were of light-microscopic preparations. Immunologists determined that histamine correlated with basophils, of all formed elements of blood (Graham et al., 1955), and that histamine could be released from sensitized basophils-reactions termed immediate hypersensitivity (Dvorak, 1988a). A new immunoglobulin, IgE, was found to be responsible for this release reaction (Ishizaka et al., 1970; Ishizaka and Ishizaka, 1975). In the 1970s,improved light-microscopic methods of Giemsa-stained plastic sections and electron microscopy contributed to the recognition that basophils were an important component of the inflammatory exudate in cell-mediated immunity (Dvorak 1987, 1988a; Dvorak et al., 1970; Monahan and Dvorak, 1985), and biochemical studies of AND and mediator release accelerated (Dvorak, 1988a). In the early 1980s, ultrastructural analysis of AND stimulated by several triggers was facilitated by the development of reliable methods for purifying peripheral

CELL BIOLOGY

OF THE BASOPHIL

111

blood basophils (Dvorak, 1988a; MacGlashan and Lichtenstein, 1980; MacGlashan et al., 1982a; Dvorak et al., 1974~).

6. Newer Tools 1. Tools That Increase Basophil Supplies

In addition to earlier methods for purifying peripheral blood basophils (Dvorak, 1988a), new methods produced basophils for studies. Mouse basophils were successfully sorted from bone marrow and spleen on the basis of the presence or absence of cell surface markers (Seder et al., 1991a; Dvorak et al., 1993a, 1994a). Specific cytokines, which were determined to be basophilopoietins, successfully produced immature and mature human basophils in various culture systems (Dvorak and Ishizaka, 1995) and increased circulating basophils in monkeys in vivo (Dvorak et al., 1989a). The cytokines useful for this purpose are available in recombinant forms. Thus, IL-3 induced human basophils to develop in vitro (but not mast cells) (Dvorak et al., 1989b; Saito er aZ., 1988); SCF induced human mast cells to develop in vitro (small numbers of basophils also developed in this suspension culture system) (Dvorak and Ishizaka, 1995); IL-3 induced circulating basophils in monkeys (Dvorak et al., 1989a). Neither IL-3 nor SCF alone (or in combination) induced mouse basophils to develop or survive in vitro, but each factor supported mouse mast cells in vitro (Dvorak et al., 1994a). Identification of these various lineages was accomplished by routine ultrastructural methods. 2. Ultrastructural Kinetic Models of Basophil Secretion

We performed extensive ultrastructural analyses of human basophil secretion based on the kinetics of mediator secretion induced with two secretogogues (Dvorak et al., 1991a, 1992a). The two releasing agents were FMLP-a bacterial peptide with rapid biochemical release kinetics (Warner et al., 1989)-and TPA-a tumor-promoting phorbol diester with slow biochemical release kinetics (Schleimer et al., 1981, 1982). Morphologic changes, ascertained with routine ultrastructural methods, provided a solid basis for future studies with specific ultrastructural tags and tracers.

a. FMLP-Induced D eg r a d a tio n of Human Basophils We examined the kinetics of morphological change induced by stimulation of human basophils with FMLP, using purified cells from normal donors (Dvorak et al,, 1991a). Samples were prepared for electron microscopy at 0,10,20, and 30 s and 1,2,5, and 10 min poststimulation with FMLP. The ultrastructural

112

ANN M. DVORAK

morphology of basophils induced by FMLP stimulation was unique. FMLPstimulated basophils, for example, initially emptied granules, the containers of which remained in the cytoplasm in situ. Many of these empty containers enlarged dramatically before accumulated intragranular membranes and vesicles were released along with the extrusion of the empty container membranes. These early events (occurring 0-20 s poststimulation) were morphologically analogous to PMD of human basophils in viva In both cases, large numbers of cytoplasmic vesicles were present, and in the FMLPstimulated model, vesicle numbers increased in conjunction with acquisition of the morphology of PMD and preceded the development of the morphology of granule extrusions. Vesicle numbers decreased dramatically in cells displaying the morphology of AND. The extrusion of full granules (and the membranes of granules previously emptied by PMD) coincided with the half-maximum histamine release reported for this model at 1.3 min poststimulation (Warner et al., 1989). Shedding of intragranular concentric dense membranes and vesicles, surface membranes and processes, and extrusion of formed, intragranular CLCs accompanied the exocytosis of granules. Extraordinary membrane shifts occurred and persisted over the 10-min kinetic interval examined poststimulation and coincided primarily with the later time frame (3.5 min) within which LTC, was generated and released from human basophils (Warner et al., 1989). Granule exocytosis, which peaked at 1 min in this model, extended from 20 s to 2 min in the ultrastructural samples studied after FMLP stimulation. The extrusion of granules took place through multiple pores in the plasma membrane; extruded intragranular materials remained associated with cul-de-sacs formed by the irregular surfaces of releasing basophils. These materials included rounded, granule-shaped, membranefree aggregates of granule particles, spherically shaped homogeneous structures identical to intragranular CLCs, and masses of concentric dense membranes. Elongated cell processes, cells actively shedding membranous surface materials, and completely degranulated, granule-free cells were prevalent between 20 and 120 s. Thus, a degranulation continuum of morphologic change occurred when human basophils were stimulated with FMLP. Essentially, in morphologic kinetic studies, a continuum of PMD occurred early and progressed to AND at later time points, coincident with the rapid release of histamine.

b. TPA-InducedDegranulation ofHuman Basophils We also performed an ultrastructural kinetic analysis of tumor-promoting phorbol diesterinduced degranulation of human basophils (Dvorak et al., 1992a), a substance known to elicit histamine release (but not LTC, release) (Schleimer el al., 1981). Unlike the rapid kinetics associated with IgE-mediated histamine release (15 min) or FMLP-mediated histamine release (2 min), the hista-

CELL BIOLOGY OF THE BASOPHIL

113

mine release stimulated from human basophils by the phorbol ester TPA is slow, reaching a maximum by 1 h (Schleimer eta/., 1981). We prepared, for ultrastructural analysis, TPA-stimulated and control samples of human basophils at multiple intervals, which were selected to precede and include maximum histamine release for this secretogogue. We found that, as in biochemical studies (Schleimer et aL, 1981, 1982), TPA was a unique ultrastructural secretogogue for human basophils. For example, extensive PMD was evident in multiple samples, achieving -50% granule alteration by 45 min poststimulation. This evidence of empty granules was associated with, and preceded by, a rapid, extensive, and sustained elevation in particlecontaining cytoplasmic vesicles, compared to buffer-incubated controls at all time points examined (p <0.001) (Dvorak et al., 1992a). Essentially, ultrastructural kinetic studies of the morphology expressed by the human basophils stimulated by TPA revealed three major findings (Dvorak et al., 1992a). There was minimal classical exocytosis, and this was not associated with significant reductions in human basophil granule numbers, over a 45-min period. Completely granule-free cells were absent-a feature that is regularly present at peak histamine release times after FMLP stimulation of replicate samples from the same donors (Dvorak et a/., 1991a). There was extensive PMD, characterized by a change in the ratio of altered to unaltered granules, from 1 to 4 (in controls) to 1 to 2 by 45 min after TPA (p < 0.001). In concert with these findings, a fivefold increase in the number of cytoplasmic vesicles containing particles occurred in TPA samples, compared to that in unstimulated cells (p <0.001), at multiple times sampled, including 45 min after TPA. At no time did empty vesicles increase to levels observed in FMLP-stimulated samples from the same donors, nor did extensively enlarged empty granule containers appear. The total number of vesicles (after TPA) was stable, indicating balanced vesicular traffic between the cell surface and granules accompanying PMD and extending to 45 min after stimulation of basophils. Thus, a coordinate secretion of histamine (Schleimer et aZ., 1981) was associated with the morphology of PMD (Dvorak et aL, 1992a) in this model. The third finding in the TPA-stimulated human basophil model has been termed a forme fruste of AND. Thus, we found prominent, previously unobserved changes in human basophils which were stimulated with TPA. These were distinctive individual granule-plasma membrane interactions. That is, multiple individual granules that were fused to plasma membranes were associated with variable, overlying membrane blebs, and individual underlying granules pushed into these joined membranes to produce bulging (membrane-covered) granule structures (up to one-half the length of granules) beyond the cell surface perimeter. When such structures opened to the cell surface and released granule contents, they failed to become externalized, leaving a granule-shaped, scalloped cell surface contour. It is

114

ANN M. DVORAK

possible that this forme fruste of AND reflects changes in membrane fluidity (or other important changes in membrane lipids), changes that result in the failure of granule and plasma membranes to fuse properly or to externalize emptied granule containers. Such effects might down-regulate AND as a major histamine release mechanism by TPA-stimulated human basophils, whereas PMD continues, or is upregulated, in the absence of the onset and completion of a major AND phase in the morphological continuum of which human basophils are capable (Dvorak et al., 1991a). 3. Newly Established Basophil Secretogogues

Studies of the direct stimulation of histamine release from basophils include those utilizing IgE, small peptides, calcium ionophore, or phorbol esters. Agents that influence the stimulation of histamine release may act indirectly by priming for histamine release, which is induced later by exposure to a direct secretogogue (MacDonald et al., 1989), and IgE-dependent histamine release may vary as a function of IgE heterogeneity (MacDonald et al., 1987). In the early 1970s, extensive morphologic studies of basophil-rich tissue reactions led to the suggestion that products of specific mononuclear cells might attract and activate nonspecific basophils in these sites (Dvorak et al., 1970, 1974b; Dvorak and Dvorak, 1993). Such reactions, termed CBH in guinea pig models (Dvorak et al., 1970), were associated with PMD exclusively. The first reports of mononuclear cell products with histamine releasing activity (HRA) supported the suggestion that mononuclear cell products activate human basophils (Thueson et al., 1979a,b). The H R A of histamine releasing factor(s) (HRF)-containing cellular products is now attributed to multiple cytokines, and these have been identified from a variety of sources (MacDonald, 1993; Dvorak, 1992a). Specific triggering events for PMD are just being identified. Likely candidates include various cytokines or bacterial and viral products associated with inflammation. We have observed the morphology of PMD in two circumstances where IL-3 might be responsible, either directly or indirectly. For example, we have observed PMD (characterized by unenlarged empty granule containers in the cytoplasm of basophils, which developed in rhIL-3-containing cultures of human cord blood mononuclear cells (Dvorak et al., 1989b), and in circulating mature basophils, which developed in monkeys infused with rhIL-3 (Dvorak et al., 1989a). There is also evidence implicating IL-3 in the activation of basophils, as determined biochemically (Alam et al., 1989a; Haak-Frendscho etal., 1988a; Kurimoto ef al., 1989; MacDonald et al., 1989). The IgE dependency of histamine release from human peripheral blood basophils was shown to be important in several circumstances (Liu et al., 1986; Warner et al., 1986; MacDonald et al., 1987). For example, human

CELL BIOLOGY OF THE BASOPHIL

115

lung macrophage-derived H R A and HRF, generated in cutaneous IgEmediated late-phase reactions, are both examples of such IgE-dependent factors (Liu et al., 1986; Warner et al., 1986). Similarly, nasal wash fluids contain an H R F that is IgE-dependent but is active only in some donors (MacDonald et al., 1987). Schulman etal. (1988) cultured human peripheral blood monocytes and demonstrated a potent H R A for human basophils in the supernatant within 24 h of culture. A role for food hypersensitivity in the generation of H R F from mononuclear cells was demonstrated by Sampson etal. (1989). These authors also showed that this activity provoked release from basophils of food-sensitive individuals but not from normal controls and that this activity was IgE-dependent (Sampson et al., 1989). Other cells also generate cytokines with histamine-releasing properties, as assayed with human basophils. For example, platelets stimulated to undergo release reactions by a variety of triggers produce an H R F that stimulates human basophil release either by IgE-associated mechanisms or alone (White et al., 1988; Orchard et al., 1986). Neutrophils also release a histamine-releasing activity into supernatant fluids (White and Kaliner, 1987; White et al., 1988). This material has been assayed on human peripheral blood basophils (White and Kaliner, 1987; White et al., 1988) and on rat basophil leukemia cells (White and Kaliner, 1987). Recently, recombinant human neutrophil-activating peptide from E. coli has been shown to induce histamine and leukotriene release by IL-3-primed basophils (Dahinden et al., 1989). Early studies implicated interferon in the enhancement of IgE-mediated release from human basophils (Hernandez-Asensio et al., 1979; Ida et al., 1977, 1980). Initially, this augmentation of histamine release was shown when certain viruses were incubated with human leukocytes and was also shown to require new RNA synthesis (Hernandez-Asensio et al., 1979). Examination of supernatant fluids from antigen-containing, sensitized leukocyte cultures showed a correlation between histamine release-enhancing activity and interferon (Ida et al., 1977). Further studies showed that the soluble factor responsible for stimulating human basophils to release histamine was indeed interferon (Ida et al., 1980). Recently, the effect of interferon on basophil releasibility was evaluated using short-term basophil cultures of mature basophils purified from the peripheral blood (Schleimer et al., 1989). In this case, enhancement of IgE-mediated histamine release (-200% of control) was established (Schleimer et al., 1989). These studies, in aggregate, demonstrate the involvement of the cytokine, interferon, in increased release of mediators from human basophils, an event of possible importance in viral disorders. The advent of the application of molecular biology techniques to the field of cytokine research has been instrumental in providing pure recombinant materials in quantity for experimentation. A number of these have now

116

ANN M. DVORAK

been evaluated for secretogogue action in human basophils purified from blood (Alam et al., 1989a; Haak-Frendscho et al., 1988a; Kurimoto et al., 1989; MacDonald et al., 1989; Schleimer et al., 1989; Hirai et al., 1988), monkey basophils in vivo (Dvorak et al., 1989a; Mayer et al., 1989), and human basophils developing from cultured cord blood cells (Dvorak et al., 1989b). There is now evidence regarding the capability of inducing histamine release by a number of recombinant interleukins and cytokines. The biochemical studies of mediator release show that rhIL-3 stimulates histamine release from human basophils (Tedeschi et al., 1995; Okayama and Church, 1992; Valent et al., 1989; Alam et al., 1989a; Haak-Frendscho et al., 1988a; Kurimoto et al., 1989; MacDonald et al., 1989; Schleimer et al., 1989; Hirai et al., 1988). Recombinant human IL-3 enhanced IgE-mediated release (Schleimer et al., 1989;Hirai et al., 1988) but, in some studies (HaakFrendscho et al., 1988a), also stimulated histamine release in the absence of cooperating triggers given simultaneously. Recombinant human IL-3 enhanced human basophil histamine release induced with other secretogogues as well. For example, FMLP-mediated (Schleimer et al., 1989; Hirai et al., 1988), ionophore-A23187-mediated (Hirai et al., 1988), MBPmediated (Sarmiento et al., 1995), and platelet-activating-factor-mediated (Brunner et al., 1991) histamine release and CSa-mediated leukotriene release (MacGlashan and Hubbard, 1993) were enhanced. Basophils from patients with atopic disease responded to a greater extent to rhIL-3 than did basophils from nonatopic basophil donors (MacDonald et al., 1989). A novel function in the secretory processes of human basophils was demonstrated by the release of newly generated LTCQin large quantities when rhIL-3 and C5a were used to trigger cells (Kurimoto et al., 1989;MacGlashan and Hubbard, 1993). This established that the mediators released can be modified by this cytokine, since neither IL-3 nor C5a alone stimulates the release of LTC4 from human basophils (Kurimoto et al., 1989; MacGlashan and Hubbard, 1993). In these studies, it was also found that IL-3 enhanced the release of histamine by other stimuli but that IL-3 alone did not cause significant release over that of control spontaneous release (Kurimoto et al., 1989). Interestingly, however, it was also noted that IL-3 caused a low, continuous release of histamine over several hours in basophils from some atopic donors (Kurimoto et al., 1989). To us, this profile for histamine release suggests a biochemical correlate of PMD. Human recombinant GM-CSF was shown to be an effective releaser of histamine from human basophils by several groups (Alam et al., 1989a; Haak-Frendscho et al., 1988a; Hirai et al., 1988), but another group did not confirm this (Schleimer et al., 1989). The circumstances of study differed, however, in that basophils were used either immediately (Alam et al., 1989a; Haak-Frendscho et al., 1988a; Hirai et al., 1988) or after 24 h in culture (Schleimer et al., 1989).

CELL BIOLOGY OF THE BASOPHIL

117

Recombinant human IL-5 was also tested for secretogogue activity and was found to release histamine from basophils in 2 of 14 donors tested (Haak-Frendscho et al., 1988a), whereas tests of rhIL-3 and GM-CSF given together released histamine in 8 of 10 and 12 of 14 donors, respectively (Haak-Frendscho et al., 1988a). More recently, it has been determined that IL-5 enhances histamine release from human basophils evoked by anti-IgE, FMLP, or ionophore A-23187 (Hirai et al., 1990; Bischoff ef al., 1990) as well as histamine release and leukotriene generation by C5A (Bischoff ef al., 1990). IL-1 has also been found to have histamine-releasing capabilities (Subramanian and Bray, 1987; Massey et al., 1989; Haak-Frendscho et al., 1988b). Different sources of IL-1 were tested on isolated human peripheral blood basophils or mast cells from adenoids (Subramanian and Bray, 1987). These included purified IL-1 from stimulated human monocytes and rhIL-1. Both were found to induce histamine release from basophils and mast cells (Subramanian and Bray, 1987). Studies from Lichtenstein’s laboratory showed that either rhIL-la or rhIL-lb potentiated IgE-mediated histamine release from human peripheral blood basophils but direct stimulation of histamine release alone was not demonstrated (Massey et al., 1989). Others have shown that basophils released histamine when stimulated by IL-1 in the presence of D 2 0 , a histamine release-enhancing factor (Alam et al., 1989a). IL-2 has a priming effect on the release of histamine from human basophils in cells stimulated with ionophore A-23187, FMLP, or anti-IgE (Morita et aZ., 1987) and enhanced IgE-mediated or HRF(s) (nasal washing origin)stimulated histamine release from human basophils (White et al., 1992). IL-8 was found to enhance IL-3-mediated histamine release from human basophils (Dahinden et al., 1989; Krieger et al., 1992). A newly subcloned protein, rp21, is an IgE-dependent H R F (MacDonald et al., 1995) that causes histamine release from human basophils similar to the previously described H R F found in vivo (MacDonald et al., 1987). The kinetics of histamine release with rp21 is similar to that stimulated by antiIgE (MacDonald et al., 1995). We recently found ultrastructural evidence for PMD in association with rhIL-3 in two circumstances (Dvorak ef aL, 1989a,b). Recombinant human IL-5 was also found to be associated with images of PMD in human basophils (Dvorak et al., 1989b). The studies of human basophils were done on cells that developed from human cord blood mononuclear cell cultures containing rhIL-3 or rhIL-5. With each recombinant interleukin, the ultrastructural correlate of PMD was present in viable, mature basophils (Dvorak et al., 1989b). We also examined monkey peripheral blood basophils that were induced to increase by continuous infusion of rhIL-3 (Dvorak et aZ., 1989a). Images of PMD were also present in these circulating mature

118

ANN M. DVORAK

basophils in vivo. Recently, monkeys that were infused with either rhIL-3 or rhGM-CSF (individually or combined) were shown to have elevated plasma histamine levels (Mayer et al., 1989). Values for the combined infusion of rhIL-3 or rhGM-CSF exceeded those that were recorded for either cytokine alone (Mayer et al., 1989). Chemokines are chemotactic proteins of cellular origin with a functional role in allergic inflammation, often by activation of eosinophils and basophils. Some of these are potent secretogogues for human basophils (MacDonald, 1993). One of these is the chemokine monocyte chemotactic protein-1 (MCP-1) (Yoshimura et af., 1989; Matsushima et al., 1989; Zachariae et al., 1990; Furutani et al., 1989) (also called monocyte chemotactic and activating factor). MCP-1 is a low-molecular-weight chemokine, the most potent histamine-releasing cytokine described, with a potency equivalent to that of anti-IgE (Bischoff et al., 1992; Alam et al., 1992a; Kuna et al., 1992a). It acts rapidly, with 80% histamine release by 30 s of stimulation (Alam et af., 1992a) without further release after 1 min (Kuna et af.,1992a). Recently, it has been shown that deletion of the NH2-terminal residue converts MCP-1 from an activator of basophil mediator release to an eosinophi1 chemoattractant (Weber et al., 1996). New reports of basophil secretogogue action by chemokines include those for monocyte chemotactic proteins (MCP)-2 and -3 (Weber et al., 1995; Alam et al., 1994; Proost et af., 1996; Dahinden et af., 1994). Of the three monocyte-derived factors, MCP-1, MCP-2, and MCP-3, the latter is equivalent to MCP-1 for release of histamine frcm human basophils (Dahinden et af., 1994). Several other chemokines are known to stimulate human basophil secretion as well. These include the macrophage inflammatory proteins l a and 1p and RANTES (regulated on activation, normal T expressed and secreted) (Alam et al., 1992b; Bischoff et al., 1993; Kuna et al., 1992b). Cytokines that inhibit the release of histamine from human basophils may also be produced in inflammatory reactions. For example, Alam et nl. have identified such a factor which is produced when peripheral blood mononuclear cells are stimulated with histamine or mitogens, such as concanavalin A (Alam et al., 1988, 1989b). These investigators found that this histamine release inhibitory factor (HRIF) was produced by both B and T cells as well as monocytes (Alam et af., 1988). Both CD4- and CD8- T cell subsets produced the factor (Alam et af., 1988). A similar HRIF has also been identified in supernatants of cultured, sensitized spleen cells (Alam et al., 1988). These studies, in aggregate, clearly show that cytokines can stimulate, enhance, or inhibit the secretion of histamine from human basophils.

CELL BIOLOGY OF THE BASOPHIL

119

4. Ultrastructural Subcellular Tags

a. The CLC Protein The CLC protein (Ackerman et al., 1982) is a granule-associated protein that has been imaged by the postembedding immunogold staining of human basophils (Dvorak and Ackerman, 1989). This protein is also present in human eosinophils (Ackerman et al., 1982). It exhibits lysophospholipase activity (Weller et al., 1984; Zhou et al., 1992), has recently been cloned and expressed in COS and Chinese hamster ovary cells (Ackerman et al., 1993; Zhou et al., 1992), and has had its threedimensional structure resolved by X-ray crystallography (Leonidas et al., 1995). The latter studies revealed that the CLC protein is a unique protein with a dual functional role based on structural topology similar to galectins1and -2 and identification of a putative lysophospholipase active site (Leonidas et al., 1995). The galectins bind carbohydrates (Barondes et al., 1994), and, like the CLC protein, their functional role in biology is not known. The presence of the structurally similar CLC protein in similar cells (basophils, eosinophils) and in similar sites in these cells (nucleus, cytosol) suggests possibilities that include a role in allergic inflammation and nuclear RNA processing (Dagher et al., 1995; Frigeri et al., 1993; Truong et al., 1993). An ultrastructural immunogold postembedding method was used to localize the CLC protein initially in human peripheral blood eosinophils (Dvorak et al., 1988); later studies localized subcellular sites of the CLC protein in human eosinophils and macrophages in vitro (Dvorak et al., 1991b, 1992c, 1994d), in macrophages, eosinophils, and tumor cells in vivo (Dvorak et al., 1990a,b), and in peripheral blood (Dvorak and Ackerman, 1989) and cultured basophils (Dvorak 1996; Dvorak et al., 1994c) (Fig. 5A). The method was straightforward and worked on routinely fixed and processed samples containing glutaraldehyde, osmium, and uranyl, with embedment in Epon-all reagents that are needed for optimal visualization of subcellular structures but that notoriously destroy antigens. The immunogold procedure and necessary specificity controls were done as follows. Fifty- to seventy-nanometer sections were placed on gold grids and floated on 50-pl drops of reagent at 25°C on covered petri dishes containing dental wax. The following sequence of reagents was used: (a) 4% sodium metaperiodate (Sigma Chemical Co.), 15 min; (b) three washes, 10 min each, in 0.2 pm of Millipore-filtered (Fischer Scientific, Indiana) 20 mM Tris(hydroxymethy1)aminomethane buffer containing 0.9% saline, 0.1% globulinfree bovine serum albumin (BSA),pH7.6 [Tris-bufferedsaline (TBS)-BSA]; (c) 5% normal goat serum in TBS-BSA, 1 h; (d) primary rabbit polyclonal affinity chromatography purified anti-CLC (150 p g h l in TBS-BSA containing 1% Tween-20 and 1%normal goat serum), 2 h at 25°C; (e) three washes, 10 min each, in TBS-BSA; (f) secondary gold-labeled antibody

120

ANN M. DVORAK

FIG. 5 In A, a mature human basophil, which developed in a 7-week suspension culture of cord blood cells supplemented with stem cell factor-containing fibroblast culture supernatant and prepared with immunogold to detect the CLC protein, shows extensively labeled cytoplasmic granules. In B, a mature peripheral blood human basophil, recovered from shortterm culture 6 h after stimulation with FMLP and prepared with enzyme-affinity gold to detect histamine, shows extensively labeled, reconstituted cytoplasmic granules (G). N, nucleus (A, reproduced with permission from Dvorak et al., J . Histochem. Cytochem. 42 251-263, 1994 ~B, ; with permission of Karger, Basel, from Dvorak et al., 1995a). Bars: (A) 0.6pm; (B)

0.04pm.

[l :20 dilution of 10 nm (Janssen Life Sciences Products, Bromma, Sweden), 20 nm (EY Laboratories, San Mateo, CA), or 30 nm (Janssen) of colloidal gold conjugated to goat anti-rabbit IgG in TBS-BSA containing 0.1% Tween-20, 0.4% gelatin, and 1%normal goat serum, 1 h; (g) two washes, 10 min each in TBS-BSA; and (h) two washes, 10 min each, in distilled water. Controls included the following alterations of the standard sequence: (a) omission of primary antibody, (b) substitution of nonimmune normal

CELL BIOLOGY OF THE BASOPHIL

121

rabbit IgG (150 pg) for the specific primary antibody, and (c) substitution of solid-phase CLC protein Sepharose-absorbed primary antibody for unabsorbed specific primary antibody.

b. Histamine We developed a new cytochemical method to detect histamine (Fig. 5B) in routinely prepared electron-microscopicsamples (Dvorak et al., 1993b) so that fine structural details would be preserved, and, thus, one could assess release and recovery events in histamine-rich mast cells (Dvorak 1995a, 1997;Dvorak and Morgan, 1996,1997;Dvorak et al., 1994e, 1995b, 1996~).The method is based on the affinity of the enzyme diamine oxidase (DAO) for its substrate, histamine (Beaven, 1976; Schayer, 1959; McBride et al., 1988; Buffoni, 1966; Zeller, 1965). It is a postembedding enzyme-affinity-gold technique based on principles described by Bendayan and Frens (Bendayan, 1981,1984; Frens, 1973). Complete technical details and a large number of controls for specificity have been reported (Dvorak et al., 1993b). The procedure for making the DAO-gold reagent, staining sections on grids, and key specificity controls is as follows. A colloidal suspension of gold was prepared according to the method of Frens (1973). Four milliliters of an aqueous 1%solution of sodium citrate was added to a boiling aqueous solution of 100 ml 0.01% tetrachloroauric acid and allowed to boil for 5 min before cooling on ice. The pH of the colloidal gold suspension was adjusted to 7 with 0.2 M potassium carbonate. Preparation of the DAO-gold complex was according to the method of Bendayan (1984). Three milligrams of DAO was dissolved in distilled water and placed in a polycarbonate ultrafuge tube with 10 ml of the gold suspension. The mixture was centrifuged at 25,000 rpm for 30 minutes, 4"C, in a Beckman ultracentrifuge with a no. 50.2 Ti rotor. The DAO-gold complex formed a red sediment that was carefully recovered and resuspended in 3 mlO.1 M phosphate-buffered saline (PBS) containing 0.02% polyethylene glycol, pH 7.6 (final concentration, 1 mg DAO/ml). For cytochemical labeling, section-containing grids were inverted and floated on PBS drops for 5 min, followed by incubation on a drop of DAO-gold at 37°C for 60 min. The grids were vigorously washed in distilled water and stained with dilute lead citrate for 10 min before viewing by electron microscopy. Specificity controls for the enzyme-affinity-gold method included prior digestion of samples with DAO or absorption of the DAO-gold reagent by solid-phase histamine. This newly developed method has been applied to cell biological studies of human basophils (Dvorak, 1995a, 1997; Dvorak et al., 1994b, 1995a, 1996f). 5. Ultrastructural Subcellular Tracers

Tracers that are either inherently electron-dense or that can be rendered so by an enzyme reaction are useful in following endocytotic traffic of

122

ANN M. DVORAK

exogenous soluble proteins or to trace intracellular spaces in continuity with the extracellular space. In endocytotic studies, the tracer is administered before fixation, either in vivo or ex vivo; for tracing continuities between intracellular spaces and the exterior of cells, the tracer is administered after fixation. These simple techniques when applied to basophil cellbiological studies have provided much new data. Specific procedures are as follows.

a. Diaminobenzidine (DAB) Reaction to Demonstrate Peroxidase Activity Fixed cell suspensions, or tissue blocks, are washed with buffer (0.05 M Tris-HC1, p H 7.6) three times, following which the tissue blocks are chopped into 40-pm sections using a Sorvall TC-2 tissue chopper. Cells or 40-pm sections are incubated while on a metabolic shaker for 1 h in Graham and Karnovsky's medium containing 5 mg of 3-3' diaminobenzidine tetrachloride (DAB) and 0.01% hydrogen peroxide in 10 ml of 0.05 M Tris-HC1 buffer, p H 7.6 (Dvorak, 1987; Karnovsky, 1967). Following this, the samples are again washed three times in Tris-HC1 buffer for 5 min per wash. They are then postfixed in 1.5% collidine-buffered osmium tetroxide for 2 h, 4"C, dehydrated in a series of graded concentrations of alcohol, infiltrated, and embedded in a propylene oxide-Epon sequence. We have used this cytochemical procedure to demonstrate endocytosis of horseradish peroxidase in small vesicles and its subsequent storage in guinea pig basophil secretory granules (Dvorak et af., 1972, 1980d) as well as endocytosis and secretory granule storage of EPO by guinea pig basophils (Dvorak etaf., 1985a) and human basophils (Dvorak et af.,1985b). b. Cationized Ferritin Tracer Method Fixed samples are incubated with 1ml of Hanks' balanced salt solution containing 0.5 mg of cationized ferritin on a rotary shaker set at low speed for 30 min at room temperature (Danon et al., 1972). Following washes in 0.1 M sodium cacodylate buffer, samples are processed with routine ultrastructural methods (Dvorak, 1987). Cellbiological studies of guinea pig (Dvorak et af., 1981b, 1982c) and human basophils (Dvorak et al., 1980b, 1981c, 1984b; Findlay et al., 1981) have benefited from this tracer technique.

VI. Vesicles as Prominent Transport Organelles in the Cytoplasm of Basophils A. Cytoplasm of Basophils in Multiple Species

We found small, smooth membrane-bound vesicles to be a prominent feature of the cytoplasm in basophils from the four species we have studied

CELL BIOLOGY OF THE BASOPHIL

123

in detail (Dvorak, 1978, 1988a,b; Dvorak et al., 1982a, 1989a; Dvorak and Dvorak, 1975) (Figs. 6 and 7). These structures were generally larger than Golgi area vesicles (when these were visible), and they had two primary locations in the cytoplasm: (i) adjacent to secretory granules (Fig. 6A) and (ii) in the peripheral, granule-free cytoplasm near the plasma membrane (Figs. 6A and 6B). The appearance of these vesicles was variable, from

FIG. 6 The cytoplasm of mouse (A) and monkey (B) basophils shows large numbers of perigranular vesicles. The mouse basophil (A) was present in the Fc,R-positive cells sorted from the bone marrow of mice injected with goat anti-IgD; the monkey peripheral blood basophil was obtained from an animal that was perfused with rhIL-3. Many vesicles are also present in the peripheral cytoplasm of this cell (A, with permission, from Dvorak eta/., 1989a; B, with permission, from Dvorak et al., 1993a). Bars: (A) 0.6 pm; (B) 0.8 pm.

124

ANN

M. DVORAK

FIG.7 Electron micrographs of guinea pig (A) and human (B) basophils show vesicles attached to secretory granules (arrows). In both instances, the vesicle is budding from the granule in the direction of the overlying cell surface. Most of the perigranular vesicles, as well as the attached vesicle in the guinea pig basophil, are electron-lucent (A); granule particles are present in the attached and perigranular vesicles in the human basophil (B) (A, with permission, from Dvorak and Dvorak, 1993; B, with permission, from Dvorak, 1988b). Bars: (A) 357 nm; (B) 290 nm.

electron-lucent to electron-dense (Fig. 7). In human basophils, a number of the vesicles contained particles similar to granule particles (Fig. 7B). Basophils in tissue locations (guinea pig, human) often showed large num-

CELL BIOLOGY OF THE BASOPHIL

125

bers of cytoplasmic vesicles accompanying partially empty or empty, nonfused cytoplasmic granules, features of PMD. Some vesicles were accompanied by elongated tubules, a process that develops, by our hypothesis, when the rate of vesicular traffic increases to the point where discrete vesicles fuse to form elongated structures, which may interconnect granules to each other and to the plasma membrane (Dvorak and Dvorak, 1975). Through the use of electron microscopy, diligent searches have documented attachment of vesicles to individual secetory granules in basophils (Fig. 7). Such images demonstrate fusion of these structures, as one would expect if the smaller vesicles were transporting materials from (or to) the granules.

B. Early Experiments Early experiments with guinea pig and human basophils identified vesicular transport in these granulocytes. The ultrastructural similarity of vesicles and their abundance in basophils of at least four species suggest that these organelles are important to the function(s) of basophils. Additionally, the emptying of granules (with container retention in the cytoplasm of activated basophils which migrate into tissues, a process termed PMD) invokes the necessity for a vesicular transport mechanism to effect this mode of secretion (Dvorak and Dvorak, 1975). Our initial examination of the issue of vesicular trafficking in basophils revealed that guinea pig basophils internalized horseradish peroxidase (HRP) in small, smooth membrane-bound vesicles in vivo (Dvorak et al., 1972) and in vitro (Dvorak et al., 1980d). This electron-dense tracer enzyme was delivered to and stored in the cytoplasmic secretory granules, thus defining for the first time a new function for secretory granules as repositories for exogenous soluble proteins (Dvorak et al., 1972,1980d). The biological relevance of this newly described function for secretory granules was enhanced when we demonstrated that EPO trafficked to guinea pig (Dvorak et al., 1985a) and human (Dvorak et al., 1985b) basophils by vesicular transport. Wash-out experiments in vitro demonstrated that HRP was removed from granule repositories by vesicular transport in guinea pig basophils (Dvorak et al., 1980d), but EPO bound sufficientlytightly to these granules so that wash-out did not occur (Dvorak et al., 1985a).Theoretically, in these circumstances, an appropriate stimulus for regulated secretion to the EPO-loaded guinea pig basophils would result in the release of an eosinophil product from basophils by exocytosis. C. Vesicular Transport in Cell Biology

Exocytotic, endocytotic, and transcytotic cellular traffic routes effected by vesicular transport are the subjects of extensive studies in cell biology (Barr

126

ANN M. DVORAK

et al., 1992; Bean et al., 1994; Brodsky, 1988; Calakos et al., 1994; de Curtis and Simons, 1989; Ferro-Novick and Jahn, 1994; Fine, 1989; Goldstein et al., 1979; Malhotra et al., 1989; O’Connor et al., 1994; Ostermann et al., 1993; Rothman, 1994; Rothman and Wieland, 1996 Schatz and Dobberstein, 1996; Schekman and Orci, 1996; Schmid and Damke, 1995; Sudhof, 1995; Sudhof et al., 1993; Sztul et al., 1991; Takizawa and Malhotra, 1993). Numerous cellular systems are included in these works. Transport vesicles of variable size, shape, contents, coats, and functions circulate constitutively, or following stimuli, between rough endoplasmic reticulum and Golgi, between Golgi stacks, from Golgi to secretory granules and/or cell surfaces, between specific cell surfaces of polarized cells, from cell surfaces to early and late endosomes and lysosomes, or to Golgi structures. Despite a potentially bewildering array of trafficking vesicles, extensive progress has been made in the characterization of many of these important structures. Detailed review of these studies is beyond the scope of this review. However, many principles, structures, and biochemical components of these systems might apply to vesicular trafficking in basophils. Since large quantities of basophils are now obtainable from multiple species, future studies of the details of vesicular trafficking will be thereby facilitated. Some generalizations are possible regarding vesicular containers in basophils, based on currently available ultrastructural data. The vesicles involved in trafficking bidirectionally between secretory granules and plasma membranes arise by budding from these structures (Dvorak et al., 1972, 1974b, 1976a, 1996e) and do not have visible coats in routinely prepared samples. Classical clatharin-coated vesicles are extremely rare in basophils of multiple species. Basophilic myelocytes, prepared identically, have extensive Golgi structures, and small coated vesicles bud from and cluster at cisternal ends (Dvorak et al., 1972). The smooth membrane-bound vesicles in basophils are dynamically moving structures, as determined by kinetic ultrastructural studies of endocytosis of HRP or EPO (Dvorak et al., 1972, 1980d, 1985a), and of stimulated secretion of CLC protein (Dvorak et al., 1996a, 1997a,b) and histamine (Dvorak, 1997; Dvorak et al., 1996f). They also increase or decrease in number per unit size of cells during complex stimulated secretory events (Dvorak, 1997; Dvorak et al., 1981b, 1982c, 1991a, 1992a, 1997a,b). The smooth membrane-bound vesicles in basophils do not resemble typical caveolae (endothelial cell plasmalemmal vesicles) in that they are not flaskshaped and do not display the stomata and stomata1 diaphragms which typify the endothelial cell vesicles termed caveolae (Bruns and Palade, 1968; Schnitzer et al., 1995a,b). We have recently described vesiculo-vacuolar organelles (VVOs) in capillary and venular endothelial cells in several organs of multiple species (Kohn et al., 1992; Qu-Hong et al., 1995; Dvorak ef al., 1996d; Feng et al., 1996). These structures arc composed of variably

CELL BIOLOGY OF THE BASOPHIL

127

sized vesicles and vacuoles, resembling clusters of grapes, that are interconnected by stomata and diaphragms similar to those of endothelial cell capillary plasmalemmal vesicles. VVOs play a key role in the permselectivity of polarized endothelial cells, a function which is extensively upregulated by potent permeabilizing mediators such as vascular permeability factor (also called vascular endothelial cell growth factor (Feng et al., 1996; Dvorak et af., 1995c), histamine, and serotonin (Feng et al., 1996). Extensive ultrastructural studies, using electron-dense tracers, serial ultrathin sections, and three-dimensional reconstructions, show that VVOs are sessile, interconnected structures that transport large soluble proteins in concentrationdependent diffusion waves across endothelia, in the absence of individual vesicle transport (Kohn et al., 1992; Dvorak et al., 1996d; Feng et al., 1996). The selectivity of transport through these normally present endothelial cell organelles likely resides at stomata1 diaphragms (which close interconnected stomata) and is dependent on individual permeabilizing signals that initiate and enhance permeability. Vesicular transport in basophils does not involve structures resembling VVOs but does involve traffic of smooth membrane-bound vesicles. The biochemical constitution of these structures remains to be determined if effective isolation of vesicles can be accomplished. With such purified preparations, one could then model reconstitution studies with specific cytosolic components to determine their possible similarities to vesicular carriers, coated or uncoated, in other systems. Using purified human basophils, we have performed extensive kinetic studies of transport in these vesicular carriers of two basophil granule proteins, CLC protein and histamine, with secretogogues of distinctly different kinetics and secretory profiles (Dvorak et al., 1991a, 1992a, 1996a, 1997a,b).

D. CLC Protein-Loaded Human Basophil Transport Vesicles

1. FMLP Stimulation Cytoplasmic smooth membrane-bound vesicles (Figs. 8-12) with either electron-lucent [empty vesicles (EVs)] (Fig. 8) or electron-dense particles or material filling their interiors [full vesicles (FVs)] (Figs. 8 and 9) and total cytoplasmic vesicles (V) were quantitatedkellular pm2 for unstimulated cells and FMLP-stimulated samples and analyzed by both phenotypic (see Section VII1.C) and kinetic approaches (Tables II-IV). Additionally, gold label indicating the presence of CLC protein was quantitated in the vesicle population and analyzed to determine the proportions of vesicles that were carrying the CLC protein in specific phenotypes and over time after FMLP stimulation for comparison with unstimulated cells (Figs. 10-

128

ANN M. DVORAK

FIG. 8 Human basophils, stimulated for 20 s with FMLP and prepared for immunogold demonstration of CLC protein, show 30-nm gold-loaded empty vesicles in PMD-I (A) and PMD-I1 (B) cells and 30-nm gold-loaded empty and full vesicles (arrow) in a CDB cell (C). In A, the gold-labeled empty vesicle is adjacent to an unaltered, particle-filled granule; in B, the gold-labeled empty vesicle is adjacent to an altered, partly particle-filled granule that has an expanded, partly empty container; in C, gold-loaded empty and full vesicles are in the granule-free cytoplasm between the gold-labeled plasma membrane and nucleus (N) (with permission of Karger, Basel, from Dvorak er al., 1997b). Bars: (A) 140 nm; (B) 220 nm; (C) 240 nm.

12) (Table 11). The subsequent analyses of these data were done to answer the following questions regarding the role of vesicular transport in the trafficking of the CLC protein in human basophils.

a H o w Many Vesicles Are There (Number V/pM2)? Substantial numbers of vesicles exist in the cytoplasm of buffer-incubated, unstimulated basophils. Histograms of the change in vesicle numbers of basophils obtained at eight time points after FMLP stimulation are shown in Fig. 12A; similar vesicle number histograms for seven basophil phenotypes stimulated by FMLP are shown in Fig. 12K. Comparison of these two histograms reveals that extensive elevation of total V is best seen at 0 time after FMLP (Fig. 12A) and the smallest number of vesicles is present in the completely degranulated basophil (CDB) phenotype (Fig. 12K). p values for changes in total cytoplasmicvesicles in FMLP-stimulated samples compared to those of unstimulated cells and among stimulated samples for phenotypes and individual times are listed in Tables I1 and 111. b. H o w Many Vesicles Are Gold-Labeled (% VG/V)? Substantial numbers of cytoplasmic vesicles were labeled with gold indicating the presence of CLC protein (Figs. 8-12) (Table 11). Histograms of the %VG/Vfor the time analysis are presented in Fig. 12B and for the phenotypic analysis are presented in Fig. 12L. For the kinetic analysis, these data show a significant

CELL BIOLOGY OF THE BASOPHIL

129

FIG. 9 Human basophils, stimulated for 2 min with FMLP and prepared for immunogold demonstration of CLC protein, show 30-nm gold-loaded full vesicles in RB-I1 (A), CDB (B), and RB-I ( C ) cells. The granule in the RB-I cell ( C ) contains a gold-labeled formed CLC. The plasma membrane of the RB-I cell is devoid of gold label. N, nucleus (with permission of Karger, Basel, from Dvorak et a[.,1997b) Bars: (A) 100 nm; (B) 150 nm; (C) 170 nm.

increase in the %VG/V at 10 s (6%) compared to that of unstimulated cells (l%, p <0.005), at 20 s (20%) compared to that of unstimulated cells (l%, p <0.001), at 30 s (10%) compared to that of unstimulated cells (l%,p <0.001), at 1 min (29%) compared to that of unstimulated cells (l%, p <0.001), and at 2 min (14%) compared to that of unstimulated cells (l%, p <0.001). Significant differences between the unstimulated cells and the O-time, and the 5- and 10-min samples, did not exist ( p = NS). p values for comparisons of differences among the individual times are listed in Table 11. The phenotypic analysis of the %VGN revealed the highest value (also including the time analysis) to be in the CDB phenotype (37%),a phenotype appearing in greatest numbers (40%) in the l-min sample where the highest

TABLE 1 I Average Number of Smooth Membrane-BoundCytoplasmic Vesicles in Human Basophils: A Kinetic Analysis Following FMLP Stimulation, Indicating the Proportion Carrying CLC Protein

Unstimulated (average of 205, 1-min, and 10-min samples) Stimulated 0 time 10 s 20 s 30 s 1 min 2 min 5 min 10 min

Vesicles/ SE fimZ I

Gold-labeled vesicles/ p n 2 2 SE

Gold-labeled vesicles (%)

1.91 ? 0.27

0.02 2 0.20

1*

3.02 t 0.65 1.40 2 0.19 0.82 I 0.197 0.72 ? 0.19t 0.98 2 0.21f 1.45 t 0.24 0.77 2 0.31 0.97 i 0.22t

0.05 5 0.03 0.09 2 0.06 0.16 -t 0.04 0.07 i 0.02 0.28 -C 0.06 0.21 2 0.05 0.03 2 0.02 0.01 2 0.01

* p <0.005, compared to 10 s; p <0.001, compared to 20 s, 30 s, 1 min, and 2 min. ** p (0.01, compared to 10 s; p <0.001, compared to 20 s, 30 s, 1 min, and 2 min. * * * p <0.001, compared to 20 s and 1 min; p <0.01, compared to 2 and 10 min.

tp

<0.01, compared to 0 time.

tt p 10.05, compared to 30 s; p <0.001, compared

to 5 and 10 min. p <0.001, compared to 1 min; p <0.005, compared to 10 rnin. f p <0.05, compared to 0 time. $3p <0.001, compared to 2, 5 , and 10 min. $$4 p <0.01, compared to 5 min; p <0.001, compared to 10 min.

TABLE 111 Average Number of Cytoplasmic, Smooth Membrane-Bound Vesicles in Unstimulated Cells and FMLP-Stimulated Basophil Phenotypes

Vesicleslpd

t_

SE

Unstimulated

1.91 -+ 0.27*

Stimulated-Phenotypes PMD-I PMD-I1 AND-I AND-I1 CDB RB-I RB-I1

1.38 i 0.15** 0.75 t 0.17 0.82 i 0.17 0.71 ? 0.22 0.33 5 0.08*** 1.68 2 0.25 1.62 t 0.15

* p <0.001, (compared to CDB); p c0.05, (compared to AND-11). ** p <0.01, (compared to CDB). * * * p <0.001, (compared to RB-I and RB-11). 130

TABLE IV p* Values for Comparisons of the Percentage of Total Cytoplasmic Vesicles That Are Labeled with Gold Indicating CLC Protein in Unstimulated Easophils and FMLP-Stimulated Basophil Phenotypes

Phenotvoes

A

2

UN vs PMD-I p <0.001

UN vs PMD-I1 p <0.005

UN vs AND-I p 10.001

UN vs AND-I1 p <0.001

UN vs CDB p <0.001

UN vs RB-I p <0.001

PMD-I vs PMD-I1 p ~0.05

PMD-I vs AND-I p = NS

PMD-I vs AND-I1 p = NS

PMD-I vs CDB p 10.001

PMD-I vs RB-I p 10.01

PMD-I vs RB-I1 p <0.001

PMD-I1 vs AND-I p ~0.005

PMD-I1 vs AND-I1 p = NS

PMD-I1 vs CDB p <0.001

PMD-I1 vs RB-I p = NS

PMD-I1 vs RB-I1 p = NS

AND-I vs AND-I1 p = NS

AND-I vs CDB p <0.05

AND-I vs RB-I p <0.001

AND-I vs RB-I1 p <0.001

AND-I1 vs CDB p <0.001

AND-I1 vs RB-I p NS

AND-I1 vs RB-I1 p 4.005

CDB vs RB-I p <0.001

CDB vs RB-I1 p <0.001

RB-I vs RB-I1 p = NS

* 2.

UN vs RB-I1 p = NS

132

ANN M. DVORAK

FIG. 10 Relationships of numbers of granules and vesicles, the percentage of altered granules, and the percentage of vesicles that are labeled with gold for CLC protein localization are shown in samples of human basophils stimulated with FMLP and examined at multiple times thereafter. The individual phenotype composition of basophils comprising each sample is also indicated.

value for %VG/V occurred in the kinetic analysis (29%). The highest value for gold-labeled vesicles in the CDB phenotype, which has completed secretion of all cytoplasmic granules and has a high percentage of total cellular gold associated with the plasma membrane (13.8%)(Dvorak et al., 1997a), indicates that endocytosis of previously released CLC protein is the source of this label, a process prevalent in the 1-min sample (9% total cellular label for CLC protein is in the plasma membrane compartment). Other relationships for the %VG/V show that the PMD-I phenotype has significantly more labeled vesicles (14%) than do unstimulated cells (l%,p <0.001). Also, the phenotypes PMD-I1(7%), AND-I (20%),AND-I1 (12%), CDB (37%), and recovering basophil (RB-I) (7%) have gold-labeled vesicles in excess of those in unstimulated cells (l%,p c0.005, p <0.001), indicating the active trafficking of CLC protein in FMLP-stimulated basophi1 phenotypes. The %VG/V in the phenotype RB-I1 returned to unstimulated levels ( p = NS). p values for comparisons of differences among stimulated phenotypes are listed in Table IV.

133

CELL BIOLOGY OF THE BASOPHIL

I

1

Vesicles

CLC Prot. ~ o sVesicles .

I

FIG. 11 Histograms of unstimulated and FMLP-stimulated human basophils showing the relationships of the average number of cytoplasmic vesicleslpm’ (top) and the percentage of these vesicles that are labeled with gold, indicating the presence of CLC protein (bottom), for individual phenotypes, compared to unstimulated cells (*p <0.001; **p <0.005;*p ~ 0 . 0 5 ) (with permission of Blackwell Science Ltd., from Dvorak et al., 1997a).

c How Many Vesicles Are Empty (% EV/V)? We counted the number of electron-lucent, smooth membrane-bound cytoplasmic vesicles (Fig. 8) in unstimulated basophils and in FMLP-stimulated basophils and compared these values by stimulated phenotypes and times after stimulation (Fig. 12). Histograms of these values for the kinetic analysis (Fig. 12C) and the phenotypic analysis (Fig. 12M) show that the highest percentage of EV occurred in unstimulated basophils (92%). Of the FMLP-stimulated samples, the 0 time point was the closest to unstimulated cells, with 87% EVs ( p = NS). Comparisons of the number of EVs in stimulated samples by time to unstimulated cells showed a significant reduction in EVs at 10 s (48%), 20 s (67%), 30 s (67%), 1 min (71%), 2 min (49%), 5 min (50%),and 10 min (37%) (all p <0.001) after FMLP. Of the kinetic samples, the smallest number of EVs occurred at 10 rnin (37%), a value significantly smaller than that in unstimulated cells (92%, p <0.001), at 0 time after stimulation (87%,

134

ANN M. DVORAK

Kinetic Analvsis

Phenotvoe Analvsis

FIG. 12 Histograms of human basophil cytoplasmic vesicles analyzed by kinetics (A-J) and phenotypes (K-T) stimulated by FMLP. (A,K) Number of vesicles/ym2; (B,L) percentage gold-labeled vesicle/vesicles (%VG/V); (C,M) percentage empty vesicles of total vesicles (% EVIV); (D,N) percentage full vesicles of total vesicles (%FV/V); (E,O) percentage goldlabeled empty vesicles of total vesicles (%EVG/V); (F,P) percentage gold-labeled full vesicles of total vesicles (%FVG/V); (G,Q) percentage gold-labeled empty vesicles of total goldlabeled vesicles (%EVG/VG); (H,R) percentage gold-labeled full vesicles of total gold-labeled vesicles (%FVG/VG); (1,s) percentage gold-labeled empty vesicles of total empty vesicles (%EFG/EV); (J,T) percentage gold-labeled full vesicles of total full vesicles (%FVG/FV). (A-J) Unstimulated and times 0, 10, 20, and 30 s and 1, 2, 5, and 10 min for each histogram (A-J, with permission of Karger, Basel, from Dvorak et al., 1997b). (K-T) Unstimulated and PDM-I, PDM-11, AND-I, AND-11, CDB, RB-I, and RB-I1 phenotypes for each histogram.

p <0.001) and at 20 s (67%, p <0.001), 30 s (67%, p <0.001), 1 min (71%, p <0.001), 2 min (49%, p <0.025), and 5 min (50%, p c0.05) after FMLP, thus characterizing the 10-min sample as one with far fewer EVs than all previous samples. Examination of the %EV/V for individual stimulated basophil phenotypes revealed that all phenotypes had significantly diminished numbers of EVs compared to those in unstimulated cells ( p <0.001). p values for interphenotype comparisons showed that the RB-I1 phenotype had significantly smaller numbers of EVs (41%) than those in the PMD-I (60%, p <0.001), AND-I (62%, p <0.001), AND-I1 (60%, p <0.005), CDB (63%, p <0.01), and RB-I (53%,p (0.01) phenotypes. a! How Many Vesicles Are Full (%FV/V)? Smooth membrane-bound vesicles, filled with either electron-dense particles or homogeneous material

CELL BIOLOGY OF THE BASOPHIL

135

(Fig. 9), were quantitated for unstimulated cells and FMLP-stimulated basophils and related to time after stimulation or to individual phenotypes (Fig. 12). Histograms of these values are presented for time analysis in Fig. 12D and phenotypic analysis in Fig, 12N. The %FV/V did not differ between the unstimulated cells and the 0 time point ( p = NS). All of the kinetic samples studied thereafter showed significant increases in the %FV/V ( p <0.001) compared to those in unstimulated cells. Similarly significant increases were recorded for all times after FMLP compared to that at the 0 time ( p <0.001). The highest value for %FV/V in stimulated samples occurred at 10 min. This value was significantly greater than that in the 20and 30-s (33%, p <0.001), l-min (29%, p <0.001), 2-min (51%, p <0.025), and 5-min (50%, p <0.05) samples, thus characterizing the 10-min sample as one with the largest number of full vesicles among the individual time points examined. The second highest value for %FV/V occurred at 10 s (52%) after FMLP stimulation, a stimulation time which contained only basophils undergoing PMD as determined by the phenotypes present. This suggests that at 10 s the full vesicles are primarily transporting materials out of cells as an integral part of the secretory process preceding the granule extrusion phase. The phenotypic analysis of %FV/V showed that all stimulated phenotypes had more FVs than were present in unstimulated cells ( p <0.001). Interphenotype comparisons showed that the RB-11 phenotype differed significantly in the %FV/V (59%) from that found in the PMD-I (40%,p <0.001), AND-I (38%, p <0.001), AND-I1 (40%, p <0.005), CDB (37%, p <0.01), and RB-I (47%, p <0.01) phenotypes. The RB-I1 phenotype comprised 50% of the 10-min sample showing the correlation for these two analyses for the presence of FVs in basophils after FMLP stimulation.

e. How Many Vesicles Are Empty and Gold-Labeled (%EVG/V)? We determined the percentage of cytoplasmic vesicles that were empty (electron-lucent) and gold-labeled for the presence of CLC protein in unstimulated cells for comparison with these values in stimulated phenotypes and at various times after FMLP stimulation (Fig. 12). Histograms for these values for individual times (Fig. 12E) and phenotypes (Fig. 120) show that electron-lucent (empty) vesicles labeled with gold exceeded unstimulated cell values (1%)and 0 time (1%)in the 10-s (6%, p <0.005), 20-s (16%, p <0.001), 30-s (8%, p <0.001), l-min (26%, p <0.001), and 2-min (9%, p <0.001) samples. Differences in the %EVG/V between unstimulated cells and the O-time, 5-, or 10-min samples did not exist ( p = NS). Thus, the kinetic analysis clearly shows transport of CLC protein occurring in the EV population between the 10-sand the 2-min time points. Other significant inter-time point comparisons were also noted in the %EVG/V. For example, all times evaluated from 10 s to 2 min had an elevated %EVG/V compared

136

ANN M. DVORAK

to that at the 0 time ( p <0.005 for 10 s; p <0.001 for the remaining times) and the %EVG/V was substantially elevated in the 20-s sample (16%, p cO.01) and 1-min sample (26%, p <0.001) compared to that for the 10-s sample (6%); the 10-min sample had no EVG ( p CO.01 compared to that for the 10-s sample; p <0.001 compared to that for the 20-s, 30-s, 1-min, and 2-min samples; p <0.05 compared to that for the 5-min sample). Thus, transport of CLC protein in EVs occurred between 10 s and 5 min after FMLP and was absent at 10-min. The %EVG/V in FMLP-stimulated phenotypes (Fig. 1 2 0 ) shows that significant amounts of the gold label are being transported in EVs in all phenotypes except the RB-I1 phenotype, which comprised 50% of the 10min sample. The unstimulated sample (1%) showed significantly less gold in the EV portion of vesicles than in the PMD-I (12%, p <0.001), PMDI1 (6%, p <0.01), AND-I (16%, p <0.001), AND-I1 (9%,p <0.001), CDB (37%, p <0.001), and RB-I (4%, p <0.05) phenotypes. Significant interphenotype comparisons were also present. For example, the PMD-I early secreting phenotype had a smaller %EVG/V (12%) than did the CDB phenotype (37%, p <0.001), which is composed of cells having just completed secretion, thus adding support to the inward direction of vesicular traffic in the CDB phenotype. The %EVG/Vin PMD-I (12%) significantly exceeded that in the recovering phenotypes RB-I (4%, p <0.001) and RBI1 (l%,p<0.001). The secretory phenotypes AND-I (16% EVG/V,p <0.01) and AND-I1 (9% EVG/V, p <0.001) had a smaller proportion of goldlabeled EVs than did the CDB phenotype (37% EVG/V). Both of the AND phenotypes were primarily secreting by granule extrusion (not by vesicular transport), and these values support this morphology as well as substantiate the inward traffic of EVG in the CDB phenotype. The %EVG/ V in the CDB phenotype (37%) significantly exceeded that of both RB-I (4%) and RB-I1 (1%)phenotypes ( p <0.001).

f. How Many Vesicles Are Full and Gold-Labeled (%FVG/V)? The percentage of cytoplasmic vesicles that were full and gold-labeled, indicating the presence of CLC protein, was determined for unstimulated cells for comparison with the kinetic samples and individual basophil phenotypes stimulated by FMLP (Fig. 12). Histograms of these values for individual times (Fig. 12F) and phenotypes (Fig. 12P) show that vesicles containing electron-dense particles or homogeneous material did not contain gold label until 20 s following FMLP (4%, p <0.005 compared to that for unstimulated cells). Peak values for %FVG/V were obtained at 2 min (6%, p <0.001 compared to that for unstimulated cells). The %FVG/V was also significantly elevated compared to that for unstimulated cells at 1min after FMLP (3%, p <0.01). The comparisons for %FVG/Vwith the 0 time point (0%) after FMLP showed that significant elevations of FVG also occurred at

CELL BIOLOGY OF THE BASOPHIL

137

20 s (6%, p <0.005), 1 min (3%, p <0.025), and 2 rnin (6%, p <0.001). The %FVG/V in the 20-s sample (4%) was also significantly elevated compared to that at 10 s (O%, p <0.005), as was that for the 1-min (3%, p <0.025) and the 2-min samples (6%, p <0.001). The highest value for %FVG/V, occurring in the 2-min sample (6%), significantly exceeded that in the 10-min sample (l%, p c0.025); the 10-min sample did not differ from unstimulated cells in the %FVG/V ( p = NS). FVGs were elevated in several phenotypes stimulated by FMLP (Fig. 12P). Compared to unstimulated cells, which did not have any FVG (O%), the PMD-I (2%, p <0.025), AND-I (3%, p <0.01), AND-I1 (3%, p <0.025), RB-I (3%, p <0.01), and RB-I1 (2%, p <0.05) all had significant numbers of FVs transporting CLC protein; the PMD-I1 phenotype did not ( p = NS). Also, no FVGs were present in the CDB phenotype (O%), which did, however, contain the greatest number of EVG (37%) of all phenotypes. g. How Many Gold-Labeled Vesicles Are Empty (%EVG/VG)? We

counted the number of gold-labeled vesicles and determined the proportion that were also electron-lucent, or empty (EVG), in unstimulated cells, and at variable times after stimulation with FMLP and in individual phenotypes stimulated by FMLP (Fig. 12). Histograms of these data for the kinetic analysis (Fig. 12G) and the phenotypic analysis (Fig. 12Q) were of interest. The gold-labeled vesicles in unstimulated cells were all EVs (loo%), and none were FVs (see later). The majority of VGs remained EVGs in kinetic samples until 10 rnin after stimulation, where the value for the percentage EVG/VG was 0%. Statistically significant differences among the individual times examined after FMLP were present between 10 s (100%) and 2 min (62%, p <0.025), 10 s and 10 rnin (O%, p <0.001), 30 s (89%) and 10 min (O%, p <0.05), and 1 rnin (89%) and 10 rnin (O%, p C0.025). In the phenotypic analysis, differences in the percentage EVGlVG revealed that significance was achieved when several phenotypes were compared. For example, the proportion of VG that were EV in the PMD-I phenotype (83%) exceeded that in the RB-I (55%, p C0.025) and RB-I1 (38%, p (0.025) phenotypes. The PMD-I1 phenotype (88%)also contained more EVG than the RB-I1 phenotype (38%, p (0.05). The AND-I phenotype (83%) exceeded the RB-I (55%, p C0.05) and RB-I1(38%, p (0.025) phenotypes. AND-I1 cells (75%) contained a smaller proportion of EVG/ VG than did CDBs (loo%, p <0.05), whereas CDBs exceeded those in the RB-I (55%, p <0.005) and RB-11 (38%, p <0.001) phenotypes. The percentages of EVG/VG in the two RB phenotypes were not significantly different ( p = NS). h. How Many Gold-Labeled Vesicles Are Full (%FVGIVG)? We also counted the number of gold-labeled vesicles and determined the proportion

138

ANN

M. DVORAK

that were filled with electron-dense particles or homogeneous material (FVG) at variable times after FMLP stimulation and in individual stimulated phenotypes (Fig. 12). Histograms for these data are in Fig. 12H (kinetics) and Fig. 12R (phenotypes). The proportion of gold-labeled vesicles that were FVs varied from 0 in unstimulated cells to 100% at 10 min. Intertime comparison for significance showed that at 10 s (O%), the proportion of VG that were FVG was less than that at 2 min (38%, p <0.025) and that at 10 min (loo%, p <0.001); the 30-s (11%)and 1-min (11%) samples were also less than that at 10 min (100%) ( p
CELL BIOLOGY OF THE BASOPHIL

139

these cells had released all of their granules and had a large percentage of the total cellular label (Dvorak et al., 1997a) bound to the plasma membrane, the direction of traffic for CLC protein in this phenotype was likely inward in endocytotic vesicles. The major releasing phenotypes, PMD-I, AND-I, and AND-11, also had an elevated %EVG/EV, participating in the secretion of CLC protein ( p <0.001 compared to unstimulated cells); the recovering phenotypes, RB-I and RB-11, had a reduced percentage of EVG/ EV compared to that of releasing phenotypes. Statistical analysis of the EVG content in phenotypes showed that all phenotypes had more EVG than unstimulated cells ( p <0.001; p <0.005 for RB-I) except RB-IT cells ( p = NS). Interphenotype comparisons showed that the EVG fraction of EVs in PMD-I (19%) was significantly less than that in CDB (58%, p <0.001), despite the fact that the CDB cells had a smaller number of vesicles for transport than were present in the PMD-I phenotype, suggesting that reuptake of released CLC protein was more efficient in the CDB phenotype than release was from PMD-I cells. The percentage EVG/EV was substantially greater in PMD-I cells (19%) than in RB-I (7%, p <0.001) or RB-I1(3%, p <0.001). The PMD-I1 phenotype, a phenotype with unbalanced vesicular traffic leading to markedly enlarged granule chambers, had a significantly smaller (12%) proportion of EVG/EV than did AND-I cells (26%, p <0.05), which were actively forming degranulation channels and rapidly releasing granule materials. The enhanced vesicular release of CLC protein in the AND-I phenotype supports the degranulation continuum PMD + AND previously postulated (Dvorak and Dvorak, 1975). PMDI1 cells had substantially more EVG/EV (12%) than did the RB-I1 phenotype (3%, p ~ 0 . 0 5 )Of . the actively releasing phenotypes, AND-I had 26% EVG/EV, a value still smaller than that in CDBs (58%, p <0.01) but larger than that in recovering phenotypes (7%, 3%, p <0.001). AND-I1 cells, also an actively secreting phenotype, had fewer EVG/EV (15%) than did CDBs (58%, p <0.001) but a greater number than did RB-I1 cells (3%, p <0.01). CDB cells exceeded both recovery phenotypes for the %EVG/EV ( p
140

ANN M. DVORAK

content of stimulated basophils were noted between the 10-s sample (0) and the 20-s, 1-min, and 2-min samples ( l l % , p <0.001). Two of these times (20 s, 1 min) had releasing phenotypes dominating the samples (Fig. 10). The 20-s (11%) and 1-min (11%) samples were significantly richer in FVG than the 10-min sample (l%, p <0.01 and p <0.025), a sample dominated by the RB-I1 recovering phenotype (50%).The 2-min sample, containing a mixture of releasing and recovering phenotypes (Fig. 10) (ll%), also exceeded the 10-min sample (l%, p <0.01). The proportion of FVs that were transporting CLC protein (FVG) was calculated for individual phenotypes stimulated by FMLP (Fig. 12T). Statistically significant differences in the FVG content of several phenotypes included a significantly greater percentage of FVG/FV in the actively granule-extruding phenotype, AND-I1 (7%), than that in the recovering phenotype, RB-I1(4%, p c0.025). The AND-11 phenotype (7%) also exhibited significantly more FVG/FV than was found in the PMD-I1 phenotype (2%, p <0.025), a phenotype exhibiting unbalanced vesicular traffic, with inward flow exceeding outward flow, and with retention of expanded granule containers. This ultrastructural analysis of FMLP-stimulated human basophils allowed us to document considerable variations in basophil cytoplasmic vesicles. The greatest number existed at 0 time after FMLP (3.02/pm2)-viz., at the very onset of secretion-and the smallest number existed in the CDB phenotype (0.3/pm2)-viz., at the very completion of secretion. We wondered how many vesicles of the total population were empty (EV) and how many were full (FV); we discovered that all stimulated time points between 10 s and 10 min had fewer EVs than unstimulated cells, with the smallest amount of EVs at 10 min (37%). All of the activated phenotypes also displayed a smaller proportion of EVs than that in unstimulated cells. Full vesicles were significantly increased over unstimulated cells at all stimulated times, peaking at 10 min (63%). A second peak of FVs occurred at 10 s, a sample with >50% of cells showing the PMD-I phenotype (Fig. 10). This suggests that these FVs are secretory carriers in the PMD-I phenotype (see later), which has many altered granules but does not have any evidence of secretion by granule extrusion. All stimulated phenotypes had more FVs than did unstimulated cells, and the number in the RB-I1 phenotype significantly exceeded that of all other phenotypes. The 10-min sample had 50% RB-I1 cells, which showed no morphologic evidence of secretion and had a full complement of unaltered cytoplasmic granules. The FVs in this phenotype, generally occurring 10 min after stimulation, are likely to be primarily recycling CLC protein into the cell by endocytosis from the plasma membrane rather than secreting it. We also analyzed gold-labeled vesicles, indicating the presence of CLC protein, in FMLP-stimulated basophils. Initially, we wondered how many

CELL BIOLOGY OF THE BASOPHIL

141

of the total vesicle population were carrying CLC protein. This analysis showed that, by 10 s after stimulation and until 2 min, the % V G N was significantly elevated, compared to that of unstimulated cells. All phenotypes (except RB-11) also had elevated vesicles transporting CLC protein. By 5 and 10 min (in the RB-I1 phenotype, as well), values of %VG/V had decreased and were equivalent to those of unstimulated cells. The largest %VG/V occurred at 1min in the kinetic analysis and in the CDB phenotype in the phenotypic analysis, a phenotype which comprised 40% of the 1-min sample. Thus, although the CDB phenotype had the smallest number of cytoplasmic vesicles (0.3/pm2), it also displayed the largest proportion of available vesicles to be carrying CLC protein. The CDB phenotype was typified by the virtual absence of granules (and, thus, granule stores of CLC protein) and positive plasma membrane-bound CLC protein (Dvorak et al., 1997a). Therefore, in this phenotype and at this time after stimulation (1 min), the direction of vesicular transport of CLC protein is likely into cells by endocytosis of previously released plasma membrane stores of CLC protein. The histograms in Figs. 12E-J and 120-T show additional important relationships of gold loading (indicating the presence of CLC protein) of carrier vesicles in basophils stimulated by FMLP. For example, we wondered what proportion of cytoplasmic vesicles are EVs (or FVs) and are carrying CLC protein (%EVG/V; %FVG/V) in timed samples and in stimulated phenotypes. This analysis revealed that CLC protein is present in EVs in significant amounts between 10 s and 2 min after FMLP. These values were also significantly greater than the %EVG/V at 0 time, despite the fact that the greatest proportion of cytoplasmic vesicles were EVs in the 0-time sample. Therefore, the initial burst of EVs seen at 0 time was composed of EVs that were not transporting CLC protein, suggesting [as postulated in the general degranulation model (Dvorak and Dvorak, 1975)] that these EVs are primarily inward-traveling vesicles generated from the plasma membrane in response to the FMLP trigger. These endocytotic, unlabeled vesicles could provide membrane and solvent to granules, as they attach to them, thereby causing expansion and alteration of granule membranes and their contents. As the number of EVs dropped, the proportion carrying CLC protein progressively increased over time, to peak at 1 min, and then progressively fell to 0% at 10 min. EVGs were increased in all phenotypes over those in unstimulated cells except in the RB-I1 phenotype, which comprised 50% of the 10-min sample (Fig. 10).The granule-free CDB phenotype had the greatest proportion of EVG/V, significantly exceeding secretory and recovering phenotypes, thus characterizing the CDB phenotype as the most avid recycling phenotype for released CLC protein. Analysis of the %FVG/V by time after FMLP stimulus showed a lag, compared to the %EVG/V, until 20 s, when significant increases over those

142

ANN M. DVORAK

in unstimulated cells were recorded. Peak values occurred at 2 min, and by 10 min, the %FVG/V did not differ from unstimulated cell values. The analysis of %FVG/V in stimulated phenotypes shows that the releasing and recovering phenotypes all exceeded that in unstimulated cells, but that the PMD-I1 and CDB phenotypes did not. None of the gold-labeled vesicles in the granule-free CDB phenotype were FVs, adding support to the interpretation that FVs are releasing granule materials in the secretory phenotypes. We next questioned what proportion of the gold-labeled vesicles (VG) were empty or full. These data showed that most of the gold-labeled vesicles remained EVs until 10 min after FMLP, where all of them were FVs. And, the %FVG/VG increased as time passed in the kinetic continuum. Therefore, CLC protein is clearly transported in both EVs and FVs, but the predominant labeled vesicle fraction changes with time. Similarly, the %FVG/VG progressively increased across the phenotype spectrum, with the exception of PMD-I1 and CDB cells, phenotypes more representative of endocytotic cells than of secretory cells. When the proportion of EVs (or FVs) that was labeled for CLC protein was calculated (%EVG/EV; %FVG/FV), a progressive increase in the %EVG/EV occurred over time (except at 30 s) to a maximum at 1 min. The labeled proportion of EVs then dropped to unstimulated levels by 10 min. Thus, the E V population clearly showed transport of CLC protein until 1 min after FMLP. Better than half of the EVs in CDB cells (cells with high plasma membrane label for CLC protein and no granules) were labeled and were, thus, transporting CLC protein back into the cell. The efficiency of inward traffic for the CDB phenotype (58% EVG/EV) was much greater than that of the PMD-I cellular secretory traffic (19% EVG/ EV), and the efficiency of uptake for CDB cells was much greater than that in the recovering phenotypes (RB-I, 7%; RB-11, 3%). The proportion of FVs that was labeled for gold (%FVG/FV) showed significant elevations over that in unstimulated cells and 0 time samples at 20 s, 1 min, and 2 min. The 20-s and 1-min samples are dominated by secretory phenotypes, and the number of CLC protein-positive FVs at 20 s and 1 min exceeded those in the 10-min sample. The secretory phenotype AND-I1 had a significantly larger proportion of FVG/FV than both the final recovered phenotype (RB-11) and the PMD-I1 phenotype. The latter phenotype is composed of cells exhibiting unbalanced vesicular traffic, with “in” > “out,” thereby producing massive expansion of granule chambers.

2. TPA Stimulation Small, smooth membrane-bound cytoplasmic vesicles that contained either electron-lucent interiors or electron-dense particles were labeled with gold,

CELL BIOLOGY OF THE BASOPHIL

143

which indicated the presence of CLC protein, at various times after the stimulation of human basophils with TPA (Fig. 13) (Dvorak et al., 1996a). The total number of cytoplasmic vesicles/pm* and the number of vesicles/ pm2 that were gold-labeled were obtained for unstimulated cells and for each time point after TPA stimulation. When the percentage of total vesicles

FIG. 13 Human basophils stimulated with TPA for 1 rnin (A), 2 min (B), 5 min (C), 10 rnin (D,E), and 15 rnin (F) show CLC protein-gold-labeled vesicles in the cytoplasm (arrows). Some vesicles are electron-lucent; others contain particles similar to the particulate matrix of adjacent granules. CLC protein is also localized by gold particles in the nuclear matrix and nuclear membrane (C), the cytosol (A-F), the plasma membrane (A,E), a homogeneously dense primary granule (D), a formed CLC within the particulate matrix of a granule (E), and the granule membrane of an empty granule chamber (F). The arrowhead in D shows an example of the small minor granule population of human basophils that does not contain label for CLC protein (with permission, from Dvorak ef al., 1996a). Bars: (A) 150 nm; (B) 160 nm; (C,D) 240 nm; (E) 230 nm; (F) 120 nm.

144

ANN M. DVORAK

that were gold-labeled (e.g., carrying CLC protein) was calculated and compared (Fig. 14), the fraction of total vesicles that was carrying CLC protein was significantly increased at 2 rnin (27.6% vs 0.9%, p <0.05), 5 min (31% vs 0.9%,p <0.01), and 10 rnin compared with that of unstimulated cells (37% vs 0.9%, p <0.001). In addition, vesicles carrying CLC protein at 10 rnin (37%) exceeded those at zero stimulation time (6.3%, p ~ 0 . 0 1 ) as well as those at 30 rnin (19%,p <0.05) and 45 rnin (9.5%, p <0.05) after TPA stimulation. These experiments quantitatively show that the number of CLC protein-loaded vesicles changes in a kinetic sequence in TPAstimulated human basophils. We conclude that the extensive PMD of basophils, which is characterized by virtually empty cytoplasmic granule containers that no longer contain CLC protein present at 45 rnin after TPA stimulation, likely results from the vesicular translocation of granule CLC protein. These findings support a role for the vesicular transport of granule materials during the development and persistence of PMD in human basophils.

E. Histamine-Loaded Human Basophil Transport Vesicles 1. FMLP Stimulation The relationship of cytoplasmic vesicles and their contents to the complex secretory response stimulated in human basophils by FMLP was examined using a morphometric analysis of DAO-gold-labeled cells. We calculated

I

UN

0

1

2

5

10

15

30

45

Minutes FIG. 14 Percentage of total cytoplasmic vesicles that are labeled with gold, which indicates the presence of the CLC protein in unstimulated human basophils and in human basophils stimulated with TPA (0 to 45 min). * p <0.05; **p <0.01; ***p <0.001 compared with unstimulated cells (with permission, from Dvorak et al., 1996a).

145

CELL BIOLOGY OF THE BASOPHIL

the average cell area, total and average numbers of cytoplasmic vesicles, total and average numbers of gold-labeled (histamine-carrying) vesicles, and the numbers of total and gold-labeled vesicles/pm2 for unstimulated, buffer-incubated cells (20 s, 1 min, and 10 min) as well as for FMLPstimulated cells collected at 0 time and at 10, 20, and 30 s and 1, 2, 5 , and 10 min thereafter. From these data, the fraction of available total cellular vesicles carrying histamine (gold-labeled) was determined (Fig. 15). The percentage of VG/V/pm2 was significantly elevated in all FMLPstimulated samples compared to that of unstimulated cells (p < 0.001) (Fig. 15). Thus, the vesicles carrying histamine in FMLP-stimulated cells increased and remained elevated during the same time frame in which histamine release can be measured (Warner et al., 1989) and in which anatomic recovery of histamine-secreting cells takes place (Dvorak et al., 1991a). Intersample comparisons showed that the 20-s (p <0.001), 1-min (p <0.025), and 5-min (p <0.001) samples all exceeded the 0 time point of stimulated cells; the 20-s (p <0.001), 30-s (p <0.005), and 1-,2-, 5-, and 10-min (p <0.001) samples all exceeded the 10-s sample; the 1-rnin (p <0.001), 2-min (p <0.005), and 5-min 0, <0.001) samples exceeded the 30-s sample; and the 5-min (p <0.025) sample exceeded the 2-min sample. Thus, progressive significant increases in histamine-carrying vesicles occurred in FMLP-stimulated

80

FMLP

60-

?

P 8

Seconds

*

Minutes

p < .001

FIG. 15 Percentage of total cytoplasmic vesicles that are labeled with gold indicating the presence of histamine in unstimulated human basophils and in human hasophils stimulated with FMLP (0 to 10 min). * p < 0.001 compared to unstimulated cells (with permission, from Dvorak et al., 19960.

FIG. 16 FMLP-stimulated human basophils prepared with DAO-gold to detect histamine (A-C,E,F,H-L) or with specificity controls (D,G) at 0 time (A-D), 10 s (E-G), 20 s (H), 30 s (I), 1 min (J,K), and 10 min (L) after activation. The open arrowhead in all panels indicates the cell surface; G, granule; EG, empty granule (typical for PMD); and N, nucleus. A 0 time and in the same activated cell, both empty, electron-lucent (arrow in A) and full, electron-dense (arrow in B) vesicles are gold-labeled. Cytoplasmic glycogen particles are electron-dense (A). In C, a nearly empty granule (typical for PDM) retains some DAO-gold label. One cytoplasmic vesicle is also labeled. Note the vesicle attached to the empty granule. Electron-dense glycogen particles (arrows) are attached to the narrowed neck of the fused vesicle. The inset in C shows a free, electron-lucent vesicle, labeled with gold for histamine (arrow), encased in surrounding electron-dense glycogen aggregates, present in the cytoplasm of the same basophil. The specificity control in D (DAO-gold absorbed with histamineagarose before staining) is negative for histamine in the large granule and adjacent vesicles.

CELL BIOLOGY OF THE BASOPHIL

147 cells concomitant with the development of the anatomic secretory continuum PMD -+AND -+ recovery. Recovery mechanisms for human basophils following FMLP stimulation include vesicular trafficking of synthetic and endocytotic materials (Dvorak et aL, 1995a, 1996b). Ultrastructural identification of histamine-labeled cytoplasmic vesicles was done using the DAO-gold technique (Fig. 16). Gold-labeled vesicles were present in all samples of FMLP-stimulated cells. In addition to the presence of these smooth membrane-bound, gold-labeled vesicles in the perigranular and peripheral cytoplasmic areas, and in contrast to TPAactivated cells, some were also present in the cytoplasm containing Golgi structures. Some gold-labeled vesicles rested beneath the plasma membrane, through which granules had been extruded during the AND phase of the degranulation continuum (Fig. 16K). The FMLP-stimulated samples contained particle-filled granules that labeled for histamine (Figs. 16E and 16F); these samples also contained granules (partially and completely devoid of electron-dense materials) with reduced or absent histamine stores (Figs. 16C and 16F). Specificity controls for the DAO-gold technique in these samples revealed abrogation of granule and vesicle label when the DAO-gold reagent was absorbed by histamine beads prior to staining or when DAO digestion of sections was done before DAO-gold staining (Figs. 16D and 16G). Cytoplasmic vesicles that were attached by fusion to large secretory granules were evident in the 0-time F'MLP-stimulated sample (Fig. 16C). These fused vesicles were attached to virtually empty granules with reduced gold label (Fig. 16C) as well as to granules with particles and gold-labeled

Electron-dense glycogen particles (arrows) remain visible. In E, at 10 s, three subcellular sites are labeled with DAO-gold as follows: (i) granule matrix (G) but not the intragranular CLC (ii) cytoplasmic, electron-lucent, perigranular vesicle (arrow); (iii) polyamines in the nucleus (N) (Dvorak et al., 1993b). A vesicle is attached to the histamine-labeled granule (arrowhead). In F, also at 10 s, DAO-gold extensively labels the granule matrix (G) of the full granule and shows less label in the empty granule (EG) typical for PMD. The specificity control in G (digestion of the section with D A O before staining with DAO-gold) is negative for histamine in the large granules (G), adjacent perigranular vesicles, and nucleus (N). Electron-dense glycogen particles (arrow) remain visible in the cytoplasm. Granule particle-filled, DAO-goldlabeled vesicles rest just beneath the plasma membrane (open arrowheads) at 20 s (H) and 30 s ( I ) poststimulus. In J, at 1min, similar particle-filled cytoplasmic vesicles contain histamine. Also, at 1 min after stimulation (K), extrusion of granule particles to the cell surface is visible (open arrowhead). The underlying cytoplasm contains gold-labeled electron-lucent vesicles (arrows). In L, at 10 min, DAO-gold is attached to the cell surface (open arrowhead), within underlying electron-lucent and granule particle-filled vesicles, and to an adjacent recovered granule (G) (with permission, from Dvorak et al., 19960. Bars: (A) 150 nm; (B) 100 nm; (C) 170 nm; (D) 360 nm; (E) 200 nm; (F) 185 nm; (G) 370 nm; (H-K) 87 nm; (L) 130 nm.

148

ANN

M. DVORAK

histamine (Fig. 16E). Some cytoplasmic vesicles containing gold label for histamine were surrounded by glycogen aggregates (Fig. 16C, inset). The principal findings and interpretations for the DAO-gold labeling of cytoplasmic vesicles in secreting human basophils stimulated by FMLP are as follows; (i) histamine-labeled vesicles in all stimulated samples exceeded those in unstimulated samples (thus, histamine is transported in vesicles in cells releasing histamine and undergoing PMD -+ AND -+ recovery); (ii) comparisons of timed samples generally revealed increases in later samples compared to earlier samples. For example, all samples between 20 s and 10 min exceeded the 10-s value. The increased %VGN indicating histamine transport was pirmarily accompanied by the morphology of PMD at early times (10 s-1 mh), indicating stimulated secretory transport of vesicle-packagedhistamine. At later times (2-10 min), the increased %VGNwas associatedwith the morphology of recovery of granule contents,indicatingtransport of vesicle-packagedhistamine from two possible sources (endocytosis of released histamine, synthesis in Go@ structures) as mechanisms for effecting granule content reconstitution.

2 TPA Stimulation We used a similar morphometric analysis of DAO-gold-labeled, TPAstimulated cells to quantitate vesicular transport of histamine in TPAstimulated human basophils. We calculated the average cell area, total number and average number of cytoplasmic vesicles, total number and average number of gold-labeled (histamine-carrying) vesicles, and the number of total vesicles/pm’ and of gold-labeled vesicleslpm’ for unstimulated, buffer-incubated cells (20 s, 1min, and 10 min) as well as for TPS-stimulated cells collected at 0 time and 1 ,2 ,5 , 10, 15, 30, and 45 min thereafter. From these data, the fraction of available total cellular vesicles carrying histamine (gold-labeled) was determined (Fig. 17). The percentage of VGlV was similar in unstimulated cells and in TPAstimulated cells obtained at time 0 (p = NS). At 1 rnin stimulation with TPA, 30% of the available vesicles were gold-labeled (JJ (0.025 compared to unstimulated cells, 22%); at 2 min, 29% were gold-labeled (p <0.05, compared to unstimulated cells); at 10 min, 45%; at 15 min, 56%; at 30 rnin, 39%; and at 45 min, 55%-values all significantly greater than those of unstimulated cells (p <0.001). Thus, the vesicles carrying histamine in TPAstimulated cells increased and remained elevated in the same time frame during which histamine release was measured (79.1%; 66.2% at 60 min in each of two experiments after TPA stimulation). Intersample comparisons showed that all stimulated samples from 10 min on had a significantly greater %VG/V than that at 0 time (p
149

CELL BIOLOGY OF THE BASOPHIL

60-

un

I

0

1

2

5

10 15 30 45. J

Minutes

* p < ,025

**

p < .05

m p < .001

FIG.17 Percentage of total cytoplasmic vesicles that are labeled with gold indicating the presence of histamine in unstimulated human basophils and in human basophils stimulated with TPA (0 to 45 min). *p <0.025; **p c0.05; ***p <0.001 compared to unstimulated cells (with permission, from Dvorak et af., 1996f).

(p <0.001) samples exceeded the 2-min sample; all samples from 10 min on (p <0.001) exceeded the 5-min sample; the 15-min (p c0.025) and 45-min (p c0.05) samples exceeded the 10-rnin sample; and the 45-min (p ~ 0 . 0 1 )sample exceeded the 30-min sample. Thus, progressive significant increases in histamine-carrying vesicles occurred in TPA-stimulated cells concomitant with the development and persistence of PMD without evidence of recovery of basophils either by synthetic or endocytotic mechanisms or by a mixture of these processes (Dvorak et al., 1995a, 1996b). Ultrastructural identification of histamine-labeled cytoplasmic vesicles was possible using the DAO-gold enzyme-affinity technique (Fig. 18). At all times following exposure to TPA, it was possible to identify gold-loaded cytoplasmic vesicles primarily in the perigranular and peripheral cytoplasmic areas. These vesicles were bounded by smooth membranes and either displayed electron-lucent interiors or contained electron-dense particles analogous to the particles filling the much larger secretory granules. Some gold-labeled electron-lucent vesicles were surrounded by masses of glycogen (Fig. 18B). Specificity controls showed abrogation of label in vesicles and granules (data not shown). Rarely, fusion of gold-labeled vesicles with the plasma membrane was visible (Fig. 18G). Gold-loaded, peri-

FIG. 18 TPA-stimulated human basophils prepared with DAO-gold to detect histamine studied at 0 time (A), 2 min (B-D), 5 rnin (E-G), 10 min (HJ), 15 min (J), 30 min (K), and 45 min (L) after activation. In A, at 0 time, the basophil granule (G) is labeled with DAO-gold. In B, at 2 min, three cytoplasmic vesicles near the cell surface (open arrowhead) are labeled for histamine. One gold-labeled vesicle is electron-lucent and is encased in eletron-dense glycogen particles (solid arrowhead). Another gold-labeled vesicle also contains granule particles (long arrow), and a third gold-labeled vesicle is electron-lucent (short arrow). In C , at 2 min, note the gold-labeled vesicle filled with granule particles adjacent to the labeled granule (G). In D, also at 2 min, the electron-lucent vesicle contains DAO-gold. In E-G, at 5 min, the cell surface is indicated by open arrowheads. Note in E and F the granule particle-filled, DAO-gold-labeled perigranular vesicles (open arrows) adjacent to the cell surfaces. The adjacent granules (G) also label for histamine; the granule in F has a focal electron-lucent region (arrow) typical for PMD. Cytoplasmic glycogen particles (arrows in E) are electrondense and generally larger than the -20-nm gold label. In G, at 5 min, a DAO-gold-labeled vesicle is fused to the cell surface (open arrowhead). In H and I, at 10 min, electron-lucent (H) and particle-filled (I) cytoplasmic vesicles (arrows) contain histamine. In H, a large empty granule (EG) devoid of gold label and granule particles is typical for PMD. In I, the cell surface is coated with cationized ferritin (used in cell processing for electron microscopy) and

CELL BIOLOGY OF THE BASOPHIL

151

granular cytoplasmic vesicles adjacent to particle-packed granules (Figs. 18C, ME, and 18F) as well as those adjacent to empty granules devoid of their electron-dense contents (Figs. 18H and 18L) were noted, persisting to the 45-min time interval after TPA activation (Fig. 18L). The principal findings and interpretations of the specific enzyme-affinity gold labeling of cytoplasmic vesicles in secreting human basophils stimulated by TPA are as follows: (i) the fraction of gold-labeled vesicles (indicating histamine) of the total vesicles was elevated in all stimulated samples compared to that of unstimulated samples (thus, histamine is transported in vesicles in cells undergoing PMD and releasing histamine; (ii) comparison of timed samples revealed a progressive increase in gold-labeled vesicles over time persisting to 45 min, except in the 30-min sample. The value at 45 min was equivalent to that of the 10- and 15-min samples. Thus, as histamine release increased and persisted and as PMD developed, so did the transport of histamine-loaded vesicles. AND began in the 30-min sample, and basophils revealing this anatomy had diminished cytoplasmic vesicles (Dvorak et al., 1981b, 1991a). The proportion of these vesicles carrying histamine at 30 min still significantly exceeded values for controls and early samples, however (e.g., 0-5 min).

3. Comparison of Vesicular Transport of Histamine Stimulated by FMLP and TPA Vesicular transport of histamine in basophils of the same donors has different kinetics, analogous to biochemically measured release of histamine, when TPA and FMLP activation are compared. In Table V, comparisons of the percentage of VG/V/pm2are listed for similar (and several disparate) times tested when the basophils from the same donors were stimulated by either TPA (slow kinetics) or FMLP (fast kinetics). Significantly larger numbers of total vesicles were gold-loaded in the FMLP-triggered cells than in the TPA-triggered cells Cp <0.001 for 0 time and 1-, 2-, and 5-min samples, andp <0.01 for the 10-min samples) (Fig. 19).Thus, a secretogogue that rapidly induced the anatomic continuum of PMD + AND + recovery also induced more histamine-loaded vesicles, compared to similar times

is indicated by the open arrow. The granule particle-filled cytoplasmic vesicles are labeled with DAO-gold (arrows) at 15 (J), 30 (K), and 45 (L) min after TPA activation. The cell surface is indicated by open arrowheads in K and L; an empty granule (EG) typical for PMD is present at 45 min poststimulation in L (with permission, from Dvorak et al., 19960. Bars: (A,B) 180 nm; (C) 83 nm; (D) 150 nm; (E,K) 140 nm; (F) 160 nm; (G) 125 nm; (H) 150 nm; (1,J) 87 nm; (L) 185 nm.

152

ANN M. DVORAK

TABLE V Comparisons of Histamine-LoadedVesicles in Human Basophils from the Same Donors Stimulated with TPA or FMLP

Condition time

Stimulus

% VG/V/,umZ

Significance

~~

0 0

TPA FMLP

24 59

p <0.001

1 min 1 min

TPA FMLP

30 67

p <0.001

2 min 2 min

TPA FMLP

29 62

p (0.001

5 min 5 min

TPA FMLP

21 72

p <0.001

10 min 10 min

TPA FMLP

45 56

p <0.01

15 rnin 20 s

TPA FMLP

56 72

p <0.001

45 min 5 min

TPA FMLP

55 72

p <0.001

45 min 10 min

TPA FMLP

55 56

p = NS

after stimulation, than a secretogogue that slowly induced PMD, persisting to involve -50% of the cells by 45 rnin and showing only minor amounts of AND and no morphologic evidence of recovery. Comparison of the fraction of total vesicles carrying histamine at dispartate times after stimulation with TPA or FMLP follows (Table V). For example, the peak value for the percentage of VG/V/pm2 for the slow trigger, TPA, was at 15 min. This value (56%) was significantly less than the peak value (72%) for the fast trigger, FMLP, which occurred at 20 s after stimulation (p <0.001). Thus, speed of secretion was associated with greater histamine-loaded vesicular traffic at peak histamine-loaded vesicle times. TPA stimulation induced elevated VGIV/pm* at 45 rnin (55%) in samples predominantly still undergoing PMD (and with small amounts of AND and with no anatomic signs of recovery), whereas predominantly recovering cells (studied 5 rnin after FMLP stimulation) had significantly more VG/V/pm2 (73%, p <0.001). End times of kinetic samples for each

153

CELL BIOLOGY OF THE BASOPHIL

60-

b

2

40-

b? 200-

un

, 1

0

1

2

5

10

.

I

Minutes FIG. 19 Percentage of total cytoplasmic vesicles that are labeled with gold indicating the presence of histamine in unstimulated human basophils and in human basophils stimulated with FMLP or TPA for similar times. *p cO.01; **p <0.001 for comparisons between stimuli at similar times (with permission, from Dvorak er al., 1996f).

secretogogue showed no significant difference in the percentage of VG/V/ pm2 (TPA, 45 min 55% vs FMLP, 10 min 56%; p = NS). Thus, elevated histamine vesicular traffic persists in cells that are still secreting by PMD but not recovering (e.g., TPA stimulation) as well as in cells that are recovering granule materials by endocytosis and synthesis but no longer are secreting by PDM (e.g., FMLP stimulation). These quantitative comparisons of the fraction of total cytoplasmic vesicles loaded with histamine in these two models of human basophil activation were also important, since the samples for each secretogogue were aliquots of peripheral blood basophils purified from the same donors and on the same days. Therefore, interindividual donor and daily variations in secretory response were eliminated in this analysis. These studies establish for the first time that an important proinflammatory mediator, histamine, traffics from secretory granules to the extracellular milieu in small cytoplasmic vesicles in stimulated human basophils. The association of this process with the ultrastructural release reaction defined as PMD primarily produced by TPA and, in part, by FMLP establishes vesicular transport as the mechanism for effecting this type of regulated secretion. Vesicular transport of histamine was also significant in the more complex stimulated secretory and recovery model produced by exposure of human basophils to the bacterial peptide, FMLP.

154

ANN M. DVORAK

F. Comparison of Vesicular Transport of CLC Protein and Histamine Comparison of the percentage of VG/V, e.g., the proportions of total vesicles that are carrying histamine or CLC protein, between two unique secretogogues for human basophils (Fig. 20) are of interest and are facilitated by ultrastructural cytochemical morphometric kinetic analyses (Dvorak et al., 1996a,f, 1997a,b). FMLP, an extremely rapid secretogogue, induces simultaneous peaks (percentage of VG/V) of vesicles loaded with CLC protein or histamine, values that return to zero, in the case of CLC protein, by early recovery at 10 min. This coincides with reconstitution of formed CLCs within granules at 10 rnin (Dvorak et al., 1997a,b) (see later). The percentage of VG/V for histamine does not return to baseline, however, but at 10 rnin is still elevated over that for unstimulated cells. Thus, the behavior of vesicle transport for these two cellular products differs considerably in FMLP-stimulated cells over a 10-min interval. The elevated percentage of VG/V for histamine at 10 min most likely is a combination of uptake and synthesis (see later), since histamine release, measured biochemically, is essentially over much earlier than at 10 min after stimulation with FMLP (Warner et al., 1989). TPA (a slow secretogogue with morphologica1 criteria indicating extensive and continuing PMD by 45 min, little evidence of AND, and none of recovery) shows peak values of percentage of VGiV for CLC protein and

TPA

FMLP

I

Jn.

I

I

I

I

0 1 2 5 10 15 3045

Minutes FIG. 20 Percentage of total cytoplasmic vesicles that are labeled for gold indicating the presence of histamine ( 0 )or CLC protein (0)in unstimulated human basophils and in human basophils stimulated with FMLP (0-10 min) or TPA (0-45 min).

CELL BIOLOGY OF THE BASOPHIL

155

histamine between 10 and 15 min, also corresponding to peak histamine release times for this trigger (Schleimer et al., 1981, 1982). As for FMLP, the percentage of VG/V for CLC protein drops extensively by 45 min, but the percentage of VG/V for histamine remains high. Since there is no morphologic evidence for recovery (and therefore synthesis) in TPAstimulated basophils at 45 min, and little evidence of AND, the elevated vesicular traffic for histamine likely reflects ongoing PMD. These findings are consistent with ultrastructural evaluation of granule contents at 45 min after TPA. For example, granule number is not reduced and nearly 50% of the granules are empty and are represented only by their granule-sized containers, and neither particle-filled nor empty containers have residual formed CLCs. Thus, a granule source for histamine continues to exist at 45 min poststimulation, but one for CLC protein is virtually absent.

VII. Proof of a Degranulation Model Identified in Human Basophils in 1975 In 1975, we proposed a degranulation model to explain progressive losses, occurring over days, of granule contents from human basophils in experimentally induced contact allergy skin lesions (Dvorak et al., 1974b, 1976a; Dvorak and Dvorak, 1975). Essentially, we postulated that closely coupled endocytotic-exocytotic traffic of small, smooth membrane-bound vesicles effected the emptying of secretory granule containers, in the absence of granule fusion and extrusion-a process characterized by the retention of granule containers of undiminished size in the cytoplasm. We further postulated that this steady-state secretion would be altered in an important way if either the rate or the amount of vesicular traffic was changed. For example, we envisioned that a faster rate of vesicular traffic would result in fusions of vesicles which would create channels between granules and plasma membrane, thus producing the anatomy of regulated secretion, or AND. The cytoplasmic channels that form could contain multiple membrane-free granules (degranulation sacs or channels) in situ as well as provide communication between a single granule and the plasma membrane-events necessary for exocytosis directly through membrane pores to the external milieu. The amount of vesicular traffic might also vary. If the inward flow of endocytotic vesicles exceeded the outward flow of exocytotic vesicles, granule chambers would become enlarged, a prevalent feature of early samples in the kinetic morphologic studies of FMLP-induced human basophil secretion (Dvorak et al., 1991a). If the reverse were true, one might expect

156

ANN M. DVORAK

granule chambers to become diminished in size-a feature of some human basophils that we have examined (Dvorak, 1988a, 1992a, 1993). A degranulation continuum, essentially like the one predicted by this model, was established for human basophils upon stimulation by FMLP (Dvorak et al., 1991a). Essentially, in morphologic kinetic studies, a continuum of PMD occurred early and progressed to AND at later time points, coincident with the rapid release of histamine. The ultrastructural morphologic events characterizing this degranulation continuum are as follows. (1) A burst of small cytoplasmic vesicles, visible at 10 s, spanned a 30-s interval. Present were cells with vesicles, which appeared empty and were located in the peripheral cytoplasmic area, cells with mixtures of full and empty vesicles, and cells with perigranular, particle-filled vesicles. At later time intervals, cytoplasmic vesicles decreased in number in completely degranulated basophils or in basophils actively extruding granules through membrane pores. (2) Cytoplasmic granules emptied over the same time frame as the burst of vesicles (10-30 s). This process involved the loss of granule particles associated with container enlargement, initially without fusion to other granules or to the plasma membrane. These discrete, giant structures often contained large amounts of concentric dense membranes, irregular membranes, and small, lucent vesicles. (3) Between the 20- and 60-s time intervals, large, deep clefts coursed through the cytoplasm, often from the plasma membrane to as deep within the cell as the nucleus. These open clefts were filled with concentric dense membranes, vesicles, and CLCs. The general absence of granule particles, the morphologic configuration, and the contents suggest that these clefts represent the main resolution route of large, particle-depleted, empty granules after FMLP stimulation. The emergence of these clefts was associated with resolution of cytoplasmic vacuolar structures (i.e., empty granules) of all sizes. Loss of cytoplasmic vacuoles overlapped with the emergence of completely degranulated basophils. (4) Interconnected, granule particle-filled degranulation channels formed within the cytoplasm of a small number of basophils, between 20 and 60 s poststimulus. ( 5 ) Extrusion events typified the 20- to 120-s time interval. Extrusion occurred through multiple pores in the plasma membrane. Materials that were extruded individually or en masse remained associated with cul-de-sacs of the irregular surfaces of releasing basophils. These included membrane-free particle granules, homogeneously dense, spherical CLCs, and masses of concentric dense membranes. Kinetic ultrastructural morphometric studies of TPA-stimulated human basophils (Dvorak et al., 1992a) also support the predicted degranulation model (Dvorak and Dvorak, 1975). For example, this slow secretory model primarily shows vesicular transport of histamine or CLC protein and the anatomy of PMD. A minor component of AND images occurred, but most attempts at granule extrusion (i.e., AND) were characterized by a forme

CELL BIOLOGY OF THE BASOPHIL

157

fruste of AND, whereby granules bulged into plasma membranes and beyond the peripheries of cells, but the granule membranes were unfused with plasma membranes and many granules had not undergone extrusion throughout the 45-min interval examined (Dvorak et al., 1992a). Granule numbers also did not decrease-a concomitant event in cells undergoing AND (Dvorak et al., 1992a). Classically, secretion biologists have categorized secretion as either regulated (granule exocytosis) or constitutive (vesicle transport without storage or stimuli) (Dvorak, 1993; Palade, 1975; Kelly, 1985). Our definition of PMD holds that stimuli produce secretion (in the absence of exocytosis of granules) that is mediated by vesicle transport of granule materials to the extracellular milieu. Specific stimuli that produce PMD in human basophils are now being identified. Among these are FMLP, a bacterial peptide (Dvorak et al., 1991a, 1996f, 1997a,b), TPA, a tumor-promoting phorbol ester (Dvorak et al., 1992a, 1996a,f), IL-3, an interleukin with basophil activation properties (Dvorak et al., 1989b), MCP-1, a chemokine (Dvorak et al., 1996e), and a newly cloned cytokine, rp21, an IgE-dependent histamine-releasing factor (Dvorak et al., 1996e). As understanding of the cell biology of basophil secretion progresses, it has become apparent that newer paradigms for secretion in other cell systems have blurred the once classical definition of secretion (Palade, 1975; Kelly, 1985; Castle, 1990; Matsuuchi and Kelly, 1991; Chavez et al., 1996; Cleves et al., 1996). Thus, evidence is accruing for nongranular secretion from acinar exocrine cells (Castle, 1990; Login et af., 1994, 1996), stimulated constitutive secretion that differs from nonstimulated basal secretion from endocrine cells (Matsuuchi and Kelly, 1991), and a regulated secretory pathway in constitutive secretory cells (Chavez et al., 1996).

VIII. Charcot-Leyden Crystal Protein Distribution in Actively Degranulating Human Basophils A. Temporal Changes in CLC Protein Distribution

The amount and distribution of CLC protein differed in FMLP-stimulated human basophils from those in unstimulated, freshly prepared cells (Table VI) (Fig. 21) (Dvorak and Ackerman, 1989). These values were distinctive for individual time points sampled for immunogold analysis following stimulation. For example, the average total number of gold particles indicating the presence of CLC protein was 1.25/pm2 in unstimulated, bufferincubated cells. This value. was not significantly different from that of cells studied at the 0 time point after FMLP stimulation (2.97 gold particles/

ANN M. DVORAK

158

TABLE VI Total Average Cellular Gold Label Indicating CLC Protein in Basophils: A Kinetic Analysis Following FMLP Stimulation

Total average cellular gold particleslpm’ 2 SE

Total average Epongold particles/pm2 ? SE

Unstimulated

1.25 i- 0.34

0.10 f 0.02

Stimulated 0 time 10 s 20 s 30 s 1 min 2 min 5 min 10 min

2.97 t 0.31 7.90 2.43* 6.26 -t 0.91** 1.76 2 0.39 8.76 ? 1.45*** 4.95 2 0.63t 2.47 2 0.62 2.09 t 0.27

0.13 0.28 0.35 0.06 0.19 0.15 0.13 0.12

0.01/pm2 z 0.005 0.24/prn2 -+ 0.05

0.01/prn2 ? 0.005 0.22/pm2 -t 0.05

0.16/pm2 i 0.04

0.08/prn2 2 0.03

Specificity controls 1. Omit primary antibody 2. Substitute nonimmune IgG for primary antibody 3. Absorption control

_f

2 2 2 2

0.06 0.04 0.08 0.03 ? 0.09 2 0.35 i 0.03 t 0.02

* p <0.05, compared to unstimulated. ** p <0.01, compared to unstimulated; p <0.05, compared to 30 s. ***p <0.001, compared to unstimulated; p (0.01, compared to 30 s. t p <0.05, compared to unstimulated.

pm2; p = NS). By 10 s following stimulation, a significant increase in gold label for CLC protein was evident (7.90/pm2;p <0.05). Additionally, CLC protein label exceeded that of unstimulated cells at 20 s (6.26/pm2;p<0.01), 1min (8.76/pm2;p <0.001), and 2 min (4.95/pm2;p c0.05) following stimulation with FMLP. Values at 30 s, 5 min, and 10 rnin poststimulus were not significantly increased over those of unstimulated cells. Specificity controls and nonspecific background label for the immunogold method were all low (Tables VI and VII). Unstimulated, buffer-incubated cells expressed CLC protein in the nucleus, cytoplasm, granules, vesicles, and plasma membrane as well as in formed CLCs within granules. The percentage of total cellular gold label for CLC protein in cellular compartments varied in unstimulated cells compared to that at individual times sampled after stimulation (Fig. 21). The nuclear compartment of unstimulated cells contained 31.2% of the total label for CLC protein, a value significantly greater than this compartment in cells studied at 0 time after stimulation (14.6%, p <0.001) and at 2 rnin (19.1%, p <0.005), 5 min (8%,p <0.001), and 10 rnin (10.2%, p <0.001) thereafter. The cytoplasmic compartment of unstimulated cells (37.6%) differed significantly from three stimulated times, e.g., 2 rnin (24.7%, p

159

CELL BIOLOGY OF THE BASOPHIL W50-

Nucleus c

FormedCLC

Granule

Cytoplasm

-

-

Un 0 10' 20' 30' 1' 2 5' 1 0

FIG. 21 Histograms showing the percentage of total cellular gold label for CLC protein in individual cellular compartments in unstimulated cells and at 0, 10, 20, and 30 s and 1, 2, 5 , and 10 min after stimulation with FMLP (with permission of Karger, Basel, from Dvorak et

al., 1997b).

<0.005), 5 min (23%,p
160

ANN M. DVORAK

TABLE VII Total Average Cellular Gold Label Indicating CLC Protein in Unstimulated Basophils and FMLP-Stimulated Basophil Phenotypes

Total cellular gold particles/pm* 2 SE Unstimulated

1.25 2 0.34

Stimulated-Phenot ypes PMD-I PMD-I1 AND-I AND-I1 CDB RB-I RB-I1

9.71 2.28 4.17 3.98 4.11 3.48 4.40

t.1.42* 2 0.89** t 0.83 2 1.39 ? 1.20*** 2 0.55 2 0.74

Total Epon background gold particleslpm’ 2 SE 0.10 t 0.02

0.30 0.19 0.20 0.16 0.15 0.10 0.15

0.05 0.06 f 0.07 2 0.03 2 0.09 2 0.02 2 0.03 -C -C

*p

<0.001, (compared to unstimulated). * * p <0.01, (compared to PMD-I). *** p <0.05, (compared to PMD-I).

intervals after stimulation. At 0 time (24.5%),2 rnin (26.3%), 5 rnin (41.4%), and 10 min (31.8%) after FMLP, all values were significantly greater (p <0.001) for this compartment compared to those of unstimulated cells. The vesicle compartment contained a small proportion of the total cellular gold in unstimulated cells. One and four-tenths percent (1.4%) of the total cellular gold was present in vesicles. The plasma membrane compartment of unstimulated cells contained 3.5% of the total cellular label, a value significantly less than that of cells stimulated with FMLP for 10 s (9.7%, p <0.025), 1 rnin (9%, p <0.05), 2 rnin (9.7% p <0.025), 5 rnin (9.5%, p <0.05), or 10 min (11.5%,p <0.01). Degranulation channel membranes and lumens and extracellular granules were absent from unstimulated cells. When they appeared in stimulated samples, a small proportion of the total cellular gold was associated with these structures. The distributions of total cellular gold in cellular compartments differed from one another when these compartments were compared at different times after FMLP stimulation (Fig. 21). The nuclear compartment label of 0-time cells (14.6%), for example, contained significantly less than after 10 s (34.5%), 20 s (30%), 30 s (29.4%), or 1 rnin (28.5%) (all p <0.001) and did not differ from the amount of label after 2, 5, or 10 rnin ( p = NS). The nuclear label for the 10-s sample (34.5%) was greater than that for the 20-s (30%, p <0.025), 1-min (28.5%, p <0.005), 2-min (19.1%, p <0.001), 5-min (8%, p <0.001), and 10-min (10.2%, p <0.001) samples. The 20-s sample (30%) was greater than the 2-min (19.1%, p <0.001), 5-min (8%,

CELL BIOLOGY

OF THE BASOPHIL

161

p <0.001), and 10-min (10.2%, p <0.001) samples; the 30-s sample (29.4%) exceeded the 2-min (19.1%, p <0.001), 5-min (8%, p <0.001), and 10-min (10.2%, p <0.001) samples; the 1-min sample (28.5%) exceeded the 2-min (19.1%, p <0.001), 5-min (8%, p <0.001), and 10-min (10.2%, p <0.001) samples; and the 2-min sample (19.1%) exceeded the 5-min (8%, p <0.001) and 10-min (10.2%, p <0.001) samples. The nuclear compartment label for the 5- and 10-min-stimulatedbasophils was similar ( p = NS). Similar comparisons for the distribution of total cellular gold in the cytoplasmic compartment showed a significant elevation in label in this compartment compared to the 0 time (36%) after stimulation with FMLP at 1 min (46.3%, p <0.005), and reductions at 2 rnin (24.7%, p <0.001), 5 min (23%, p <0.001), and 10 min (19.3%, p <0.001). When 10-s sample cytoplasmic label (41.2%) was compared, significantly less was present at 20 s (34.6%, p <0.001), 2 rnin (24.7%, p <0.001), 5 rnin (23%, p
162

ANN

M. DVORAK

The 2- and 5-minute samples did not differ in the gold content of granules ( p = NS). The 2-min (15.3%) and 5-min (16%) samples contained less than the 10-min sample did in granules (25.9%, p <0.001, p ~ 0 . 0 0 5 ) . The formed CLC compartment also showed differences in the amount of total cellular label at different times after stimulation with FMLP. This compartment at 0 time (24.5%) exceeded all times ( p <0.001) prior to 2 rnin (26.3%,p = NS) after stimulation, but was significantly less than that after 5-min (41.4%, p <0.001) and 10-min (31.8%, p <0.05) intervals. The 10-s sample (4.4%) contained label similar ( p = NS) to the 20-s (4.9%) and 1-min (4.5%) times but significantly less than that in the 30-s (9%, p
CELL BIOLOGY OF THE BASOPHIL

163

( p C0.005) the 10-s value. No differences between the 1-, 2-, S-,and 10-min samples existed compared to 10 s ( p = NS). The amounts of plasma membrane label for 20- and 30-s samples were similar ( p = NS). The 20-s interval (10.5%) showed more label in plasma membranes than the 1-min (9%, p <0.001), 2-min (9.7%, p <0.001), and S-min (9.5%, p <0.001) intervals, but less than that at 10 min (11.5%, p <0.001). The 30-s sample (7.8%) had less plasma membrane label than did the l-min (9%, p <0.005), 2-min (9.7%, p <0.005), 5-min (9.5%, p <0.01), and 10-min (11.5%, p <0.001) samples. The plasma membrane label was similar among the 1-, 2-, 5-, and 10-min samples ( p = NS). Two peak elevations of gold-labeled CLC protein occurred in the kinetic samples, as follows: 10 s, 1 min-i.e., a marked increase in cellular CLC protein occurred after the peak vesicle increase and the peak increases of labeled CLC protein coincided exactly with the peak elevation of altered granules (10 s) as well as with maximum secretion by extrusion of granules (1 min). The relative distribution of gold-labeled CLC protein in cellular compartments of FMLP-stimulated basophils showed significant translocations of this protein in activated cells at variable times after stimulation. Some relationships for statistically significant changes in CLC protein distribution in cellular compartments compared to unstimulated, bufferincubated cells are summarized as follows: (i) nucleus: UN >O time and 2, 5 , and 10 min; (ii) cytoplasm: UN >2, 5, and 10 min; (iii) granules: UN >lo, 20, and 30 s and 1,2, and S min; (iv) formed CLCs: UN
6.Granule and Vesicle Numbers in FMLP-Stimulated Cells The earliest change elicited by FMLP in human basophils was increased cytoplasmic vesicles (average, 3.02/pm2) (Fig. 10, Table 11), a value which peaked at the earliest possible time of an extremely rapid release reaction (0 time after stimulus) at which samples can be recovered for electron microscopy. Numbers of vesicles then decreased to a low at 30 s after stimulation (0.72 vesicles/pm2; p <0.01, compared to 0 time), when a large number of actively degranulating and completely degranulated basophils prevailed (Fig. 10) (Dvorak et al., 1997b). The average number of cytoplasmic vesicles in FMLP-stimulated cells was also significantly depressed, relative to that at 0 time point, 20 s (0.82/km2; p <0.01), 1 min (0.98/pm2;

164

ANN M. DVORAK

p <0.05), and 10 min (0.97/pm2; p <0.01) after stimulation (Table 11; Fig. 10). The morphologic kinetic sequence stimulated by FMLP in human basophils was accompanied by changes in granule numbers within cells (Fig. 10) (Dvorak et al., 1997b). The decreased numbers observed gave rise to a virtual absence of granules in degranulated cells, most prevalent at 1rnin poststimulus ( p <0.001 compared to 0 time) (Fig. lo), to recovery of cytoplasmic granules, observed later ( p = NS) at 10 rnin compared to 0 time). Granule numbers were also depressed in FMLP-stimulated cells, relative to 0 time, at 20-s ( p cO.01) and 30-s ( p <0.001) intervals. These changes in granule numbers were accompanied by qualitative alterations of granule contents (Fig. 10). For example, the percentage of altered granules with reduced amounts of dense particles was maximum at 10 s post-stimulus [52%, p <0.001 compared to unstimulated cells (29%) or to cells at 0 time of stimulation (30%)], values which decreased gradually thereafter, until recovery at 5 and 10 rnin (Fig. 10) (28%,p = NS compared to unstimulated cells). Thus, the increase in cytoplasmic vesicles after stimulation (0 time) preceded the maximum alteration of granules at 10 s; both events preceded the maximum loss of entire granules occurring at 1 min poststimulation (Fig. lo), thereby substantiating a sequential degranulation continuumproceeding from PMD to AND. Biochemical studies showed that halfmaximum histamine release occurred at 1.3 rnin in this model (Warner et al., 1989).

C. Morphologic Phenotypes of Basophils We classified FMLP-stimulated human basophils using ultrastructural criteria, regardless of time after stimulation, into seven different morphologic phenotypes (Dvorak et al., 1997a). These phenotypes were readily recognizable using these ultrastructural criteria and generally corresponded to adjacent, overlapping times in a kinetic analysis (Dvorak et al., 1997b). The number of 30-nm gold particles representing expression of CLC protein was enumerated, and a gold profile (percentage of total cellular gold in cellular compartments) for each phenotype assembled. In addition to unique morphologic criteria for these FMLP-stimulated phenotypes, the CLC protein profile, as expressed by the gold labeling index, was also unique and differed considerably from a similar analysis of unstimulated human basophils. 1. CLC Protein Distribution in Buffer-Incubated, Unstimulated Control Basophils

Charcot-Leyden crystal protein (CLC protein) was localized to the particlefilled matrix (or to formed CLCs within this matrix) of secretory granules

CELL BIOLOGY OF THE BASOPHIL

165

in unstimulated peripheral blood basophils. Rarely, electron-lucent vesicles in the perigranular cytoplasmic areas were also labeled. CLC protein was present in small amounts in several other cellular compartments of the buffer-incubated control samples. The compartments included nucleus, cytoplasm, and plasma membrane. Approximately one-half of the unstimulated basophils had no altered granules. The remainder were classified as PMD-I phenotype (see later) with the difference that fewer altered granules were present in the PMD-I phenotype found in the buffer-incubated controls than in the PMD-I phenotype present at all times poststimulation. Thus, the average number of particle-filled granules in the control cells was 1.06/pm2of the cell, and 70.8% of these did not show visible evidence of content diminution or alteration (Fig. 22). Rarely, small paranuclear granules with non-particulate content (Hastie, 1974) were also visible. Unstimulated basophils displayed 50- to 150-nm vesicles (average number, 1.9 vesicles/pm2 of the cell) in their cytoplasm (Table 111; Fig. 22). Basophil nuclei were polylobed and condensed; the cytoplasmic contents and surface architecture conformed to previous ultrastructural descriptions (Dvorak and Ackerman, 1989; Dvorak et al., 1980b). Quantitation of CLC protein by counting gold particles in six major cellular compartments revealed the following gold profile (percentage distribution of total cellular gold particles) for unstimulated basophils: nucleus, 31.2%;cytoplasm, 37.6%; granule, 26.2%; vesicle, 1.4%;plasma membrane, 3.5%;formed CLCs, 6.4%.

FIG. 22 Relationships of altered granules (% of total granules) and cytoplasmic vesicles (average number/pm*) in unstimulated cells and FMLP-stimulated human basophil phenotypes. p values for comparisons to unstimulated cells: * p (0.001; **p <0.05; + p <0.05. Bars: % altered granules. (0) Average number/pm2 of cytoplasmic vesicles (with permission of Blackwell Science Ltd., from Dvorak et al., 1997a).

166

ANN M. DVORAK

2. Piecemeal Degranulating Basophil I (PMD-I) The PMD-I phenotype was characterized by focal and complete losses and alterations of the dense particle contents of the major granule population. Rarely, several granules were fused, but fusion events were not a general feature of this phenotype. Rather, numerous cytoplasmic, smooth membrane-bound vesicles (many containing electron-dense granule particles) were noted. Some granules had either vesicles with electron-lucent interiors or dense particle- or dense content-filled vesicles budding from their membranes. The total number and size of cytoplasmic granules were similar to those of unstimulated basophils, but 45.1% of the granules showed content losses and alterations ( p <0.005) (Fig. 22). The number of cytoplasmic vesicles did not significantly differ from that of unstimulated cells (Fig. 22; Table 111); they were, however, significantly greater in number than that in the CDB phenotype ( p <0.01) (Table III). The typical, condensed, polylobed nucleus was unchanged, as was true for all phenotypic categories. The surface architecture resembled that of unstimulated cells, consisting of blunt, irregular protrusions. Thirty-nanometer gold particles, representing CLC protein, showed changes from unstimulated cells. There was an increase in the average total cellular gold label (9.71 gold particles/pm2 in the PMD-I phenotype; 1.25 gold particles/pm2 in unstimulated cel1s;p <0.001) (Table VII). The greatest percentage of labeling in PMD-I cells was in the cytoplasm (40.1%), with the next highest amount in the nucleus (33.1%). The granule compartment contained 13.3%, a decrease from the unstimulated state (26.2%, p <0.001); the plasma membrane was labeled (9.2%), an increase compared to unstimulated cells (3.5%, p <0.025) (Fig. 23A). The remainder of the phenotypic gold profile of the PMD-I phenotype showed 1.9% of the total gold label in vesicles ( p = NS compared to unstimulated cells), and 2.1% in formed CLCs (decreased compared to unstimulated basophils, 6.4%, p C0.005). Overall, the gold profile in PMD-I cells stimulated by FMLP showed losses of CLC protein from formed CLCs and granules in this stimulated phenotype and a significant gain in the plasma membrane label for the CLC protein. Since granule extrusion and channel formation were not present in PMD-I cells, the relocation of CLC protein from granule to plasma membrane may be mediated by vesicular transport (Dvorak et al., 1997b). The formed CLC cellular compartment in the FMLP-stimulated basophil PMD-I phenotype was of interest (Fig. 24). Formed CLCs consisted of either typical bipyramidal and hexagonal structures or irregular (or rounded) but discrete masses within granules, granule chambers, degranulation channels, cytoplasm, nucleus, or in the extracellular, periplasma membrane location in the various phenotypes. When these structures were present in cytoplasmic granules or in emptying-enlarging granule chambers,

CELL BIOLOGY OF THE BASOPHIL

167

FIG. 23 FMLP-stimulated human basophils, recovered at 10 s (A), 1 min (B), and 2 min (C), show high magnifications of the PMD-I (A), AND-I1 (B), and RB-I1 (C) phenotypes. In A, the PMD-I basophil shows extensive labeling of peripheral cytoplasm and plasma membrane with 30-nm gold particles representing CLC protein. In B, the AND-I1 basophil shows 30-nm gold label in the nucleus, in the cytoplasm, and in extruded, formed CLCs adjacent to the cell surface (arrowheads). An electron-lucent cytoplasmic vesicle is also labeled (arrow). In C, the RB-I1 basophil shows 30-nm gold labeling of formed CLCs within particle-filled granules. Perigranular cytoplasmic label is present, but the plasma membrane is devoid of gold label (with permission of Blackwell Science Ltd., from Dvorak et al., 1997a). Bars: (A$) 0.6 pm; (B) 0.8 pm.

ANN M. DVORAK Unstim.

PMD-I I

rn CLC-G

PMD-I1

AND4

AND-I1

CDB

I

CLC-cylo

RB-I

RS-II

*

CLC-CH

CLC-EC

FIG. 24 Percentage of total formed CLC protein gold label indicating CLC protein in formed CLCs, which are located in variable cellular or extracellular locations of unstimulated cells and FMLP-stimulated human basophil phenotypes. CLC-G, CLC granule; CLC-cyto, CLC cytoplasm; CLC-CH, CLC channel; CLC-EC, CLC extracellular.p value comparisons are to unstimulated cells. * p <0.001; ' p <0.05 (with permission of Blackwell Science Ltd., from Dvorak et al., 1997a).

the gold counts indicating the presence of CLC protein were included in the total granule gold counts. Therefore, in the gold profile of PMD-I cells (where nearly all formed CLCs were located in granules), most of the gold label, indicating CLC protein, was associated with diffuse particle granule contents (11.2%) rather than with formed CLCs in granules (2.1%), reflecting intragranular solubilization of CLCs in the PMD-I phenotype.

3. Piecemeal Degranulating Basophil I1 (PMD-11) The most characteristic feature of this phenotype was the presence of nonfused, empty (or emptying) granule chambers in the cytoplasm, many of which showed marked enlargement when compared to unaltered specific granules. The total number of granules was less than that in the PMD-I phenotype; the percentage of altered granules (61.8%) was considerably greater (45.1%,p <0.005; Fig. 22). The average number of cytoplasmic vesicles was somewhat decreased compared to that of the PMD-I phenotype and to that of unstimulated cells (Table 111;Fig. 22). The surface architecture of PMD-I1 cells was more smooth, with fewer processes than was true for either unstimulated or PMD-I cells. Formed CLCs were present in empty granule chambers and very rarely in extracellular cul-de-sacs associated with the plasma membrane. The gold profile, representing the location of CLC protein in PMD-I1 cells, showed a reduction of total cellular label (2.28/pm2) compared to the PMD-I phenotype (9.751~111~; p <0.01). The cytoplasmic compartment contained 35.1% of the total gold label, a value significantly greater than

CELL BIOLOGY OF THE BASOPHIL

169

that in either the RB-I (26.4%, p C0.005) or the RB-I1 (18.8%, p <0.001) phenotypes. The granule compartment contained 23.8%, a value significantly greater than that in granules of the PMD-I (13.3%,p <0.001), ANDI (3.5%, p <0.001), AND-I1 (1.2%, p <0.001), and CDB (O%, p <0.001) phenotypes. Nuclear content was 18%, a marked decrease compared to that of PMD-I (33.1%, p <0.001) and unstimulated cells (31.2%, p <0.005). Formed CLCs contained 15.7% of the total gold label, all of which was in either empty granule chambers or an extracellular location adjacent to the plasma membrane. This value exceeded that for formed CLC label in unstimulated cells (6.4%, p <0.01) or in the PMD-I (2.1%, p <0.001) and CDB (6.5%,p <0.001) phenotypes. The plasma membrane alone had 4.7% of the total gold label-a value similar to that of unstimulated cells (3.5%, p = NS) and less than that found in PMD-I cells (9.2%, p C0.005). The percentage of total gold in vesicles was similar to that of the PMD-I phenotype, both of which were slightly increased compared to that of unstimulated cells ( p = NS). Overall, the gold profile of PMD-I1 cells stimulated by FMLP showed an amount in the formed CLC compartment that exceeded that of unstimulated cells, with some reduction in the nuclear compartment compared to that in unstimulated cells. Both the granule and the formed CLC compartments contained considerably more of the total gold label than was present in these compartments in the PMD-I phenotype, whereas the plasma membrane label was less than that in the PMD-I phenotype and was similar to the plasma membrane of unstimulated cells. Nuclear label was considerably reduced in the PMD-I1 phenotype compared to that of the PMD-I phenotype and unstimulated cells.

4 Anaphylactic Degranulating Basophil I (AND-I) The characteristic morphologic feature of this phenotype was the presence of intracytoplasmic degranulation channels formed by fusion of granule membranes. These degranulation channels contained altered granule matrix particles, concentric intragranular membranes, formed CLCs, or they appeared empty. Some residual, unaltered, or partially altered granules remained in the cytoplasm. The total number of granules, while less, did not differ significantly from that of unstimulated cells or either PMD-I or PMDI1 cells. The percentage of altered granules was the greatest (64%) of all phenotypes stimulated by FMLP (PMD-I, 45.1%, p c0.005; AND-11, 27.8%, p <0.001; CDB, 0%, p <0.05; RB-I, 33.1%, p <0.001; RB-11, 19.3%, p <0.001), differing significantly from all phenotypes except PMD-I1 (Fig. 22) and exceeding those in unstimulated cells (29.2%, p <0.001). The average number of cytoplasmic vesicles did not differ significantly from that of unstimulated cells or from that of the PMD-I, PMD-11, AND-11, CDB,

170

ANN M. DVORAK

RB-I, and RB-I1 phenotypes (Table 111; Fig. 22). The cell surface showed increased processes when compared to unstimulated cells or to PMD-I and PMD-I1phenotypes. Formed CLCs were primarily present in the cytoplasm and granule matrix; small numbers were present in d e g ra d a tio n channels and adjacent to the cell surface. The total number of gold particles of the AND-I phenotype (4.17/pm2) was statistically similar to that of other stimulated phenotypes (Table VII). The gold profile of the AND-I phenotype, however, showed a different distribution. Nuclear label (18.1%) was identical, to that of the PMD-I1 phenotype (18%). Nuclear label was considerably less than that in unstimulated cells (31.2%,p <0.001) or that in the PMD-I (33.l%,p <0.001), ANDI1 (29.5%, p <0.001), and CDB (36.3%,p <0.001) stimulated phenotypes but exceeded that of the RB-I (12.5%,p <0.01) and RB-II(11.9%,p <0.005) recovering phenotypes. The cytoplasmic compartment (34.5%) was less than that in the PMD-I (40.1%, p (0.025) and the CDB (40.3%,p C0.05) phenotypes and greater than that in the RB-I (26.4%, p <0.01) and the RB-I1 (18.8%,p <0.001) phenotypes. The granule compartment had 3.5% of the total gold in the AND-I phenotype. Thus, a significant reduction of label for the CLC protein was evident in this compartment compared to that in unstimulated cells (26.2%, p <0.001) and to that in the PMD-I (13.3%,p <0.001) and PMD-11 (23.8%,p <0.001) stimulated phenotypes, despite no significant reduction in granule numbers among them. Granule label for CLC protein in the AND-I phenotype still exceeded that for the AND-I1 (1.2%,p <0.005) and CDB (O%,p <0.001) stimulated phenotypes, however. The percentage of granule gold label of the total cellular label was significantly less in AND-I cells than that in the recovering phenotypes (RB-I, 21%, p <0.001; RB-I1,28%, p <0.001). The amount of gold in the formed CLC compartment (21.9%) was greater than that in the PMD-I (2.1%, p <0.001) and PMD-I1 (15.7%, p (0.025) phenotypes or that in unstimulated cells (6.4%, p <0.001). The distribution of this formed CLC component differed, however, from that of these phenotypes (Fig. 24). That is, in PMD-I and PMD-I1 cells, the formed CLCs were in granule spaces, whereas in AND-I cells, formed, gold-labeled CLCs were primarily present as focal, rounded masses in the cytoplasm, thus contributing to the high gold index for the cytoplasmic compartment. For example, cytoplasmic gold in PMD-I and PMD-I1 cells was diffuse, whereas a large amount was in formed cytoplasmic CLCs in AND-I cells. Thus, with an increased cytoplasmic concentration, crystal formation (to produce formed CLCs in the cytoplasm) was prevalent. The percentage of total cellular gold in vesicles (3.9%) was greatest in AND-I cells, exceeding the percentage in the PMD-I (1.9%, p <0.01) and RB-I1 (1.3%, p (0.005) phenotypes. The percentage of total gold label of the plasma membrane (12.2%) was greater than that in unstimulated cells

CELL BIOLOGY OF THE BASOPHIL

171

(3.5%, p <0.005) and in either PMD-I (9.2%, p <0.05) or PMD-I1 (4.7%, p <0.001) cells. Two new compartments-the degranulation channel membrane and channel lumen (which typify AND-I cells)-together contained 5.5% of the total gold label. If all cell membrane label was combined-i.e., plasma membrane (12.2%) and channel membrane (4.5%)-the total membrane label equaled 16.7% for AND-I cells, a value identical to that of plasma membrane label in the AND-I1 phenotype (16.5%, p = NS). Thus, the greatest amount of cell membrane-associated CLC protein was seen in the two phenotypes which represented the most active externalization of intracellular granule membranes and materials-AND-I and AND-11-and was considerably greater than that of the PMD-I1 phenotype (4.7%, p <0.001). The CDB phenotype also expressed a high plasma membrane label (13.8%). Phenotypes PMD-I and RB-I were equivalent for plasma membrane label ( p = NS), and RB-I1 cells had a low amount of gold label in the plasma membrane compartment (4.5%), a value similar to that for unstimulated cells (3.5%, p = NS) and stimulated PMD-IT cells (4.7%, p = NS). Overall, the gold profile of AND-I cells stimulated by FMLP showed amounts in vesicle, plasma membrane, and formed CLC compartments in excess of those in unstimulated cells. Gold label in the granule and nuclear compartments was reduced compared to that in unstimulated cells. A large portion of the formed CLC gold label was associated with cytoplasmic CLCs. And, newly formed degranulation channels and their membranes-structures not present in unstimulated cells-stained for CLC protein in AND-I cells.

5. Anaphylactic Degranulating Basophil I1 (AND-11) This phenotype was characterized by multiple extrusion events. Various intragranular contents were extruded multiply and circumferentially around the perimeter of the cell through multiple, narrow pores in the plasma membrane. The materials released consisted of membrane-free granule particles, intragranular concentric membranes, intragranular vesicles, and formed, homogeneous, spherical CLCs (Fig. 23B). Residual unaltered or partially altered granules remained in the cytoplasm. The total number of granules, 0.25/pm2, was, however, the lowest of all phenotypes (PMD-I, 1.25/pm2,p <0.05; RB-I, 1.76/pm2,p <0.001; RB-11, 2.44/pm2, p (0.001) (except in granule-free, completely degranulated basophils). Only 27.8% of the total remaining granules in AND-I1 cells were altered, a value similar to that of unstimulated basophils (29.2%, p = NS) (Fig. 22). The average number of cytoplasmic vesicles (0.71/pm2) was significantly less than that in unstimulated cells (1.91/pm2, p c0.05) (Table 111). The cell surface of AND-I1 cells was the most complex of all phenotypes stimulated by FMLP. Most of the extraordinary surface complexities and elongated processes

172

ANN M. DVORAK

were the result of externalization of granule and channel membranes as the contents of these spaces were extruded to the extracellular space. While all granule contents were extruded to the extracellular milieu in the ANDI1 phenotype, concentric and vesicular membranes, CLCs, and particulate contents remained adjacent to the plasma membrane, generally at separate locations. The location of formed CLCs in the AND-I1 phenotype was virtually all extracellular in close association with the plasma membrane in cul-de-sacs created by granule membrane additions to the plasma membrane (Fig. 24). The AND-I1 phenotype was the only one stimulated by FMLP for which this was the primary location of formed CLCs. By contrast, intragranular CLCs in AND-I1 cells were virtually absent, and formed CLCs in the cytoplasm were not present. The number of total gold particles in the AND-I1 phenotype was 3.98/ pm2, a value statistically similar to that of all stimulated phenotypes as well as to that of unstimulated cells (Table VII). The gold profile for CLC protein location in the AND-I1 phenotype showed the largest amount in the cytoplasmic (36.3%) compartment; but in contrast to AND-I cells, all of the cytoplasmic label in the AND-I1 phenotype was diffuse and unassociated with formed CLCs free in the cytoplasm. The plasma membrane of AND-I1 cells had 16.5% of the total gold label, representing the highest labeling of plasma membrane of all phenotypes stimulated by FMLP (PMD-I, 9.2%, p <0.001; PMD-II,4.7%,p <0.001; AND-I, 12.2%,p C0.05; CDB, 13.8%, p = NS; RB-I, 7.7%, p <0.001; RB-11, 4.5%, p <0.001); the plasma membrane label also exceeded that for unstimulated cells (3.5%, p <0.001). Granule gold label was the lowest for AND-I1 cells (1.2%), except for the granule-free, CDB phenotype (O%, p CO.01) and was significantly lower than that in unstimulated cells (26.2%, p <0.001). Extracellular granules, present only in the AND-I1 phenotype, also contained some gold label, indicating the presence of CLC protein (0.9%). Formed CLCs had 13.5% of the total gold label, which exceeded this value for unstimulated cells (6.4%, p <0.025) and for the stimulated phenotypes PMD-I (2.1%, p <0.001) and CDB (6.5%, p <0.001) but was less than that in either recovering phenotype (RB-I, 29%, p <0.001; RB-11, 35.6%, p <0.001). Eighty and one-half percent (80.5%)of the total formed CLC label was associated with extracellular CLCs (Figs. 23B and 24). AND-I1 cells had 2.1% of the total gold label in vesicles, a value similar to that in other stimulated phenotypes. Overall, the gold profile of AND-I1 cells stimulated by FMLP showed amounts in the plasma membrane and formed CLC compartments far in excess of those of unstimulated cells. Gold label associated with granules was the lowest of the phenotypes containing granules, including control unstimulated cells, and was approximately equally distributed between unaltered cytoplasmic and extracellular extruded granules. Virtually all of the

CELL BIOLOGY

OF THE BASOPHIL

173

gold label associated with formed CLCs in the AND-I1 phenotype was in the extracellular, plasma membrane-associated location (Figs. 23B and 24). 6. Completely Degranulated Basophil (CDB)

This human basophil phenotype stimulated by FMLP was characterized by the virtual absence of specific cytoplasmic granules, following their stimulated release, and no altered granules remained in the cytoplasm (Fig. 22). The average number of vesicles in the CDB phenotype (0.33/pm2) was the lowest of all phenotypes (PMD-I, 1.38/pm2,p <0.01; RB-I, 1.68/pm2, p <0.001; RB-11, 1.62/pm2,p <0.001), including the number in unstimulated cells (1.91/pm2, p <0.001) (Table 111). Regardless of the diminishing numbers of these structures, gold particles were found within them. CDBs were distinguished by large aggregates of cytoplasmic glycogen and increased numbers of mitochondria. Their nuclei, like those of all phenotypes, remained polylobed. Cell surfaces were either irregular or smooth, subsequent to shedding of portions of cellular processes (Dvorak et al., 1991a). Formed CLCs were virtually absent; when present, they were located near plasma membranes in the extracellular space. The total cellular gold label for the CDB phenotype was 4.11/pm2, a value significantly less than that in the PMD-I phenotype (9.71/pm2, p <0.05) (Table VII). The gold labeling profile for CLC protein of the CDB phenotype showed the largest amounts in the cytoplasm (40.3%) and nucleus (36.3%). The next most highly labeled compartment was the plasma membrane (13.8%), a value far in excess of that of unstimulated cells (3.5%, p <0.001) and of the PMD-I1 (4.7%, p <0.001), RB-I (7.7%, p <0.001), and RB-II(4.5%,p <0.001) phenotypes. The CDB phenotype granule gold was 0%, significantly less than that in all other categories as well as that in unstimulated cells. The formed CLC gold (6.5%) was lower than that in stimulated phenotypes (PMD-11, 15.7%, p <0.001); AND-I, 21.9%, p
174

ANN M. DVORAK

7. Recovering Basophil I (RB-I)

The RB-I phenotype was characterized by large numbers of cytoplasmic granules, a polylobed nucleus, and a simplified surface architecture (not unlike that of unstimulated cells). The average total number of granules was 1.76/pm2,a value in excess of all phenotypes except the RB-I1 phenotype; 33.1% of the total granules in RB-I cells had an altered matrix pattern, not differing significantly from this value in unstimulated cells (29.2%,p = NS) (Fig. 22). Altered granules were, however. significantly less in the RBI phenotype (33.1%)than in the PMD-I (45.1%, p <0.005), PMD-I1 (61.8%, p <0.001), and AND-I (64%,p <0.001) cells but significantly greater than in the RB-I1 phenotype (19.3%,p <0.001). The altered granules in RB-I cells were not enlarged, and dense concentric membranes were rare within them. The average number of cytoplasmic vesicles (1.68/pm2) (Table 111) was higher than that in the CDB phenotype (0.33/pm2; p <0.001) stimulated by FMLP; formed CLCs, as in unstimulated cells, were, once again, plentiful in specific granules. In contrast to unstimulated cells, however, the RB-I phenotype displayed rounded, cytoplasmic, formed CLCs-a feature characteristic also for the AND-I and RB-I1 phenotypes, but not for the remaining phenotypes. The average total gold particles was 3.48/pm2 for the RB-I phenotype (Table VII). The gold profile of the RB-I phenotype showed the most label in the formed CLC (29%) and granule (21%) compartments. The formed CLC compartment label was significantly increased compared to that in unstimulated cells (6.4%,p <0.001) and to that in the stimulated phenotypes PMD-I (2.1%, p <0.001), PMD-I1 (15.7%, p <0.001), AND-I (21.9%, p <0.005), AND-I1 (13.5%,p <0.001), and CDB (6.5%, p <0.001) but was significantly less than that found in the RB-I1 phenotype (35.6%, p <0.025). Similarly, the granule compartment label for RB-I cells (21%) exceeded that of the PMD-I (13.3%, p <0.001), AND-I (3.5%, p <0.001), AND-I1 (1.2%,p <0.001), and CDB (O%, p <0.001) phenotypes and was similar to that in unstimulated cells (26.2%,p = NS) and PMD-I1 cells (23.8%,p = NS). As in the formed CLC compartment, the granule compartment label in RB-I cells (21%)was less than that in the RB-I1 phenotype (28%,p C0.005). The plasma membrane gold label was 7.7% of the total cellular gold label, a value significantly greater than the plasma membrane label of the RB-I1 phenotype (4.5%, p <0.025) and significantly less than that in the AND-I (12.2%,p <0.025), AND-11 (16.5%, p <0.001), and CDB (13.8%, p (0.001) phenotypes. Three and three-tenths percent (3.3%) of the total cellular gold label was in the vesicle compartment, a value significantly greater than that in the PMD-I phenotype (1.9%, p <0.05). Amounts of CLC protein in the cytoplasm (26.4%) and nucleus (12.5%) were reduced compared to those in unstimulated cells (37.6%,p (0.01; 31.2%,p <0.001).

CELL BIOLOGY OF THE BASOPHIL

175

A large portion of the cytoplasmic gold label was present in formed cytoplasmic CLCs, structures that were absent from control unstimulated cells. In the absence of morphologic evidence of ongoing release in the RB-I phenotype, the persistence of plasma membrane CLC protein could lead to internalization of CLC protein in gold-labeled, smooth membrane-bound endocytotic vesicles in the RB-I phenotype. Overall, the gold profile of the RB-I phenotype stimulated by FMLP was distinguished by decreased label in the nuclear and cytoplasmic compartments and increased label in the vesicular, plasma membrane and formed CLC compartments compared to that in unstimulated cells.

8. Recovering Basophil I1 (RB-11) A distinctive feature of this phenotype was the presence of large, uniformly dense, membrane-bound granules which were completely devoid of the particulate, concentric membranous and formed CLC substructural components which typify the major human basophil granule population. Also characteristic of RB-I1 cells was the presence of a full complement of the large (1-2 pm) particle-filled granules, which are typical for human basophils. Many of these granules had formed CLCs within them (Fig. 23C). Intragranular concentric membranes were rare. The average number of granules of RB-I1 cells (2.44/pm2) was the greatest of all phenotypes (PMD-11, 0.78/pm2, p <0.05; AND-I, 0.60/pm2, p CO.01; AND-11, 0.25/ pm2,p <0.001; CDB, 0.02/pm2,p <0.001). Fewer of these granules showed either complete or focal alterations (19.3%) than was true for the RB-I phenotype (33.1%,p <0.001). Also, the percentage of altered granules in the RB-I1 phenotype (19.3%) was less than that in the stimulated phenotypes (PMD-I, 45.1%, p <0.001; PMD-11, 61.8%, p (0.001; AND-I, 64%, p <0.001) but did not differ significantly from that of the AND-I1 (27.8%, p = NS) or CDB (O%, p = NS) phenotypes (Fig. 22). The average number of cytoplasmic vesicles for RB-I1 cells (1.62/pm2) equaled that of RB-I cells (1.68/pm2; p = NS), exceeding that of the CDB phenotype (0.33/pm2, p <0.001) (Table 111; Fig. 22). The distribution of formed CLCs was virtually completely within the granule compartment in RB-I1 cells; a small component was present in the cytoplasm, as in the RB-I phenotype. The average total cellular gold for the RB-I1 phenotype was 4.4/pm2, a value not significantly different from that of unstimulated cells or from that present in all stimulated phenotypes (Table VII). The gold profile of the RB-I1 phenotype, which reflects the distribution of gold label, showed differences, however. The largest amount of label was in the formed CLC compartment (35.6%).This compartment contained the greatest percentage of total cellular gold when compared to that of unstimulated cells (6.4%, p <0.001) or to that of the stimulated phenotypes PMD-I (2.1%, p <0.001),

176

ANN

M. DVORAK

PMD-I1 (15.7%, p <0.001), AND-I (21.9%, p <0.001), AND-I1 (13.5%, p <0.001), CDB (6.5%,p <0.001), and RB-I (29%,p <0.025). Of the granule gold label, most was associated with formed intragranular CLCs, with another large proportion of granule gold present in the granules that were filled with homogeneously dense contents, similar t o the primary granules of eosinophils (Dvorak et al., 1988). While the numbers of these homogeneously dense granules were small, they contained a uniform distribution of large numbers of gold particles. The gold label in the granule compartment (28%) was nearly the same as that in the unstimulated cells (26.2%, p = NS) and significantly exceeded the granule compartment gold label in the stimulated phenotypes PMD-I (13.3%, p <0.001), AND-I (3.5%, p <0.001), AND-I1 (1.2%, p <0.001), CDB (O%, p <0.001), and RB-I (21%, p <0.005), except in the granules of PMD-I1 cells (23.8%,p = NS). The nuclear (11.9%) and cytoplasmic (18.8%) compartments of RB-11 cells contained significantly less gold label than that in unstimulated cells (nuclear,

C Plasma Membrane 30

FIG. 25 Histograms showing several interrelationships of unstimulated and FMLP-stimulated human basophils. The percentages of total cellular gold label, indicating CLC protein, in the granule (A), formed CLC (B), plasma membrane (C), and vesicle (D) cellular compartments are shown for unstimulated cells and for individual FMLP-stimulated basophil phenotypes. p value comparisons refer to unstimulated cells. * p <0.001; **p <0.005; + p <0.01; " p < 0.025 (with permission of Blackwell Science Ltd., from Dvorak er a / . , 1997a).

CELL BIOLOGY OF THE BASOPHIL

177

31.2%, p <0.001; cytoplasmic, 37.6%, p <0.001). The percentage of total cellular gold in the plasma membrane and vesicle compartments was not, however, significantly different.The percentage of total cellular gold in the vesicle compartment (1.3%)was significantly less than that in this compartment in the AND-I phenotype (3.9%, p <0.005). Overall, the gold profile of RB-I1 cells stimulated by the bacterial peptide FMLP showed an amount in the formed CLC compartment in excess of that of all stimulated phenotypes as well as that of unstimulated cells; the amounts in the nuclear and cytoplasmic compartments were less than those in unstimulated cells, and the vesicle and plasma membrane compartments were labeled to degrees equivalent to those in unstimulated cells. Since FMLP stimulation caused a complex morphologic secretory event which occurred with extraordinarily rapid kinetics [the earliest changes being visible in the O-time samples (Dvorak, A.M. et al., 1997b)], and since this event was asynchronous, we examined the same experimental material spatially and temporally (Dvorak, A.M. et al., 1997a, 1997b). These two analyses were useful in further elucidating the traffic pattern(s) of CLC protein. Basophils, incubated as buffer controls over the 10-min interval, showed some degree of cellular perturbation and new sites of CLC protein expression compared to freshly isolated peripheral blood samples (Dvorak and Ackerman, 1989). These changes, by morphologic and morphometric analyses, were considerably less, however, than were present in all samples of FMLP-stimulated basophils. For example, the percentage of altered granules in the unstimulated control was 29.2%, whereas the earliest morphologically identified phenotype, PMD-I, contained 45.1% altered granules, and the peak number of altered granules (64%) occurred in the ANDI phenotype. By time, the largest number of altered granules was recorded in the 10-s sample (52.3%) (Dvorak et al., 1997b). Spatial analysis of CLC protein expression was important in this rapid release reaction. Morphologic analysis of FMLP-stimulated human basophils enabled us to define seven individual phenotypes of stimulated cells. We quantitated CLC protein expression in subcellular compartments of each activated phenotype and compared these data with the individual time points at which these images were evaluated (Dvorak et al., 1997a,b) and with unstimulated cells. By doing so, we were able to extract important localization information from a rapidly evolving, asynchronous degranulation process. For example, kinetic labeling data (Dvorak et al., 1997b) were clearly affected by phenotypic smear over individual time points. The phenotype PMD-I was defined by the presence of unenlarged or minimally enlarged, partially empty, and empty granule containers. Cells of this phenotype expressed the highest number of total cellular gold particles (9.71/pm2) of all phenotypes. Moreover, this value was higher than the number of total cellular gold particles present at each time point examined,

178

ANN M. DVORAK

all of which contained cells of multiple phenotypes (Dvorak et al., 1997b). PMD-I cells were present in the 20-s and 1-min samples (Dvorak et al., 1997b). The PMD-I basophil phenotype characteristically had more altered granules (than unstimulated cells) and had a gold labeling profile of cell compartments in the following rank order: cytoplasm > nucleus > granule > plasma membrane > formed CLCs > vesicles. Of these labeled compartments, granular and formed CLC labels were diminished and plasma membrane label was increased (compared to that of unstimulated cells). An additional characteristic of cells comprising this phenotype was that most of the gold label in granules was diffuse and not associated with formed intragranular CLCs, indicating that solubilization of these crystals had occurred. Additionally, both the percentage of total cellular gold label in the plasma membrane and the percentage of total cellular vesicles that contained gold label (indicating the presence of CLC protein) were markedly increased over those in unstimulated cells. In summary, the PDM-I phenotype is characterized by vesicle-rich basophils actively transporting packets of granule materials to the plasma membrane in the absence of granule extrusion. The phenotype PMD-I1 was defined by the presence of enlarged, partially empty and empty, nonfused granule chambers. Cells of this phenotype expressed the lowest number of total cellular gold particles (2.28/pm2) of all stimulated phenotypes but about twice that of unstimulated cells. This phenotype was distributed over the lo-, 20-, and 30-s and 1-min time intervals (Dvorak et af., 1997b). The PMD-I1 basophil phenotype also had more altered granules than unstimulated cells. The rank order of the gold labeling profile in cellular compartments was cytoplasm > granule > nucleus > formed CLCs > plasma membrane > vesicles. An important characteristic of these cells was the retention of granule and formed CLC gold label, exceeding several actively releasing phenotypes (e.g., PMD-I, CDB). Also, the percentage of gold label in the plasma membrane compartment did not differ from that of unstimulated cells and was considerably less than that of actively releasing PDM-I cells. The percentage of cytoplasmic vesicles containing CLC protein gold label was increased over that of unstimulated cells, decreased compared to that of the actively releasing phenotypes (PMD-I, AND-I, and CDB), and not significantly different from that of the recovering phenotypes (RB-I and RB-11). These data suggest that an early, unbalanced vesicular influx has occurred and that these vesicles have fused with and expanded the granule chambers but that vesicular transport of granule contents out of the cell, although small in amount, is also in progress. In summary, the PMD-I1 phenotype is characterized by basophils experiencing greater vesicular traffic in than out, leading to expanded, partially empty, and empty granule chambers.

CELL BIOLOGY OF THE BASOPHIL

179

The phenotype AND-I was morphologically defined by the presence of intracytoplasmic degranulation channels formed by intergranule fusions (Dvorak, 1988a; Dvorak et al., 1980b; Findlay et al., 1981). The resultant chambers contained mixtures of granule contents, including particles, dense concentric membranes, and formed CLCs. Cells of this phenotype expressed a level of total cellular gold particles (representing CLC protein) that was not significantly different from that of unstimulated cells. This phenotype was present in 20-s and 1-, 2-, 5,and 10-min samples (Dvorak et al., 1997b). The percentage of altered granules peaked in the AND-I phenotype (64%), and the number of compartments labeled with gold increased to include newly formed degranulation channels and their membranes. The rank order of gold label in cellular compartments was cytoplasm > formed CLCs > nucleus > plasma membrane > degranulation channel membrane > vesicles > granules > degranulation channel lumens. The percentage of total cell gold label in the plasma membrane (12.2%) of the AND-I phenotype was high, reflecting an actively secreting phenotype. When the plasma membrane and degranulation channel membrane labels were combined, the total membrane label of AND-I cells (with cytoplasmic degranulation chambers) was equivalent to the plasma membrane label of AND-I1 cells (which have externalized all degranulation channel membranes to the plasma membrane). The total number of cytoplasmic vesicles available for transport diminished in AND-I cells compared to that of unstimulated cells. However, 20% of the declining available vesicles contained CLC protein compared to unstimulated cells (1%)or stimulated cells at time 0 (1.6%) (Dvorak et af., 1997b). This elevated peak of gold-loaded vesicles in ANDI cells (20%) exceeded that of PMD-I cells (14%) and AND-I1 cells (12%). These findings suggest that increased vesicle transport of CLC protein beyond that of the PMD-I phenotype occurs in the AND-I phenotype, as might be expected if vesicle flow was faster in these cells (Dvorak and Dvorak, 1975). As postulated in the generalized degranulation model (Dvorak and Dvorak, 1975), this faster flow would facilitate fusions of granules to form the observed degranulation chambers in the AND-I phenotype. A process similar to this has been documented during AND of guinea pig basophils (Dvorak et af., 1981b). In summary, the AND-I phenotype, an actively releasing cell, is characterized by basophils with continued, rapid vesicular traffic out of cells concomitant with the formation of intracytoplasmic degranulation chambers by intergranule and vesicle fusions. As expected, the plasma membrane was positive for CLC protein in these actively and rapidly secreting basophils. The phenotype, AND-11, was defined by the extrusion of membranefree granules through plasma membrane pores in the entire circumference of the cell. The granule contents that were extruded included dense particles, dense concentric membranes, and CLCs (Dvorak et al., 1991a). Cells

180

ANN

M. DVORAK

of this phenotype also expressed a level of total cellular gold particles similar to that of unstimulated cells. The AND-I1 phenotype spanned the 20-s, 30-s, 1-min, 2-min, and 10-min samples (Dvorak et al., 1997b). The number of altered granules remaining in the cytoplasm of cells in the ANDI1 phenotype decreased to values similar to those of unstimulated cells and to those of cells in recovering phenotypes (RB-I, RB-11). The total number of granules in this phenotype was the lowest of all phenotypes except the CDB phenotype. The rank order of percentage of total gold label in cellular compartments was cytoplasm > nucleus > plasma membrane > formed CLCs > vesicles > granule. The proportion of total cell gold residing in the granule compartment (2.1%) was the lowest of all stimulated phenotypes except that of the granule-free CDB phenotype. A significant proportion of the total cell gold was in the plasma membrane (16.5%) in this actively releasing AND-I1 phenotype. The number of total available cytoplasmic vesicles was diminished in AND-I1 cells (0.71/~m2)-12% of these were transporting CLC protein. In summary, the AND-I1 phenotype was actively undergoing AND characterized by extrusion of granule contents; vesicular transport of CLC protein was also evident in this phenotype. The phenotype CDB was defined by the absence of cytoplasmic granules. These cells were activated basophils which had just completed AND by extrusion of all of their granules. Cells of this phenotype expressed a level of total cell gold representing expression of CLC protein that was significantly less than that of the PMD-I early-releasing phenotype. CDB cells were present in the 20-s, 30-s, 1-min, 5-min, and 10-min samples, with the greatest concentration in the 30-s and 1-min samples (Dvorak et al., 1997b). There were no altered granules in CDB cells. The gold labeling profile demonstrated the following rank order for cellular compartments: cytoplasm > nucleus > plasma membrane > formed CLCs > vesicles. Granule label for CLC protein was absent. Of the total cellular gold label, 13.8% was associated with the plasma membrane, providing a ready source of CLC protein for endocytosis of this recently released granule protein back into the same cell. Evidence for this occurrence was supported by a substantial peak (37%) in gold-loaded vesicles (all of which were devoid of visible granule contents) in cells which expressed the smallest number of available cytoplasmic vesicles (0.33/km2) of all stimulated phenotypes. In summary, the CDB phenotype has completed release of all particle-filled granules, contains no newly evident, homogeneously dense granules, and is actively engaged in the endocytosis of previously externalized, plasma membranebound CLC protein. Thus, the primary event in the CDB phenotype, established by immunogold analysis of CLC protein, is the recycling into the cell of CLC protein by vesicular transport. The phenotype RB-I was defined by the presence of substantial numbers of reconstituted particle-containing granules, occurring at later time points

CELL BIOLOGY OF THE BASOPHIL

181

[2-10 min and predominating at 5 min (60%) (Dvorak et al., 1997b)l after FMLP stimulation. These reconstituted granules were filled with dense particles and formed CLCs; a substantial number of incompletely filled granules of normal size and smaller full granules were present in RB-I cells. RB-I cells had an amount of total cell gold label not significantly different from that in unstimulated cells. The RB-I phenotype characteristically had significantly smaller numbers of altered granules than the releasing phenotypes PMD-I, PMD-11, and AND-I but significantly more altered granules than the later recovering phenotype, RB-11. The gold labeling profile rank order of cellular compartments in RB-I cells was formed CLCs > cytoplasm > granule > nucleus > plasma membrane > vesicles. Thus, the formed CLC and granule compartments of RB-I cells had gold-labeled values in excess of or similar to those of unstimulated cells and all preceding phenotypes; only RB-I1 cells had values of gold label in excess of these two compartments in the RB-I phenotype. Of the total cell gold label, 7.7% was still bound to the plasma membrane in the RB-I phenotype. The number of cytoplasmic vesicles was increased to values similar to those of unstimulated cells. The percentage of these vesicles that were gold-loaded, however, was still greater than that in unstimulated cells. This value (7%) was considerably decreased compared to the highest value in the preceding CDB phenotype (37%). While many CLC protein-containing vesicles were electron-lucent in the RB-I phenotype, many gold-loaded vesicles filled with electron-dense material were also present. Transport of CLC protein from the plasma membrane, in electron-lucent vesicles, as well as from intracellular synthetic sites, in granule content-containing vesicles, may be occurring simultaneously in the RB-I (recovering) phenotype. In summary, the RB-I phenotype has reconstituted many cytoplasmic granules and is transporting CLC protein in electron-lucent, gold-loaded vesicles into the cell from the plasma membrane and within the cell (perhaps from synthetic sites) in gold-loaded vesicles filled with electron-dense material. Granules and their contained CLCs account for most of the CLC protein gold label in this recovering phenotype. The phenotype RB-I1 was defined as a cell phenotype with completely reconstituted granules and no evidence of secretion. A distinctive finding in the RB-I1 phenotype was the emergence of a homogeneously dense granule population that contained large numbers of gold particles indicating CLC protein (Dvorak et al., 1994c, 1997a). RB-I1 cells expressed levels of total cell gold similar to those of unstimulated cells. RB-I1 cells were distributed among 2-, 5, and 10-min samples (Dvorak et al., 1997b). The percentage of altered granules (of a total number of granules similar to that in unstimulated cells) was significantly less than that in the unstimulated cells that were incubated for similar times in buffer, as well as that in the

182

ANN M. DVORAK

recovering phenotype, RB-I. The gold labeling profile rank order for cellular compartments was formed CLC > granule > cytoplasm > nucleus > plasma membrane > vesicle. Of these, the formed CLC compartment exceeded that in unstimulated cells, and the granule compartment was equivalent. These values also significantly exceeded the same compartment label values in all previous phenotypes (except that of the granule label in PDM-I1 cells). The plasma membrane gold label was not significantly different from that of unstimulated cells, and the number of cytoplasmic vesicles had returned to control levels. The percentage of gold-loaded vesicles had also returned to that of unstimulated cells. More of the gold-loaded vesicles were, however, filled with visible electron-dense material, representing a reversal of visible vesicle contents, in contrast to other stimulated phenotypes and unstimulated cells. While the source of some vesicular transport of CLC protein could continue to be from the plasma membrane, synthetic (full) vesicles could also be carrying CLC protein to newly recovering granules. Alternatively, recovery of CLC protein and dense granule contents by endocytosis could be responsible for some granule recovery in the RB-I1 phenotype. In summary, the RB-I1 phenotype has reconstituted its granules and also contains a new granule population, heavily stained for CLC protein, that resembles eosinophil primary granules (Dvork et al., 1988).Most of the gold label in the RB-I1 phenotype is granule- and formed CLC-associated; plasma membrane label and labeled cytoplasmic vesicles have decreased to control levels. Some interrelationships of morphological findings and immunogold labeling (indicating CLC protein) in phenotypic subsets of human basophils responding to FMLP are illustrated graphically in Figs. 22, 24, and 25. Figure 22 shows that the number of cytoplasmic vesicles drops in stimulated phenotypes expressing altered granules (from control levels) and rises in phenotypes displaying less evidence of granule change. The exception is the CDB phenotype, which has the least number of altered granules (since by definition they have all been extruded); these cells also have few cytoplasmic vesicles. Figure 24 shows the relationship of CLC protein gold label distribution in formed CLCs in unstimulated cells and in stimulated phenotypes. Phenotypes that show increased amounts of extruded formed CLCs to the extracellular space (or into intracytoplasmic degranulation chambers) display a diminution of CLC protein label in the formed CLCs within granules, with a concomitant increase of CLC protein label in the formed extracellular CLCs. Recovering phenotypes (RB-I and RB-11) shift label in formed CLCs back to granules, and no label remains in the extracellular location. Figure 25 shows relationships of CLC protein label in four cellular compartments of unstimulated and stimulated cells. The compartments (granule, formed CLC, plasma membrane, and vesicle) are interrelated and

CELL BIOLOGY OF THE BASOPHIL

183

generally show that releasing phenotypes (PMD-I, AND-I, AND-11, and CDB) lose CLC protein from granules (Fig. 25A) and that recovering phenotypes (RB-I and RB-11) restore CLC protein into the granule compartment. The formed CLC compartment gains CLC protein in all phenotypes over that of unstimulated cells, except in the PDM-I phenotype, where formed CLC label diminishes, and in the CDB phenotype, which is equivalent to that of unstimulated cells (Fig. 25B). Releasing (PMD-I, AND-I, AND-11, and CDB) phenotypes have CLC protein in the plasma membrane compartment (Fig. 25C) that returns to baseline levels in recovering phenotypes (RB-I and RB-11). The PDM-I1 phenotype has plasma membrane label equivalent to that of unstimulated cells, indicating that they are transporting minor amounts of this protein to the plasma membrane. While the percentage of total cellular label of CLC protein in vesicles is small (Fig. 25D), gold-labeled vesicles are present in both releasing and recovering phentoypes (Fig. 11). When the percentage of total cellular vesicles that are gold-labeled (indicating transport of CLC protein) is calculated for unstimulated and FMLP-stimulated cells (Fig. 11),it becomes clear that the releasing phenotypes PMD-I (14%), AND-I (20%), AND-I1 (12%), and CDB (37%) and the early recovering phenotype RB-I (7%) all have significant transport of CLC protein in vesicles (Fig. 11) compared to that of unstimulated cells (1%;p <0.001). However, the RB-I1 phenotype (3%) does not have a significantly greater number of gold-labeled vesicles when compared to that in unstimulated cells 0, = NS). It is also interesting that the CDB phenotype, poised at the interface between complete release and the onset of recovery, has 37% of its cytoplasmic vesicles labeled with gold (p <0.001 compared to unstimulated cells, PMD-I, PMD-11, AND-11, RB-I, and RB-11phenotypes;p c0.05 compared to AND-I phenotype) (Fig. ll),despite having the smallest number of cytoplasmic vesicles available for such transport (Fig. 11). The PMD-I1 phenotype clearly shows differences compared to other activated phenotypes, indicating that, as seen by their morphology, they retain large amounts of granule and formed CLC-associated CLC proteini.e., this phenotype is not primarily a releasing phenotype for this protein after FMLP stimulation.

D. FMLP-Induced Redistribution of Formed CLCs and CLC Protein 1. Kinetic Analysis CLCs are visible as formed entities in several locations within or associated with FMLP-stimulated basophils (Dvorak and Ackerman, 1989; Dvorak et

184

ANN M. DVORAK

al., 1997a,b). These sites include intragranular (Fig. 23C), cytoplasmic (Fig. 26), within degranulation channels, and extracellular sites (Fig. 27). We quantitated the portion of total formed CLC gold label (indicating the presence of CLC protein) that resided in these individual locations in unstimulated cells and in cells after FMLP stimulation (Fig. 28). One hundred percent (100%) of the gold label of formed CLCs was located within granules in unstimulated cells and in basophils at 0 time after FMLP stimulation. The value dropped to a low of 16% at 1 rnin (p <0.001 compared to unstimulated and 0-time samples) and returned to unstimulated levels at 5 rnin (p = NS). Reciprocally, the percentage of formed CLC gold label associated with extracellular CLCs rose to peak levels by 30 s (81.8%) and 1 rnin (82%) after FMLP (p <0.001 compared to unstimulated and 0time levels). Amounts of gold label associated with formed CLCs in the cytoplasm were significantly elevated at 2 rnin (58.6%,p <0.001) compared to unstimulated cells, 0 time and at 5 min (7.4%, p <0.025), and at 10 min (24.7%, p <0.001) compared to 0 time. Gold label associated with formed CLCs in degranulation channels was elevated at 20 s (20.8%), p C0.025 compared to unstimulated cells; p <0.001 compared to 0 time; p <0.001 compared to 10 and 30 s and 1, 2, 5 , and 10 min) and at 1 rnin (2%) after FMLP stimulation (p <0.005 vs 2 min;p <0.025 vs 5 min;p <0.05 vs 10 rnin). The amount of total cellular gold label for CLC protein in formed CLCs at 0 time and later (2, 5, and 10 min) exceeded label in similar structures in unstimulated cells (Fig. 21). When the proportion of gold-labeled CLC protein in formed CLCs at various subcellular locations was followed by time after stimulation, it was clear that most of these unique crystalline structures resided within granules immediately after stirnulation between 0 time and 10s, that they and their label were secreted by extrusion between 20 s and 2 min, and that they reformed within granules in cells prepared for electron-microscopic study between 2 and 10 rnin after stimulation. These studies of the changing distributions of label indicating CLC protein over time after FMLP stimulation of human basophils document release mechanics and recovery potential for this granule protein in activated basophils.

2. Phenotypic Analysis Formed CLCs were regularly but variably present in the main particle granules of mature basophils (Fig. 23C) in situ or developing in vitro (Dvorak and Ackerman, 1989; Dvorak el al., 1994~).Redistribution of these structures and/or their contents was induced by FMLP (Dvorak et aL, 1991a). We noted by routine ultrastructural analysis that CLCs were extruded to the exterior of cells through pores in the plasma membrane in parallel with extrusion of secondary granules (Fig. 27). This occurred regu-

FIG. 26 RB-I basophils, prepared for immunogold demonstration of CLC protein 2 min after FMLP (A) or for immunogold specificity control by substituting normal rabbit serum for the primary antibody (B), show large, formed CLCs (CLC) in the cytoplasm. These typical structures are round, non-membrane-bound, and homogeneously dense. In A, the cytoplasmic CLC is labeled with 10- and 30-nm gold particles; the control (2) is negative (B). Note the large number of granule particle-filled cytoplasmic vesicles adjacent to the cytoplasmic CLC in A (with permission of Blackwell Science Ltd., from Dvorak er al., 1997a). Bars: 0.6 pm.

FIG,27 Extruded, formed CLCs are homogeneously dense, spherically shaped structures (open arrowheads) adjacent to the cell surface, commonly present in the AND-I1 phenotype (A, 1min, and B, 20 s after FMLP). Also extruded from the main particle granule population are dense concentric membranes (closed arrowheads, A,B) and membrane-free granule particles (arrow in B). The irnmunogold preparation for CLC protein in A shows a 30- and 10-nm gold-labeled, extracellular CLC; the plasma membrane is also labeled with the 30-nrn goldlabeled secondary antibody. The extruded, dense concentric membranes are not labeled. In B, a CLC-absorbed primary antibody was used (control 3), and all extruded structures and plasma membrane are negative (with permission of Blackwell Science Ltd., from Dvorak et al., 1997a). Bars: (A) 0.4 pm; (B) 0.6 pm.

CELL BIOLOGY OF THE BASOPHIL

187

larly in the AND-I1 phenotype. CLCs were also released into degranulation channels in AND-I cells. When CLCs reached the exterior of the cell, they became associated with the cell surface and were regularly identified in this extracellular location between 20 s and 2 min following FMLP stimulation (Figs. 23B and 27). Formed CLCs were also identified as large, nonmembrane-bound, rounded structures in the cytoplasm of FMLP-stimulated basophils (Fig. 26). These cytoplasmic structures developed by 30 s poststimulus and could be found throughout the 10-min interval we studied, but with variable frequency. Infrequently, similar, rounded formed CLCs developed in the nuclei of stimulated cells. We quantitated gold labeling of these differentially located formed CLC structures in phenotypes induced by FMLP stimulation (Fig. 24). The percentage of total formed CLC gold labeling in the individual sites of (1) intragranular formed CLCs (Fig. 23C), (2) extracellular formed CLCs (Figs. 23B and 27), (3) cytoplasmic formed CLCs (Fig. 26), and (4) degranulation channel formed CLCs is shown by phenotype in Fig. 24. Dramatic shifts in the location of formed CLCs stimulated by FMLP were accompanied by changes in the percentage of total formed CLC gold label associated with them. For example, 100% of the gold label for formed CLCs was in the intragranular location in unstimulated control cells. The amount for this intragranular location was markedly decreased in the AND-I1 phenotype and returned to peak levels in the RB-I1 phenotype. Gold-labeled extracellular CLC was increased in the AND-I1 and CDB phenotypes and absent in the RB-I and RB-I1 phenotypes. A peak in gold-labeled formed CLCs in degranulation channels coincided with the actively extruding AND-I1 phenotype. The AND-I phenotype, which was also releasing formed CLCs from degranulation channels to the exterior, had extraordinary amounts of gold-labeled formed CLCs in the cytoplasm. Similarly, the RB-I phenotype, which contained large amounts of gold-labeled, intragranular formed CLCs, also had a large amount of gold-labeled formed CLCs in the cytoplasm. The RB-I1 phenotype had diminished amounts of gold-labeled formed CLCs in the cytoplasm.

E. Profiles of FMLP-Stimulated Human Basophils Gold profiles of FMLP-stimulated human basophils, analyzed by time and morphologic phenotypes, show important differences in expression of CLC protein by an asynchronous cell population undergoing a complex secretory1 endocytotic event. The rapidity of the release reaction stimulated in human basophils by FMLP (Hook et al., 1976; Siraganian and Hook, 1977;Warner er al., 1989), the asynchronous response of individual cells within the cell population (Dvorak er al., 1997a,b), and the short but significant cell han-

188

ANN M. DVORAK

dling time required following stimulation prior to fixation for electronmicroscopic analysis all contribute to the complexities of analysis of an asynchronous cell population undergoing a complex secretory/endocytotic event. For example, the 0 time point after stimulation already contained cells undergoing changes stimulated by FMLP. This was evident in several ways. In buffer-incubated unstimulated samples, 50% of the basophils showed no morphologic change, and 50% were classified as of the PMD-I phenotype, based on minor losses of dense particles from several granules per cell (Fig. 10). At 0 time following stimulation with FMLP, this ratio had already shifted to 80% PMD-I phenotype and 20% no change in morphology. Also, documentation of the temporal evolution of changes in basophils stimulated by FMLP required analysis of the 0 time point, since a significant expansion in the mean number of cytoplasmic smooth membrane-bound vesicles, compared to that of unstimulated cells, occurred at this time point only and preceded the significant increase in altered granules at 10 s which characterized the PMD-I phenotype. The phenotypic composition of the 10-ssample consisted of 60% PMD-I cells and 40% PMDI1 cells (Fig. 10). The later phenotype is morphologically characterized by cells with greatly expanded, nonfused, partially empty, and empty granule containers in the cytoplasm. The 20-s sample contained a mixture of phenotypes which impacted on events documented at this time poststimulus (Fig. 10). Half of the cells were still of the PMD-I phenotype, and 17% were PMD-I1 cells, representing a considerable decrease of the later phenotype. The three phenotypes that typify the combined morphologies of AND were all represented. These include AND-I (17%), AND-I1 (8%), and CDB (8%).These phenotypes are recognized primarily by the formation of intracytoplasmic d e g ra d a tio n channels (AND-I), the extrusion of granules and associated granule contents (AND-11), and virtually complete absence of granules (CDB). The impact of these phenotypes on the 20-s sample was reflected in a significant loss of granule and vesicle numbers. Elevated proportions of altered granules among the remaining granules reflected continuing PMD (50% PMDI phenotype). The 30-s sample contained a different mixture of the same phenotypes (Fig. 10). Thus, 20% were PMD-I cells, 20% PMD-I1 cells, 30% AND-I1 cells, and 30%CDBs. Conspicuously absent from this time point were ANDI cells. Continued significant increases in altered granules and decreases in the number of vesicles and granules, compared to the 0 time point, persisted. By 1 min after FMLP (Fig. lo), a nadir was reached in granule number, reflecting the large proportion of the CDBs present (40%). Other represented phenotypes included 10% PMD-I, 20% PMD-11, 10% AND-I, and 20% AND-11.

CELL BIOLOGY OF THE BASOPHIL

189

The 2-min sample was the first to contain a mixture of actively degranulating phenotypes (25% AND-I; 17% AND-11) and recovering phenotypes (33% RB-I; 25% RB-11) (Fig. 10). The impact of the later phenotypes on the 2-min samples was reflected in increases in cytoplasmic granules and vesicles (to levels not significantly different from those in unstimulated or 0-time samples). The majority of basophils in the 5- and 10-min samples were of the recovering phenotypes (60% RB-I, 10% RB-I1 at 5 min; 8% RB-I, 50% RB-I1 at 10 min). Both samples did, however, contain releasing phenotypes as well (5 min: 20% AND-I, 10% CDB; 10 min: 17% AND-I, 17%AND-I1,8% CDB). The numbers of cytoplasmic vesicles and granules, as well as the percentage of altered granules, were all not significantly different from unstimulated and 0-time samples. The phenotypic smear over the individual times examined in the kinetic study of human basophils stimulated with FMLP also impacted on the label data for CLC protein. For example, the greatest number of gold particles per cell occurred in the PMD-I phenotype (9.71/pm2) (Dvorak et al., 1997a), a value not equalled at any time point in the kinetic analysis. The percentage distribution of total gold label for CLC protein in individual cellular compartments was also impacted by phenotypic smear in the kinetic analysis. For example, in the phenotypic analysis of FMLP-stimulated samples, phenotypes displaying the greatest percentage of total label for five compartments were as follows: nuclear, CDB, 36.3%; cytoplasmic, PMD-I, 40.1%, CDB, 40.3%; granular, PMD-II,23.8%; plasma membrane, AND-II,l6.5%; formed CLCs, AND-I, 21.9% (Dvorak et al., 1997a). Inspection of the same data for the kinetic analysis reveals that the greatest proportions of total cellular label for these compartments were as follows: nuclear, 10 s, 34.5% (a time point which contained no CDB cells); cytoplasm, 1 min, 46.3% [within which both PMD-I (10%) and CDB (40%) phenotypes resided); granular, 10 min, 25.9% (which contained no PMD-I1 cells); plasma membrane, 10 min, 11.5% (which contained 17% AND-I1 cells); formed CLCs, 5 min, 41.4% (which contained 20% AND-I cells). Other differences are apparent in the relative distribution of gold label for the CLC protein when the phenotypic and kinetic analyses are compared. Understanding of both the impact of time and the changing morphologies on the relative distributions of CLC protein during FMLP stimulation of basophil secretion is facilitated by this dual approach for immunogold analysis. The time course of the relative distribution of gold label indicating CLC protein in subcellular locations of formed CLCs in FMLP-stimulated basophils was also affected by phenotypic smear over collected time points (Figs. 24 and 28). Good correlation was evident between the nadir for goldlabeled formed CLCs in granules occurring at 1 min and that in the CDB phenotype. The 1-min sample contained the highest percentage of this phenotype (40%).Similarly, the 30-s sample (30% CDB cells) had low levels

190

ANN M. DVORAK

FIG. 28 Percentage of gold label representing CLC protein in formed CLCs at variable locations of human basophils stimulated with FMLP and in buffer-incubated control cells analyzed by time. CLC-G, CLC granule location; CLC-EC, CLC extracellular location; CLCCyto, CLC cytoplasmic location; CLC-Channel, CLC degranulation channel location (with permission of Karger, Basel, from Dvorak et al., 1997b).

of label in formed CLCs in the granule location, a proportion that was also influenced by 30% AND-I1 cells in the 30 s sample. The peak elevation in gold-labeled, formed CLCs in the extracellular site was shared between the 30-s and the 1-min samples and peak levels in the phenotypic analysis occurred in the prevalent phenotypes AND-I1 and CDB. Other relationships among stimulated morphologic phenotypes and the kinetic analysis are evident (Figs. 24 and 28). For example, a peak in gold-labeled formed CLCs in degranulation channels occurred at 20 s, the time point where the phenotype AND-I (cells with the onset of degranulation channel formation) first appeared; this peak in gold-labeled formed CLCs in degranulation channels preceded peak levels of gold-labeled formed CLCs that had been extruded to the extracellular space by 30 s and 1 min (samples containing 60% of the actively extruding phenotypes, AND-I1 and CDB). The maximum number of cytoplasmic vesicles occurred in basophils at 0 time after FMLP, a value that plunged thereafter to the lowest value at 30 s after FMLP. The maximum loss of entire granules and their membranes by fusions and extrusions occurred at 1 min after FMLP; the maximum loss of granule particles in the absence of granule extrusion occurred at 10 s after FMLP. The sequential documentation of these anatomic events identifies an expanded pool of vesicular carriers available for transport (0 time) preceding the maximum emptying of granule containers at 10 s after stimulation. Both of these events preceded maximum granule extrusion at 1 min. The analysis of activated phenotypes stimulated by FMLP (Dvorak

CELL BIOLOGY OF THE BASOPHIL

191

et al., 1997a) showed that the PMD phenotypes were present at 0 time and 10 s, but AND phenotypes showing secretion by granule extrusions were absent. Similarly, at 1 min after FMLP, the PMD phenotypes were absent and AND phenotypes prevailed. Thus, the kinetic analysis shows that PMD, characterized by increased vesicles and altered granules, precedes AND, which is characterized by decreased vesicles and granules.

F. FMLP-Activated Morphologic Phenotypes and Kinetics Analysis of FMLP-activated morphologic phenotypes and multiple kinetic time points collected after stimulation is complementary for understanding basophil release and recovery responses. Temporal and spatial study approaches contribute to the understanding of complex secretory events of which human basophils are capable. The speed of secretion of performed stores of biochemicals from FMLP-stimulated basophils and the fact that an asynchronous responding cell population constitutes each time point collected for ultrastructural and immunogold analysis contribute to the complexity of this system for interpretation. Analyzed together, these complexities can contribute to our understanding of responding cellular populations obtained at frequent intervals preceding, encompassing, and extending beyond peaks of secretion and generation of biochemical mediators by the cells under study. For example, we considered the impact of time after stimulation, and of changing phenotypic morphologies, on the rapid secretion and recovery processes associated with CLC protein localizations in human basophils. The temporal analysis revealed important data which were obtained only by this route-i.e., maximum vesicle levels at 0 time after stimulation-and the spatial analysis revealed that the PMD-I phenotype had the highest cellular label for CLC protein of all the defined activated phenotypes (Dvorak et al., 1997a). The individual activated phenotypes were roughly coincidental with the time analysis, but imperfectly so. And, as expected in biological systems, this phenotypic smear over several individual times collected often significantly impacted on the labeling data for CLC protein localizations for that time point. Thus, the dual analysis we used clearly revealed that individual points in the kinetic study were constituted by populations of cells undergoing asynchronous reactions to the secretogogue, FMLP, resulting in different phenotypic morphologies. Both the kinetics and the phenotypes of this reaction impacted on the labeling information for secretion and recovery of CLC protein. For example, the nadir of gold-labeled formed CLCs occurred at 1 min after stimulation and in the CDB phenotype, a phenotype which comprised 40% of the l-min sample. The dual morphometric analysis used here is necessary

192

ANN

M. DVORAK

to understand the impact of time and changing activation morphologies on redistribution of immunogold label in cellular compartments and in vesicle populations of cellular populations undergoing complex secretory and recovery reactions. The morphological complexities of secretion exhibited by human basophils clearly reveal cells asynchronously undergoing an ordered series of events that constitute a continuum of change; examination of individual time points poststimulation reveals cells at variable points along this continuum. The responding populations give rise to the mediator release curves that typify several secretogogue classes for the total cell population. Recently, distributions of single human basophil activation responses to a variety of stimuli have been examined (MacGlashan 1995; MacGlashan et aL, 1994b). Activation events so examined included cytosolic calcium, cell surface adhesion molecule expression, actin polymerization, cell size, and acridine orange-labeled granules. These studies confirmed that individual basophils respond in a graded fashion and could be captured at intermediate stages of activation. Thus, these cell-biological studies of single activated basophils confirm the ultrastructural, immunogold, and enzyme-affinitygold analyses reviewed in-depth herein.

IX. Histmaine Distribution in FMLP-Stirnulated Human Basophil Granules Granulocytes were once considered to be the cell source of histamine in human peripheral blood (Graham et al., 1955). The subcellular location of histamine in human leukocytes was found by subcellular fractionation to be within granules, structures shown to differ from lysosomes by their lack of P-glucuronidase (Pruzansky and Patterson, 1967). Histamine release from washed leukocytes of allergic individuals was demonstrated after incubation with specific antigen (Lichtenstein and Osler, 1964). Since these initial studies (Graham et al., 1955; Pruzansky and Patterson, 1967; Lichtenstein and Osler, 1964), it has become clear that (i) basophils represent the single intravascular cellular source of peripheral blood histamine in humans and (ii) a wide variety of secretogogues induce regulated histamine secretion from basophils. We used a new enzyme-affinity-gold ultrastructural method to detect and quantitate the density of enzyme-reactive label indicating histamine in human basophil granules following stimulation with FMLP (Dvorak ef a[., 1993b, 1994b, 1995a).

CELL BIOLOGY

193

OF THE BASOPHIL

A. Histamine Distribution and FMLP-Stimulated Histamine Release Secretory granules of FMLP-stimulated human basophils showed several major changes. These included altered granule content, characterized by less dense particle packing (which resulted in partially empty and empty granule containers), and extrusion of individual granule particulate structures to the cell surface. Granules with altered contents often contained CLCs, and CLCs were also extruded to the cell surface after fusion of individual granule membranes to the plasma membrane. DAO-gold staining of human basophils with such granule alterations showed that unaltered cytoplasmic granules were labeled for the presence of histamine and that extruded granules, attached to the cell surface, were generally devoid of histamine. CLCs within unaltered and altered cytoplasmic granules and CLCs after extrusion to the cell surface were devoid of histamine. Altered granules showing focal or complete release of granule particles did not contain histamine either in piecemeal areas of granule particle release or in granules largely devoid of their particles. We quantitated these findings (Table VIII) and showed that releasing human basophils contained a density of 30.77 gold particleslpm’ in their unaltered, particle-filled granules, and 4.08 gold particles/pm* in altered, particle-poor granules ( p c0.05). Neither the extruded particulate granules nor the granule-derived CLCs contained gold label in their extracellular location. The background gold density was 3.88/pm2, a value significantly different from that in unaltered, particle-filled cytoplasmic granules ( p <0.001) but essentially the same as that in altered, particle-poor cytoplasmic granules ( p = NS). Thus, it is clear that histamine is released from intracy-

TABLE Vlll Density of DAO-Gold Labeling in Human Basophil Granules Indicating Histamine during Secretion and Recovety

Phase Secretion Recovery

Dense granules

Altered granules

Background

30.77* 2 16.1 (10)

4.08 2 2.97 (10)

3.88** -C 0.9 (20)

33.49*** 2 4.21 (10)

5.25

?

1.24 (10)

Note. Density is expressed as the number of gold particles per square micrometer t SE. Numbers in parentheses indicate number of granules or Epon backgrounds counted. * p <0.05, (compared with altered granules). ** p <0.001,(compared with dense granules). *** p (0.05, (compared with altered granules or background).

194

ANN

M. DVORAK

toplasmic altered granules and is absent from extracellular granules at early times, concomitant with the biochemical detection of histamine release (Warner et aL, 1989; MacGlashan and Warner, 1991) in FMLP-stimulated human basophils.

6. Histamine Distribution and Recovery from FMLP-Stimulated Secretion

At various recovery time intervals, spanning 10 min to 6 h after FMLP stimulation, human basophils morphologically reconstitute granule contents and no longer contain large numbers of granules with altered contents. Some extruded granule particles and CLCs do persist at the earlier recovery times. As in the degranulation phase, these extracellular structures did not label for the presence of histamine. In the recovery phase, human basophils showed large numbers of cytoplasmic vesicles, increased granule contents, and morphologic evidence of internalization of membrane and materials from the plasma membrane location. Use of cationized ferritin in the postfixation period allowed the identification of granules and/or empty granule containers that were in continuity with the cell surface. A few remaining empty granules contained large CLCs but no granule particles, and they were not in continuity with the cell surface. Cytoplasmic vesicles and unaltered granules were labeled with DAO-gold in human basophils recovering from FMLP-stimulated secretion, indicating the presence of histamine. Large empty granules, either in continuity with the cell surface or not so-connected were devoid of histamine. CLCs within these altered and unaltered granules also did not stain with DAO-gold. Granule particles remaining on the cell surface, or in the process of reinternalization, contained very little histamine. The numerous reconstituted, unaltered granules present in recovering human basophils contained histamine. Quantitation of the DAO-gold label in unaltered dense granules of recovering human basophils (Table VIII) showed a density of 33.49 gold particles/pm2 (indicating histamine), a value significantly higher than histamine label in altered granules of actively secreting basophils (4.08/pm2; p (0.05) and identical to the unaltered dense granules in actively secreting cells (30.77/pm2; p = NS). Background density of label was 5.25 gold particles/pm*, a value significantly less than that of dense granule label of recovering basophils ( p (0.05). Significant differences in background gold particles were not present when degranulation and recovery experiments were compared ( p = NS). The area of dense granules and altered granules that was used for the DAO-gold analysis was also measured in these experiments. Dense granules, whether present in actively degranulating (0.30 pm) or recovering

CELL BIOLOGY OF THE BASOPHIL

195

(0.42 pm) basophils, did not differ in size ( p = NS). Also, neither dense granule population differed significantly in size from the histamine-free, altered granules that were present in actively degranulating cells ( p = NS). These studies confirmed that the granule stores of histamine (Graham et al., 1955) were in membrane-bound cytoplasmic granules, which were filled with electron-dense particles. The density of histamine label was virtually the same in the particle-filled granules of cells stimulated for 1 min with FMLP (i.e., 30.77 gold particles/pm2) as that in the dense granules of cells stimulated for 20 s with FMLP (28 gold particlesipm*) (Dvorak et al., 1994b), despite vastly different morphologies of the responding basophils at these two times after exposure to FMLP (Dvorak et al., 1991a). Similarly, the dense, particle-filled granules (i.e., those illustrating reconstitution in samples recovered 10 min t o 6 h after FMLP stimulation) were labeled for histamine with a granule density (33.49 gold particles/ pm2) analogous to that of the 20-s (Dvorak er af., 1994b) and 1-min samples. However, the presence of dense granule particles per se does not entirely correlate with the ability of the affinity-gold technique to label histamine, since there was little to no histamine labeling of the membrane-free, extruded granule particles which were focally attached to the cell surface. Specificity controls (histamine absorption, diamine oxidase digestion) appropriately abrogated labeling of unaltered basophil cytoplasmic secretory granules with DAO-gold. CLCs are present in basophil granules (Dvorak, 1988a; Dvorak and Ackerman, 1989;Dvorak et al., 1991a, 1994~). These structures are homogeneously dense and assume typical bipyramidal shapes (or irregular, atypical shapes) in their intragranule locations in human basophils. Using an ultrastructural immunogold technique, the major protein constituent of CLCs (CLC protein) has been precisely localized to these intragranular structures (Dvorak and Ackerman, 1989; Dvorak er af., 1994c) (Fig. 29). The enzymeaffinity-gold method to detect histamine did not label CLCs in their intragranular locations (Fig. 29) or in their extracellular location, where they remain attached to basophil cell surfaces following extrusion from FMLPstimulated cells (Dvorak et al., 1995a). Thus, the major secretory granule of human basophils is compartmental-it contains CLC protein within central crystals and histamine in the particle-rich granule matrix surrounding the central crystals. The CLCs that remained in altered, histaminelacking granules in FMLP-stimulated cells (see next) also did not stain with DAO-gold. In samples obtained early after FMLP stimulation, altered granules were present in human basophils undergoing PMD. Thus, in seconds following stimulation, focal pieces of granule materials are secreted, giving rise to focal electron lucencies within granule particles (Dvorak et af,, 1991a). Ultimately, the entire contents of granules are secreted by a vesicular

196

ANN M. DVORAK

FIG. 29 CLC protein (A-C) and histamine (D-F) localization in human basophil granules. Gold particles, indicating CLC protein in the central CLCs (C) within the particulate matrix of granules (G) and overlying the particulate matrix, are seen in A and B. Histamine, indicated by gold particles (D and E), resides in the particulate matrix and not in the homogeneous CLCs (C) within these granules (G). The nonparticulate primary granules (G) are heavily labeled for CLC protein in C but not for histamine in F (with permission, from Dvorak, 1996). Bars: (A,E) 0.1 pm; (B-D,F) 0.2 pm.

transport mechanism (Dvorak et al., 1991a), but the granule membrane (container) is retained in the cytoplasm. These altered granules did not contain DAO-gold histamine label exceeding background densities. Thus, it is certain that these altered granules no longer contain histamine. The altered mast cell granules that occurred in quantity in vivo either in the mast cell-rich inflammatory eye disease of IL-4 transgenic mice (Dvorak et al., 1994e, 1995b) or in human gut mast cells from biopsies of small bowel mucosa in patients with inflammatory bowel disease (Dvorak and Morgan, 1997) similarly had released their histamine content in vivo, as determined with the DAO-gold stain (Dvorak and Morgan, 1997; Dvorak et al., 1994e).

CELL BIOLOGY OF THE BASOPHIL

197

Thus, PMD of human basophils in vitro (Dvorak et al., 1991a), of mouse mast cells in vivo (Dvorak et al., 1994e), and of human mast cells in vivo (Dvorak and Morgan, 1997) results in the emptying of granule containers, which remain morphologically intact but are devoid of histamine. The FMLP-stimulated secretory continuum observed in human basophils resulted in the eventual extrusion of granules and their containers to the extracellular space (Dvorak et aL, 1991a). The resultant human basophils were generally devoid of unaltered granules and altered granules within 1-2 min of stimulation (Dvorak et al., 1991a), a time corresponding to biochemical detection of maximum histamine secretion (Warner et al., 1989; MacGlashan and Warner, 1991). We examined the ultrastructural morphology of granule reconstitution in human basophils recovered 10 min to 6 h after FMLP stimulation (Dvorak et al., 1996b). This process, while not complete in this early recovery study, did produce morphologic evidence of material and membrane conservation, granule content condensation, and synthesis (Dvorak et al., 1996b). DAO-gold staining demonstrated that dense, particle-filled, unaltered granules once again contained histamine but that persistent extracellular granule materials did not. Intracytoplasmic granules, in the process of condensing dense materials within them, contained histamine, and focal accumulations of vesicles in the subplasma membrane and perigranular areas, as well as in expanded Golgi structures, also contained gold label for histamine. All such vesicular accumulations were absent at peak release times (1-2 min) in FMLP-stimulated cells. Their appearance in the recovering cells is similar to that in guinea pig basophils recovering from degranulation (Dvorak et al., 1982c, 1985d, 1987). Their histamine content, as described here, suggests several possible routes for the reconstitution of granular histamine in human basophils: (i) reuptake of previously released histamine, and (ii) newly synthesized histamine in Golgi structures. As histamine from either route is reincorporated into granules, induction of granule content condensation would occur, analogous to the granule condensation properties of histamine (Curran and Brodwick, 1991; Fernandez et al., 1991; Nanavati and Fernandez, 1993). In summary, using DAO-gold, dense, unaltered granules of actively releasing human basophils, as well as similar structures that are reconstituted after FMLP-stimulated degranulation, contain histamine but a!tered granules in stimulated cells undergoing d eg rad a tio n are devoid of histamine before their membranes are extruded. Granule reconstitution is associated with DAO-gold-positive cytoplasmic vesicles in the peripheral cytoplasm, near granules, and in Golgi areas. The appearance of these vesicles suggests transport of histamine to granules, derived from new synthesis, reuptake of released histamine, or both, and is associated with striking granule condensation of dense contents that contain histamine.

198

ANN

M. DVORAK

X. Recovery of Basophils from Secretion Granulocytes are generally viewed as short-lived end-stage cells with little or no potential for recovery following secretion of stored granule proteins. Basophilic leukocytes (one of the three granulocyte lineages) have, however, been shown to recover from regulated secretion in two speciesguinea pigs (Dvorak et af., 1982c, 198Sd, 1987) and humans (Dvorak, 1996; Dvorak et af., 199Sa, 1996b,f, 1997a,b). Initially, routine ultrastructural analysis of the changing morphology of previously stimulated cells during recovery intervals established that the cells did not die, were not injured, maintained a mature phenotype, and reconstituted secretory granules by a mixture of synthetic and conservation mechanics (Dvorak et al., 1982c, 1996b). These morphologic events were more extensively probed with a number of ultrastructural methods designed to examine the distributions of basophil-specific surface antigen(s) (localized with an immunoferritin technique) (Dvorak et al., 1985d), of nonspecific esterase(s) [stained with a cytochemical method (Dvorak et af.,1987)], of a serine protease [localized by autoradiography of an isotope-labeled inhibitor of serine protease(s) (Dvorak et al., 1987)] in guinea pig basophils, and with a postfixation electron-dense tracer, cationized ferritin (Dvorak et al., 1995a, 1996b), an enzyme-affinity-gold cytochemical technique to label histamine (Dvorak et aZ., 1995a, 1996f), and an immunogold method to detect the basophil granule protein, CLC protein (Dvorak and Ackerman, 1989; Dvorak et al., 1988,1990a,b, 1991b, 1992c, 1994c,d; Zhou et al., 1992), in human basophils. Detailed ultrastructural studies of the recovery of human basophils (Dvorak, 1996; Dvorak et al., 1995a, 1996b, 1997a,b) and guinea pig basophils (Dvorak et af.,1982c, 198Sd, 1987) from secretion have recently been presented. These extensive morphologic and cell-biologic studies are beyond the scope of this review, and the reader is referred to these prior reports.

XI. Morphometric Analysis of Basophil Degranulation Morphometric analysis of human basophil degranulation stimulated by a cytokine or a chemokine reveals morphologic similarities and differences to that stimulated by anti-IgE or present in experimentally produced contact allergy skin lesions. We examined the ultrastructural morphology of isolated, partially purified peripheral blood human basophils that were stimulated to release histamine with MCP-1, a low-molecular-weight chemokine which is the most potent histamine-releasing cytokine documented to date (Bischoff et al., 1992; Alam, et al., 1992; Kuna et aZ., 1992a) or recombinant

CELL BIOLOGY OF THE BASOPHIL

199

histamine-releasing factor (rHRF) (rp21), a newly cloned, IgE-dependent histamine-releasing factor (MacDonald et aZ., 1995), and compared these events with those induced by anti-IgE and control basophils incubated without secretogogues (Dvorak et al., 1996e). Two time points for each secretogogue were selected for study: one at the half-maximum histamine release interval, and another earlier time, known to precede biochemically detectable histamine release. Four ultrastructurally defined activation events were identified and quantitated in the responding and control basophils as follows: (i) granule-vesicle attachments (GVA), as first identified in human contact allergy skin lesions (Dvorak et al., 1976a), (ii) PMD, (iii) AND, and (iv) uropod formation (Dvorak, 1991). Uropods (tails) are motile structures that have been identified in the basophils of guinea pigs (Galli et al., 1981; Dvorak et al., 1980e, 1981d) and in human basophils (Dvorak et al., 1980, 1981c, 1984b). These activated structures were numerous in preparations of basophils stimulated with HRA-containing mononuclear cell supernatants (Dvorak et al., 1984b), and they were generally found in basophils that lacked ultrastructural evidence of secretion (Dvorak et aL, 1980b, 1981c, 1984b). We found both similarities and differences in the populations of basophils responding to the three secretogogues in this study. Qualitatively, AND, PMD, GVA, and uropod formation were identical, when present. Quantitative differences were, however, evident. For example, the most effective trigger for AND was anti-IgE, whereas MCP-1 was the most effective stimulant for GVA and PMD; uropods predominated in rHRF-stimulated samples. An inverse relationship of the extent of PMD and AND in stimulated samples occurred, and the development of motile structures in basophils were associated with less morphological evidence of secretion in the cell population. These findings define for the first time the ultrastructural events that are stimulated in basophils by two new secretogogues-a chemokine, MCP1, and a cytokine, rHRF-and support the following conclusions: (i) the development of motile structures (uropods) is extensive in rHRF-stimulated samples, but not in MCP-l-stimulated samples; (ii) evidence of secretion from uropod-containing cells is minor; (iii) MCP-1 is a potent stimulant of GVA-structures important for the vesicular transport of granule materials in PMD; and (iv) MCP-1 stimulated the greatest amount of PMD and did so extensively at times preceding the established time of half-maximum histamine release.

A. Control Basophils Control cells incubated in buffer for 30 s or 7 min displayed the previously recorded ultrastructural morphology of unstimulated human basophils

200

ANN M. DVORAK

(Dvorak, 1991,1993; Dvorak and Dvorak, 1993). Rarely, one or two individual granules had altered, electron-lucent contents-these granules were not enlarged or fused. The presence of one such granule was sufficient to include the cell in the category of PMD (such a finding being the minimum requirement for entry into this category) (Table IX). For example, 9% (30 s) and 18% (7 min) of the photographed cells had one or two such altered granules and were classified in the PMD category ( p = NS). These values were significantly less than those of the 2-min sample stimulated with anti-IgE (40%) ( p ~ 0 . 0 0 5p, c0.025) and the 5-s sample stimulated with MCP-1 (55%) ( p <0.001, p <0.001). The 30-s sample stimulated with MCP-1 (28%) had significantly more PMD than the 30-s control sample (9%) ( p <0.05). The 7-min control sample had PMD values similar to both 7-min samples stimulated either with anti-IgE (12%) or with rHRF (9%) ( p = NS; p = NS). Granule-vesicle attachments (see description later), proposed as the mechanism for effecting PMD (Dvorak et al., 1976a), were similar in both control samples (14%, 9%, p = NS). We found no AND in either control, regardless of incubation time. Small numbers of cells displayed uropods (motile configurations) (6% at 30 s; 3% at 7 min; p = NS). Virtually all of the cells were round and demonstrated neither tails nor electron-lucent granules. The unstimulated control samples of partially purified human basophils that were examined in these studies qualitatively resembled peripheral blood basophils that were prepared for ultrastructural analysis without exposure to isolation procedures (Dvorak, 1988a, 1991). The changes recorded in the unstimulated basophils in this study differed quantitatively from the morphologic changes of cells associated with exposure to any of the three secretogogues examined. Each of these triggers was associated with variable amounts of PMD, AND, and GVA, for example, and two were associated with the formation of motility configurations, termed uropods. Controls did not display AND at all, and the amount of PMD, GVA, and uropods expressed was small and similar in the early (30-s) and late (7-min) control samples. An average of 13%of the basophils in the control samples had morphologies consistent with PMD. However, the extent of this process in the control cell populations examined at two time points, as well as within individual cells, was far less than it was in basophils undergoing PMD in stimulated samples.

B. Stimulated Basophils 1. Anti-IgE-StimulatedBasophils Forty percent (40%) of the basophils stimulated with anti-IgE for 2 min showed PMD, generally characterized by two to three empty or partially

TABLE IX Ultrastructural Morphology of Human Basophils Stimulated with Anti-lgE, rHRF, or MCP-1

No. granule-vesicle attachments (96)

Stimulant

Time

Total no. cells

Control Control

30 s 7 rnin

35 67

Anti-IgE Anti-IgE

2 rnin 7 min

30 52

rHRF rHRF

2 min 7 min

36 57

7 (19) 12 (21)

MCP-1 MCP-1

5s 30 s

341 150

108 (32) 71 (47)

No. PMD (%)

No. AND

(a)

No. motile structures (%)

5 (14) 6 (9)

189 (55) 42 (28)

0 (0) 49 (33)

2 (0.6) 8 (5)

202

ANN M. DVORAK

empty, nonfused, nonswollen granules ( p (0.005 compared to the 30-s control, 9%; p <0.025 compared to the 7-min control, 18%). This finding was associated with glycogen-rich granule-vesicle attachments (40%), a finding that was evident significantly more frequently than in controls obtained at 30 s (14%, p (0.025) or 7 min (9%, p <0.001) (Figs. 30A and 30B). Some empty granules contained glycogen and were surrounded by electron-lucent vesicles (Fig. 30C).Thirteen percent (13%) of the basophils that were stimulated with anti-IgE for 2 rnin showed AND. Generally, A N D consisted of extrusion of one or several granules to the cell surface (Fig. 30D) through different openings in the plasma membrane; many unaltered granules remained in these responding cells; some granules showed piecemeal granule losses. Three percent (3%)of the cells stimulated with anti-IgE for 2 rnin had motile structures-a value similar to that of both control samples ( p = NS;p = NS). Basophils that were stimulated for 7 rnin with anti-IgE showed 67% AND,a value significantly different from that of other samples with AND (i.e., 13% at 2 rnin with anti-IgE, p (0.001; 16% at 7 rnin with rHRF, p <0.001; 33% at 30 s with MCP-1,p <0.001). PMD (12%) and granule-vesicle attachment (2%) were similar to those in controls but were less than those in the 2-min anti-IgE-stimulated cells [p <0.005 (12% vs 4 0 % ) ; ~ <0.001 (2% vs 40%)]. AND in the anti-IgE stimulated cells resembled that previously reported for regulated secretion by human basophils. That is, we found evidence of extrusion of membrane-free granule contents (particles, CLCs, and dense concentric membranes) through multiple openings in the plasma membrane. Many of these granule materials remained loosely associated with cell surfaces, often amidst shed membranes and surface processes. These actively responding cells had markedly elongated surface processes and irregular shapes. They were devoid of motile structures (although we found uropods in nondegranulating cells in the same samples, 15%) and often were nearly devoid of granules. Some cells retained n o cytoplasmic granules at all. They contained glycogen aggregates, virtually no cytoplasmic vesicles, and retained polylobed nuclei. They did not show any ultrastructural criteria of injury, necrosis, or apoptosis. A few basophils formed intracellular degranulation chambers which were filled with extruded, membrane-free granule particles. These membrane-bound intracytoplasmic chambers were formed de n o w from individual granule fusions and opened to the cell surface through one degranulation pore. A small number of basophils that were stimulated for 7 rnin with anti-IgE showed PMD.These cells were characterized by grossly swollen, electronlucent, empty granules. (Cells similar to these were absent in the 7-min rHRF sample.) Other granules present in these cells showed no alterations, and AND was absent.

CELL BIOLOGY OF THE BASOPHIL

203

FIG.30 Portions of basophils (2 min after stimulation with anti-IgE) show granule-vesicle attachments (A,B), cytoplasmic perigranular vesicles (C), and exocytosis of a single granule (D). In A and B, the vesicles that are attached to granules contain altered granule contents, while the granules contain particles. The granule-attached vesicles are surrounded by large, electron-dense aggregates of glycogen and are oriented toward the overlying plasma membrane. Similar glycogen clusters, which are free in the cytoplasm (arrows), enclose electronlucent vesicles. In C, one granule has completely released its dense particle contents and contains larger, electron-dense glycogen particles. Small, smooth membrane-bound, electronlucent vesicles surround this granule pole, just beneath the overlying plasma membrane. Two granule types of human basophils are shown in D. One particle-containing granule, devoid of its membrane, has been extruded to the cell surface (arrow); a small, homogeneous granule (arrowhead) rests beside the nucleus (N) (with permission, from Dvorak et al., 1996e). Bars: (A,C,D) 0.2 pm (B) 0.1 pm.

204

ANN M. DVORAK

2. Recombinant Histamine-Releasing Factor-Stimulated Basophils

rHRF-stimulated basophils showed a trend toward more PMD at 2 min (25%) than control values of PMD by 30 s (9%) but did not achieve statistical significance ( p = NS). AND was absent at 2 min. The extent of AND at 7 min (16%) was equivalent to that induced by anti-IgE at 2 min (13%, p = NS) but was far less than that induced by anti-IgE at 7 min (67%, p <0.001). rHRF produced the largest amount of motile configurations in basophils, 49% by 7 min ( p <0.001 compared to anti-IgE at 7 min, 15%; p <0.001 compared to MCP-1 at 30 s, 5%; p <0.001, p <0.001 compared to controls at 30 s, 6%, and 7 min, 3%). Motile structures in the 7-min rHRF sample (49%) exceeded those seen at 2 min (14%) ( p <0.001), whereas motile basophils in control samples did not differ between 30 s (6%) and 7 min (3%) ( p = NS). The morphology of AND induced by rHRF was identical to that seen in anti-IgE-stimulated cells (Fig. 30D). The morphology of PMD in rHRF-stimulated samples differed from the PMD morphology in the anti-IgE 7-min sample, in that basophils with grossly enlarged, nonfused, empty granules were not present. Rather, individual cells showed one to two focally or completely empty, nonfused granule containers, which were not swollen, in their cytoplasm. Attached, glycogen-rich granule-vesicles similar to those of all stimulated samples (except for the 7-min anti-IgE sample) were seen in the rHRFcontaining samples. For example, large masses of glycogen focally contacted individual altered granules, and these masses capped vesicular protrusions containing altered granule materials. Some of the latter structures were attached at granule poles oriented toward plasma membranes. The large number of uropod-bearing basophils that were stimulated with a 7-min exposure to rHRF showed no morphological evidence of AND; however, some had focal PMD. 3. Monocyte Chemotactic Protein-1-Stimulated Basophils

Fifty-five percent (55%) of the basophils exposed for 5 s to MCP-1 showed PMD ( p <0.001, p <0.001 compared to controls; p = NS compared to anti-IgE, 2 min, 40%;p <0.001 compared to anti-IgE, 7 min, 12%;p <0.001 compared to rHRF 2 min, 25%; p <0.001 compared to rHRF, 7 min, 9%), a value that decreased significantly by a 30-s exposure to MCP-1 (28%) ( p <0.001), at which point AND began (33% compared to none in the 5-s MCP-1 sample). Also, the extent of PMD in individual cells exceeded that of controls in that altered granules were more prevalent. By 30 s after MCP-1, 28% of the cells exhibited PMD similar to the 2-min samples stimulated with anti-IgE (40%) and rHRF (25%) ( p = NS; p = NS). Some

CELL BIOLOGY OF THE BASOPHIL

205

basophils demonstrated extensive PMD, involving nearly all granules, which were markedly enlarged and which had not fused with adjacent granule or plasma membranes. These cells resembled the small number of cells undergoing PMD that remained in the anti-IgE 7-min sample, a sample primarily showing AND (67%). PMD in the 30-s MCP-1 sample (28%) significantly exceeded that in the 7-min anti-IgE (12%) and rHRF (5%) samples ( p <0.05; p C0.05). The images of basophil AND evident at 30 s after exposure to MCP-1 resembled those reported for other samples. For example, there was cellular evidence of extensive degranulation, with many extruded granules resting on the cell surface after emerging through multiple openings in the plasma membrane. Granule particles, CLCs, and dense concentric membranous arrays amidst shed cellular processes were clustered around these secreting, viable cells. Many basophils showed extrusion of a single granule with little to no alteration of the remaining granules. Some cells released multiple nonswollen, membrane-free granules through a single plasma membrane pore, which had widened to permit their passage. A small number of basophils undergoing AND had formed cytoplasmic degranulation chambers from fused granule membranes; these structures were filled with extruded, membrane-free granules. While there were no basophils undergoing AND in the MCP-1 5-s sample, 33% of the basophils in the 30-s MCP-1 sample were involved in AND. The extent of AND in the MCP-1 30-s sample (33%) was significantly greater than that present in the rHRF 7min sample (16%, p <0.025) and that in the anti-IgE 2-min sample (13%, p <0.05) but was less than that found in the anti-IgE 7-min sample (67%, p <0.001). The number of MCP-1-stimulated motile structures (5-s sample, 0.6%) was significantly less ( p <0.005) than those formed in the 30-s control sample (6%) but did not differ from that of the 7-min control sample (3%, p = NS). The 30-s MCP-1-stimulated cells had motile structure formation similar to that of both controls ( p = NS). The PMD images induced by MCP-1 in basophils were accompanied by numerous glycogen-associated granule-vesicle attachments (Fig. 31) (32%, 5 s; 47%, 30 s ) . In fact, the largest number of these structures of all samples examined were identified in the 30-s MCP-1 sample ( p <0.001, p <0.001 vs controls 14%, 9%; p = NS, p <0.001 vs anti-IgE at 2 min, 40%, and 7 min, 2%; p <0.005, p <0.001 vs rHRF at 2 min, 19%, 7 min, 21%; and p <0.001 vs MCP-1 at 5 s, 32%). Granule-vesicle attachments in the 5 s MCP-1 sample (Figs. 31D, 31F, and 31G) (32%) also exceeded those for 30-s (14%) and 7-min (9%)control samples ( p
FIG. 31 This montage of human basophils, stimulated with MCP-1 for 5 s (A-G) or 30 s (HJ), shows glycogen-rich granule-vesicle attachments which accompany PMD. Some of the attached, elongated vesicles contain electron-dense granule particles (arrows in A-C); some attached vesicles contain less dense (or electron-lucent) altered materials (arrows in D-I). Larger, electron-dense glycogen particles are interposed at the necks of tubular-vesicular granule extensions (A,B,E) or encase the outer surface of attached vesicles (D,F-I). Also

CELL BIOLOGY OF THE BASOPHIL

207

the 5-s MCP-1 sample significantly exceeded that in the 7-min anti-IgEstimulated cells (2%, p <0.001). Granule-attached vesicles were often uniquely encased in glycogen aggregates; many of these glycogen-encased, granule-attached vesicles were oriented on granule poles or sides facing plasma membranes (Figs. 31F and 311). Some granules had up to three attached vesicles (Fig. 31G). Focal piecemeal losses of granule particles were regularly noted adjacent to these attached vesicles (Figs. 31D-I). Some particle-filled vesicles were similarly hooked to granules at glycogen-rich areas (Figs. 31A-C). Glycogen aggregates that were near granules as well as those that were not apparently near granules surrounded cytoplasmic, electron-lucent, smooth membranebound vesicles and larger vacuoles. Some of these structures were filled with material similar to the altered granule substance adjacent to areas of vesicle attachments-areas which are referred to as focal piecemeal degranulation. The relationships of four activated morphologic features in basophils as a function of unstimulated or stimulated cells are graphically compared in Fig. 32. 4. Quantitative Differences in Basophils Exposed to Anti-IgE, rHRF, or MCP-1

Quantitative differences of several ultrastructural activation morphologies are induced in human basophils by exposure to anti-IgE, rHRF, or MCP1. We selected two time points at which to examine ultrastructural changes for each secretogogue-one sample was taken at approximately one-half maximum histamine release (MacDonald et af., 1995; Bischoff et af., 1992; Alam er af., 1992a; Kuna et af., 1992a; Warner et aL, 1989), and the other sample was selected to precede histamine release. Thus, the known kinetics for either anti-IgE- or rHRF-stimulated histamine release dictated sample collections at 7 min (one-half maximum histamine release) and 2 min (prior to histamine release). MCP-1 samples were collected at 30 s (one-half maximum histamine release) and 5 s (prior to histamine release). Four morphological processes-i.e., PMD, AND, GVA, and uropod forma-

noted are single (A-C,E,F,I), double (H), and triple (G) glycogen-rich vesicles attached to granules. Some granules, with vesicles attached, have a full complement of dense particles (A-D), focal, underlying piecemeal losses of granule particles (E,G-I), or diminished particles throughout (F). One empty granule (D) has individual glycogen particles within it (with permission, from Dvorak et al., 1996e). Bars: (A,C,D,H) 200 nm; (B) 170 nm; (E) 140 nm; (F,G) 160 nm; (I) 180 nm.

ANN M. DVORAK o-.--.o D--O

Control

Control MCP-1

A------A a-lgE

rHRF

v---

IE

GVA

t

PMD

tt-

40 0 80P

MCP-1

-

40 -

%

0C

a-lgE

80 -

40 0

d

80

-D

40

L

'

r HRF

L /

P

/

5u 3 0 sec.

2

7

min.

sec.

min.

Time FIG. 32 Kinetic and quantitative relationships of four activated morphologic features induced in human basophils by anti-IgE, rHRF, or MCP-1 compared to unstimulated human basophils (with permission, from Dvorak er al., 1996e).

tion-were markedly different quantitatively in samples stimulated with different secretogogues. Qualitatively, the processes were similar, with few exceptions (see later). When these events were examined in terms of time needed for half-maximum histamine release for each trigger, anti-IgE was clearly the most effective in producing AND images, surpassing the next

CELL BIOLOGY OF THE BASOPHIL

209

most effective trigger, MCP-1, by 34% ( p <0.001). Thus, a rank order for the elicitation of AND is as follows: anti-IgE > MCP-1 > rHRF, when examined at times comparable to half-maximum histamine release. The only other sample that showed AND was the 2-min anti-IgE sample, confirming our previous observation that the ultrastructural detection of AND, stimulated by an IgE-mediated mechanism, may precede the biochemical detection of histamine release (Dvorak et al., 1980b). PMD, at the time point required for half-maximum histamine release, did not exceed that in controls with either anti-IgE or rHRF stimulation. Stimulation with MCP-1 at the half-maximum histamine release time point did, however, produce PMD exceeding that of controls. Moreover, all triggers elicited extensive PMD in samples obtained before histamine release. A rank order for the effectiveness in producing PMD images is as follows: MCP-1 > anti-IgE > rHRF. GVA showed interesting differences at half-maximum histamine release times. For anti-IgE-a trigger that primarily functioned by inducing AND-the GVA formation percentage (2%) was the lowest of all samples examined, whereas for MCP-1-a trigger that induced large amounts of PMD-the greatest amount of GVA was recorded (47%). Thus, GVAs are implicated by association as a mechanism for effecting mediator release by vesicular transport from granules that empty and remain in place in the cytoplasm (e.g., as in PMD) rather than by release from granules that are physically extruded in their entirety to the cells’ exterior (e.g., as in AND). Uropods are motile structures that were previously recorded in quantity in human basophils stimulated with HRA-containing mononuclear cell supernatants (Dvorak et al., 1984b). As before (Dvorak et af., 1984b), we noted no basophils with uropods undergoing AND in any preparations, and instances of PMD were minor, involving only focal portions of granules (or only several granules) among many granules which showed no alterations. Of the triggers examined here, rHRF produced 49% basophils with uropods at the half-rnaximum histamine release interval. The rank order of uropod production was rHRF > anti-IgE > MCP-1. Clearly, this newly defined recombinant protein (MacDonald et al., 1995) produces considerable ultrastructural evidence of the anatomic structures needed for basophil migration-a feature not shared by stimulation of basophils with MCP-1. Some motile structures were detected in the half-maximum histamine release interval after anti-IgE, similar to our previous report of antigen Estimulated basophils (Dvorak et al., 1980b). 5. PMD and Histamine-Releasing Secretogogues

PMD is featured during the early exposure of human basophils to several histamine-releasing secretogogues. PMD of basophils was first described

210

ANN M. DVORAK

in tissue basophils following their migration from the vascular space in response to experimentally produced and sequentially biopsied contact allergy skin lesions (Dvorak et al., 1976a; Dvorak and Dvorak, 1993). The secretory process termed PMD is ultrastructurally defined by the progressive emptying of electron-dense granule contents without fusion to adjacent granules or to the plasma membrane. Since the early description in basophils, this secretory process has been documented in basophils and mast cells of numerous species in a variety of experimental models and diseases (Dvorak et al., 1991a, 1992a,b, 1993c, 1994e; Dvorak, 1992a). PMD is the single most frequently encountered ultrastructural event in activated tissue basophils and mast cells in human disease (Dvorak, 1992a, 1993). In contact allergy, a crescendo of basophil PMD evolves over a 3-day period in sequential biopsies, a secretory process that does not evolve into AND. In 1975, we postulated a degranulation model for basophils that encompassed a continuum of PMD + AND, dependent on the rate of vesicular traffic (Dvorak and Dvorak, 1975). This continuum has since been documented in a kinetic morphologic study of FMLP-stimulated basophils (Dvorak et al., 1991a). An evolving continuum of PMD -+ AND suggests that one might find ultrastructural evidence of PMD before AND following stimulation. Such a precursor relationship to AND, documented for FMLP, was also suggested by studies of TPA-stimulated basophils (Dvorak et al., 1992a). In this case, a forme fruste of AND prevailed (at later times) which was associated with infrequent images of fully developed AND. We also found that PMD preceded AND following stimulation with each of the three new triggers we examined (Dvorak et al., 1996e). Moreover, as the extent of PMD diminished at half-maximum histamine release times, the extent of AND increased, for all three triggers. This finding, coupled with our previous similar findings for FMLP and TPA, indicates a precursor relationship of PMD to AND, supporting the general degranulation model proposed in 1975 (Dvorak and Dvorak, 1975). Since the actual trigger(s) of basophil PMD in contact allergy tissue sites is not known, one cannot yet determine whether an incomplete trigger or an inhibitor functions to halt the progression of PMD to AND in basophils responding to contact allergens in the skin, for example. The ability to examine these processes carefully, ex vivo, with well-defined, purified stimulants (MacDonald et al., 1995; Bischoff et al., 1992; Alam et al., 1992a; Kuna et al., 1992a), as they become available, should aid in the resolution of such questions and, perhaps, contribute to therapeutic regulation of unwanted secretory events in human basophils during disease.

6. Qualitative Similarities in Basophils Exposed to Anti-IgE, rHRF, or MCP-1 The ultrastructural expression of secretion in human bsophils is qualitatively similar after exposure to anti-IgE, rHRF, or MCP-1. The general morpho-

CELL BIOLOGY OF THE BASOPHIL

21 1

logical processes of PMD and AND expressed by human basophils following stimulation with anti-IgE, rHRF, or MCP-1 did not differ qualitatively. In previous studies, we have called attention to two morphological variants-one having components of the morphology of AND and the other of PMD-that were evident upon examination of mannitol-stimulated cells (Findlay et af., 1981) or FMLP-stimulated cells (Dvorak et af., 1991a), respectively. It is important to recognize that these variants exist in secreting basophils, since, by light microscopy, one cannot distinguish them, and they evolve by quite different ultrastructural mechanisms. In the first case, basophils stimulated with hyperosmolar agents, like mannitol (Findlay et al., 1981), developed d e g r a d a t i o n sacslchannels within the cytoplasm that contained numerous membrane-free, extruded granules. This newly developed, membrane-delimited space is formed from the fused membranes of the extruded granules and ultimately opens to the extracellular space through a single pore in the plasma membrane. Such structures are integral features of AND in mast cells and basophils of many species (Dvorak, 1991). They are, for example, extensively formed during AND in stimulated guinea pig basophils (Dvorak et af., 1981b) but are quite rare in human basophils stimulated to undergo AND (Dvorak et al., 1980b). The second morphological variant occurs in PMD when inward vesicular traffic (endocytosis) exceeds outward vesicular traffic, leading to extensive enlargement of individual, nonfused granule containers (Dvorak et al., 1991a). These single, giant granule containers are often completely devoid of granule matrix, e.g., they do not arise by granule-to-granule membrane fusions, nor do they contain extruded granules. The expanded granule containers results from the addition of vesicle-membranes that traffic to individual granules from the cell surface. In the current experiments, intracytoplasmic d e g r a d a t i o n chamber formation was exceedingly rare as a component of AND induced by anti-IgE, rHRF, or MCP-1. Small amounts of PMD with enlarged, empty containers were evident at 7 min after antiIgE or at 30 s after MCP-1. A comparative analysis of the qualitative morphologic changes prevailing after stimulation of basophils with several triggers shows (i) that stimulation with anti-IgE produced extensive evidence of AND, analogous to previous IgE-mediated morphologic studies (Dvorak et af., 1980b), and (ii) that stimulation with rHRF produced less AND than did anti-IgE, with abundant evidence of motile structure formation. These findings are similar to those previously recorded for cytokine-containing mononuclear cell supernatants (Dvorak et al., 1984b). On the other hand, by 5 s, MCP-1 produced extensive evidence of PMD that preceded AND. PMD (produced by MCP1) exceeded that found in controls or in samples stimulated by rHRF or present 7 min after anti-IgE; thus, MCP-1 was the most potent trigger for eliciting the morphology of PMD in these studies.

212

ANN M. DVORAK

7. GVA-Supported Vesicle Transport in Stimulated Secretion

GVAs support a role for vesicle transport in stimulated secretion from human basophils. Significantly increased numbers of GVAs were present both in the basophil samples stimulated with MCP-1 and in the 2-min antiIgE-stimulated sample. We first described vesicles attached to basophil granules in contact allergy skin biopsies and noted that these attached structures were often connected to glycogen particles and aggregates (Dvorak ef al., 1976a). These associations were striking in the MCP-l-stimulated basophils studied here, suggesting a role for GVAs in effecting the extensive emptying of granules, characteristic of PMD. The closely associated glycogen aggregates may provide an energy source for vesicular motion. Vesicular traffic from the extracellular milieu has been documented in both human and guinea pig basophils, using electron-dense H R P or EPO, and release of previously granule-loaded HRP from guinea pig basophils in cytoplasmic vesicles was noted in wash-out studies (Dvorak, 1991). Morphologically similar cytoplasmic vesicles, present in FMLP-stimulated basophils during active histamine release, contain histamine and CLC protein, as determined with an enzyme-affinity-gold method (Dvorak et al., 1994b, 1996f) or by an immunogold method (Dvorak and Ackerman, 1989; Dvorak et al., 1997a,b), respectively. Thus, ample evidence exists for a vesicular role in uptake into and secretion from human basophils. The marked increase in vesicles attached to granules, encased in glycogen, and oriented to plasma membranes [in basophils displaying significant amounts of PMD after stimulation with MCP-1 (and anti-IgE, at 2 min)] supports this interpretation. Basophil samples expressing extensive AND (anti-IgE, 7 min) and/or motile structures (anti-IgE, 7 min; rHRF, 2 and 7 min) did not have GVAs exceeding those present in controls, further supporting their effector role in PMD as well as the precursor or nonobligatory relationships of PMD to AND and the lack of either PMD or AND in human basophils that form uropods after stimulation.

XII. Concluding Remarks A number of recent, diverse technical advances have hastened the pace of our understanding of cell-biological mechanisms of basophil biology. These rarest of granulocytes have been difficult to study directly, despite their key role(s) in cell-mediated and immediate hypersensitivity disorders. The necessary advances include identification of basophil growth factors, allowing de novo development of basophils in vitro, and purification schemata, allowing circulating and bone-marrow-derived basophils to be obtained in

CELL BIOLOGY OF THE BASOPHIL

213

quantity. All putative preparations designed for cell-biological studies of basophils require certainty of cell identification. The gold standard for the identification of basophils is ultrastructural analysis, a procedure instrumental in the recent initial identification of basophils in two species-mice and monkeys. Additional aids to basophil identification include cell surface markers, biochemical contents, and activation profiles. Advances in the understanding of basophil biology have also been facilitated by the use of electron-dense tracers, and immunogold and enzymeaffinity-gold probes for analyzing subcellular locations and events. Use of these ultrastructural tags in conjunction with timed samples stimulated by a wide variety of secretogogues has led to the realization that basophils not only are classical regulated secretory cells, which secrete large storage granules to the extracellular milieu, but that basophils also participate in finely tuned regulation of the extracellular milieu by combined endocytotic uptake and exocytotic secretory processes. For example, uptake of eosinophi1 peroxidase by endocytosis and storage in basophil secretory granules of this eosinophil granule protein, released from eosinophils in sites of allergic inflammation, provide a reservoir of eosinophil peroxidase that can be secreted from basophils in these sites. Synthetic granule stores of histamine are released from basophils by both granule extrusion and release of quanta of histamine in small cytoplasmic vesicles, which bud from the large secretory granules. Another basophil and eosinophii protein, the Charcot-Leyden crystal protein, is secreted from basophils by extrusion of granule-contained Charcot-Leyden crystals as well as by vesicular transport of quanta of CLC protein. Ultrastructural studies show that basophils do not die following secretion but undergo recovery in vitro. During recovery, both histamine and Charcot-Leyden crystal protein granule stores are replaced, depending on the secretogogue used, by a combination of conservation and synthetic processes. Detailed ultrastructural kinetic analysis of human basophils, stimulated by a variety of secretogogues, led to the identification of key morphological phenotypes typical of secreting and recovering cells. Coupled with quantitative analysis of changing subcellular sites of histamine and CLC protein during secretion and recovery, a continuum of basophil degranulation was identified, proving a hypothetical degranulation model for basophil degranulation that had been proposed in 1975 (Dvorak and Dvorak). The role of transport in small, smooth membrane-bound cytoplasmic vesicles (which are invariably present in basophils of all species examined by electron microscopy) in effecting basophil secretion was established with these new tools. When used in conjunction with a variety of secretory triggers (e.g., bacterial peptides, the tumor-promoting factor, phorbol ester, IgEmediated and -assisted stimuli, cytokines, or chemokines), it is clear that a continuum of vesicle-mediated transport from secretory granules, to extru-

214

ANN M. DVORAK

sion of entire granules and/or their emptied containers, prevails in stimulated secretory basophils. The speed and amount of such vesicular secretory traffic and granule extrusion vary within this continuum, depending on the properties of the secretogogue used, thus suggesting multiple regulatory points within this morphological continuum that may result in variable mediator profiles of release as judged by time after stimuli and content in inflammatory and allergic reactions.

Acknowledgments This work was supported by National Institutes of Health Grants CA-15136, CA-19141, CA28834, and AI-33372, representing continuous NIH support to A. M. Dvorak for cell-biological studies of basophils from 1973 to 1997. The expert assistance (with electron-microscopic studies) of Ellen S. Morgan, Rita A. Monahan-Earley, Kathryn Pyne, Patricia Fox, Linda Letourneau, Karen Bryan, and (with manuscript preparation) Peter K. Gardner has made this work possible and is greatly appreciated. Numerous collaborators, spanning the years between 1970 and 1997, have provided experimental material and meaningful discussions without which these studies would not have been done. Chief (but not inclusive) among these are Steven J. Ackerman, Robert B. Colvin, Robert E. Donahue, Harold F. Dvorak, Steven Findlay, Charity Fox, Stephen J. Galli, J. Andrew Grant, Kimishige Ishizaka, Teruko Ishizaka, Anne Kagey-Sobotka, Seymour Klebanoff, Michael Lett-Brown, Lawrence M. Lichtenstein, Susan M. MacDonald, Donald W. MacGlashan, Jr., Martin Mihm, Hideki Mitsui, Gary Nabel, William Paul, Hirohisa Saito, John T. Schroeder, Robert A. Seder, and Jane Warner.

References Abu-Ghazaleh, R. I., Dunnette, S. L., Loegering, D. A,, Checkel, J. L., Kita, H., Thomas, L. L., and Gleich, G. J. (1992). Eosinophil granule proteins in peripheral blood granulocytes. J. Leukocyte Biol. 52,611-618. Ackerman, S. J., Weil, G. J., and Gleich, G. J. (1982). Formation of Charcot-Leyden crystals by human basophils. J. Exp. Med. 155,1597-1609. Ackerman, S. J., Kephart, G. M., Habermann, T. M. Greipp, P. R., and Gleich, G. J. (1983). Localization of eosinophil granule major basic protein in human basophils. J. Exp. Med. 158, 946-961. Ackerman, S. J., Corrette, S. E., Rosenberg, H. F., Bennett, J. C., Mastrianni, D. M., NicholsonWeller, A,, Weller, P. F., Chin, D. T., and Tenen, D. G. (1993). Molecular cloning and characterization of human eosinophil Charcot-Leyden crystal protein (lysophospholipase): Similarities to IgE-binding proteins and the S-type animal lectin superfamily. J. Irnrnunol. 150,456-468. Alam, R., Grant, J. A., and Lett-Brown, M. A. (1988). Identification of a histamine release inhibitory factor produced by human mononuclear cells in vitro. J. Clin. Invest. 82,2056-2062. Alam, R., Welter, J. B., Forsythe, P. A., Lett-Brown, M. A., and Grant, J. A. (1989a). Comparative effect of recombinant IL-1, -2, -3, -4, and -6, IFN-y, granulocyte-macrophagecolony-stimulating factor, tumor necrosis factor-cu, and histamine-releasing factors on the secretion of histamine from basophils. J. Imrnunol. 142, 3431-3435.

CELL BIOLOGY OF THE BASOPHIL

215

Alam, R., Forsythe, P. A., Lett-Brown, M. A., and Grant, J. A. (1989b). Study of the cellular origin of histamine release inhibitory factor using highly purified subsets of mononuclear cells. J. Immunol. 143, 2280-2284. Alam, R., Lett-Brown, M. A,, Forsythe, P. A., Anderson-Walters, D. J., Kenamore, C., Kormos, C., and Grant, J. A. (1992a). Monocyte chemotactic and activating factor is a potent histamine-releasing factor for basophils. J. Clin. Invest. 89, 723-728. Alam, R., Forsythe, P. A., Stafford, S., Lett-Brown, M. A., and Grant, J. A. (199213). Macrophage inflammatory protein-la activates basophils and mast cells.J. Exp. Med. 176,781-786. Alam, R., Forsythe, P., Stafford, S., Heinrich, J., Bravo, R., Proost, P. and Van Damme, J. (1994). Monocyte chemotactic protein-2, monocyte chemotactic protein-3, and fibroblastinduced cytokine. Three new chemokines induce chemotaxis and activation of basophils. J . Immunol. 153,3155-3159. Arock, M., Merle-BCral, H., Dugas, B., Ouaaz, F., Le Goff, L., Vouldoukis, I., Mencia-Huerta, J.-M., Schmitt, C., Leblond-Missenard, V., Debre, P., and Mossalayi, M. D. (1993). IL-4 release by human leukemic and activated normal basophils. J. Immunol. 151, 1441-1447. Barondes, S. H., Cooper, D. N. W., Gitt, M. A., and Leffler, H. (1994). Galectins. Structure and function of a large family of animal lectins. J. Biol. Chem. 269, 20807-20810. Barr, F. A., Leyte, A,, and Huttner, W. B. (1992). Trimeric G proteins and vesicle formation. Trends Cell Biol. 2, 91-94. Barrett, K. E., and Metcalfe, D. D. (1985). The histologic and functional characterization of enzymatically dispersed intestinal mast cells of nonhuman primates: Effects of secretagogues and anti-allergic drugs on histamine secretion. J. Immunol. 135, 2020-2026. Bascom, R., Wachs, M., Naclerio, R. M., Pipkorn, U., Galli, S. J., and Lichtenstein, L. M. (1988). Basophil influx occurs after nasal antigen challenge: Effects of topical corticosteroid pretreatment. J. Allergy Clin. Immunol. 81, 580-589. Bean, A. J., Zhang, X., and Hokfelt, T. (1994). Peptide secretion: What do we know? FASEB J. 8, 630-638. Beaven, M. A. (1976). Histamine. N. Engl. J. Med. 294, 30-36. Becker, K. E., Ishizaka, T., Metzger, H., Ishizaka, K., and Grimley, P. M. (1973). Surface IgE on human basophils during histamine release. J. Exp. Med. 138, 394-409. Bendayan, M. (1981). Ultrastructural localization of nucleic acids by the use of enzyme-gold complexes. J. Histochem. Cytochem. 29,531-541. Bendayan, M. (1984). Enzyme-gold electron microscopic cytochemistry: A new affinity approach for the ultrastructural localization of macromolecules. J. Electron Microsc. Technol. 1,349-372. Ben-Sasson, S. Z., Le Gros, G., Conrad, D. H., Finkelman, F. D., and Paul, W. E. (1990). Cross-linking Fc receptors stimulate splenic non-B, non-T cells to secrete interleukin 4 and other lymphokines. Proc. Nutl. Acud. Sci. USA 87, 1421-1425. Bentfeld, M. E., Nichols, B. A,, and Bainton, D. F. (1977). Ultrastructural localization of peroxidase in leukocytes of rat bone marrow and blood. Anut. Rec. 187, 219-240. Bentfeld-Barker, M. E., and Bainton, D. F. (1980). Cytochemical localization of arylsulfatase B in rat basophils and mast cells. J . Histochem. Cytochem. 28, 1055-1061. Benveniste, J., Henson, P. M., and Cochrane, C. B. (1972). Leukocyte-dependent histamine release from rabbit platelets. The role of IgE, basophils, and a platelet-activating factor. J. Exp. Med. 136,1356-1377. Bischoff, S . C., Brunner, T., De Weck, A. L., and Dahinden, C. A. (1990). Interleukin 5 modifies histamine release and leukotriene generation by human basophils in response to diverse agonists. J. Exp. Med. 172, 1577-1582. Bischoff, S. C., Kreiger, M., Brunner, T., and Dahinden, C. A. (1992). Monocyte hemotactic protein 1 is a potent activator of human basophils. J. Exp. Med. 175, 1271-1275. Bischoff, S. C., Kreiger, M., Brunner, T., Rot, A., von Tscharner, V., Baggiolini, M., and Dahinden, C. A. (1993). RANTES and related chemokines activate human basophil granulocytes through different G protein-coupled receptors. Eur. J. Immunol. 23, 761-767.

216

ANN M. DVORAK

Bochner, B. S., and Lichtenstein, L. M. (1991). Anaphylaxis. N. Engl. J. Med. 324,1785-1790. Bochner, B. S., and Schleimer, R. P. (1994). The role of adhesion molecules in human eosinophil and basophil recruitment. J. Allergy Clin. Immunol. 94,427-438. Bochner, B. S., McKelvey, A. A,, Schleimer, R. P., Hildreth, J. E. K., and MacGlashan, D. W., Jr. (1989). Flow cytometric methods for the analysis of human basophil surface antigens and viability. J. Immunol. Methods 125, 265-271. Bodger, M. P., and Newton, L. A. (1987). The purification of human basophils: Their immunophenotype and cytochemistry. Br. J. Haematol. 67, 281-284. Bodger, M. P., Mounsey, G . L., Nelson, J., and Fitzgerald, P. H. (1987). A monoclonal antibody reacting with human basophils. Blood 69, 1414-1418. Boyce, J. A,, Friend, D.,Matsumoto, R., Austen, K. F., and Owen, W. F. (1995). Differentiation in vitro of hybrid eosinophitbasophil granulocytes: Autocrine function of an eosinophil developmental intermediate. J. Exp. Med. 182,49-57. Brodsky, F. M. (1988). Living with clathrin: Its role in intracellular membrane traffic. Science 242, 1396-1402. Brunner, T., de Weck, A. L., and Dahinden, C. A. (1991). Platelet activating factor induces mediator release by human basophils primed with IL-3, granulocyte-macrophage colonystimulating factor, or IL-5. J. Immunol. 147, 237-242. Brunner, T., Heusser, C . H., and Dahinden, C. A. (1993). Human peripheral blood basophils primed by interleukin 3 (IL-3) produce IL-4 in response to immunoglobulin E receptor stimulation. J. Exp. Med. 177, 605-611. Bruns, R. R., and Palade, G. E. (1968). Studies on blood capillaries. I. General organization of blood capillaries in muscle. J. Cell Biol. 37, 244-276. Buffoni, F. (1966). Histaminase and related amine oxidases. Pharmacol. Rev. 18, 1163-1199. Burd, P. R., Rogers, H. W., Gordon, J. R., Martin, C. A,, Jayaraman, S., Wilson, S. D., Dvorak, A. M., Galli, S. J., and Dorf, M. E. (1989). Interleukin 3-dependent and -independent mast cells stimulated with IgE and antigen express multiple cytokines. J. Exp. Med. 170,245-257. Busse, W. W., Swenson, C. A., Borden, E. C., Treuhaft, M. W., and Dick, E. C. (1983). Effect of influenza A virus on leukocyte histamine release. J. Allergy Clin. Immunol. 71,382-388. Calakos, N., Bennett, M. K., Peterson, K. E., and Scheller, R. H. (1994). Protein-protein interactions contributing to the specificity of intracellular vesicular trafficking. Science 263, 1146-1149. Castells, M. C., Irani, A.-M., and Schwarz, L. B. (1987). Evaluation of human peripheral blood leukocytes for mast cell tryptase. J. Immunol. 138, 2184-2189. Castle, J. D. (1990). Sorting and secretory pathways in exocrine cells. Am. J. Respir. Cell Mol. Biol. 2, 119-126. Chan, B. S. T., and Yoffey, 3. M. (1960). The basophil cells of guinea-pig bone marrow and their response to horse serum. Immunology 3, 237-243. Chavez, R. A., Miller, S. G., and Moore, H.-P. H. (1996). A biosynthetic regulated secretory pathway in constitutive secretory cells. J. Cell Biol. 133, 1177-1191. Clementsen, P., Jensen, C. B., Jarlov, J. O.,Hannoun, C., and Norn, S. (1988a). Virus enhances histamine release from human basophils. Agents Actions 23, 165-167. Clementsen, P., Jensen, C. B., Hannoun, C., Seborg, M. and Norn, S. (1988b). Influenza A virus potentiates basophil histamine release caused by endotoxin-induced complement activation. Examination of normal individuals and patients with intrinsic asthma. Allergy 43,93-99. Cleves, A. E., Cooper, D. N. W., Barondes, S. H., and Kelly, R. B. (1996). A new pathway for protein export in Saccharomyces cerevisiae. J. Cell Biol. 133, 1017-1026. Collin, H. B., and Allansmith, M. R. (1977). Basophils in vernal conjunctivitis in humans: An electron microscopic study. Invest. Ophthalmol. Visual Sci. 16, 858-864. Combs, J. W. (1966). Maturation of rat mast cells. An electron microscope study. J. Cell Biol. 31,563-575.

CELL BIOLOGY OF THE BASOPHIL

21 7

Combs, J. W. (1971). An electron microscope study of mouse mast cells arising in vivo and in vitro. J. Cell Biol. 48, 676-684. Conrad, D. H., Ben-Sasson, S. Z., Le Gros, G., Finkelman, F. D., and Paul, W. E. (1990). Infection with Nippostrongylus brasiliensis or injection of anti-IgD antibodies markedly enhances Fc-receptor-mediated interleukin 4 production by non-B, non-T cells. J. Exp. Med. 171,1497-1508. Creticos, P. S., Adkinson, N. F., Jr., Kagey-Sobotka, A,, Proud, D., Meier, H. L., Naclerio, R. M., Lichtenstein, L. M., and Norman, P. S. (1985). Nasal challenge with ragweed pollen in hay fever patients. Effect of immunotherapy [published erratum appears in J. Clin. Invest. 78, 1421, 19861. J. Clin. Invest. 76, 2247-2253. Curran, M. J., and Brodwick, M. S. (1991). Ionic control of the size of the vesicle matrix of beige mouse mast cells. J. Gen Physiol. 98, 771-790. Dagher, S. F., Wang, J. L., and Patterson, R. J. (1995). Identification of galectin-3 as a factor in pre-mRNA splicing. Proc. Natl. Acad. Sci. USA 92, 1213-1217. Dahinden, C. A,, Kurimoto, Y., De Weck, A. L., Lindley, I., Dewald, B., and Baggiolini, M. (1989). The neutrophil-activating peptide NAF/NAP-1 induces histamine and leukotriene release by interleukin 3-primed basophils. J. Exp. Med. 170, 1787-1792. Dahinden, C. A., Geiser, T., Brunner, T., von Tscharner, V., Caput, D., Ferrara, P., Minty, A., and Baggiolini, M. (1994). Monocyte chemotactic protein 3 is a most effective basophiland eosinophil-activating chemokine. J. Exp. Med. 179, 751-756. Danon, D., Goldstein, L., Marikovsky, Y., and Skutelsky, E. (1972). Use of cationized ferritin as a label of negative charges on cell surfaces. J. Ultrastruct. Res. 38, 500-510. de Boer, M., and Roos, D. (1986). Metabolic comparison between basophils and other leukocytes from human blood. J. Immunol. 136,3447-3454. de Curtis, I., and Simons, K. (1989). Isolation of exocytic carrier vesicles from BHK cells. Cell 58,719-727. deShazo, R. D., Levinson, A. I., Dvorak, H. F., and Davis, R. W. (1979). The late phase skin reaction: Evidence for activation of the coagulation system in an IgE-dependent reaction in man. J. Immunol. 122,692-698. Donahue, R. E., Seehra, J., Metzger, M., Lefebvre, D., Rock, B., Carbone, S., Nathan, D. G., Garnick, M., Sehgal, P. K., Laston, D., LaVallie, E., McCoy, J., Schendel, P. F., Norton, C., Turner, K.,Yang, Y.-C., and Clark, S. C. (1988). Human IL-3 and GM-CSF act synergistically in stimulating hematopoiesis in primates. Science 241, 1820-1823. Dvorak, A. M. (1978). Biology and morphology of basophilic leukocytes. In “Immediate Hypersensitivity-Modern Concepts and Developments” (M. K. Bach, ed.), Immunology Series, Vol. 7, pp. 369-405. Dekker, New York. Dvorak, A. M. (1986). Morphologic expressions of maturation and function can affect the ability to identify mast cells and basophils in man, guinea pig, and mouse. In “Mast Cell Differentiation and Heterogeneity” (A. D. Befus, J. Bienenstock, and J. A. Denburg, eds.), pp. 95-114. Raven Press, New York. Dvorak, A. M. (1987). Monograph-Procedural guide to specimen handling for the ultrastructural pathology service laboratory. J. Electron Microsc. Technol. 6, 255-301. Dvorak, A. M. (1988a). Morphologic and immunologic characterization of human basophils, 1879 to 1985. Riv. Immunol. Immunofarmacol. 8, 50-83. Dvorak, A. M. (1988b). The fine structure of human basophils and mast cells. In “Mast Cells, Mediators and Disease” (S. T. Holgate, ed.), Immunology and Medicine Series (W. G. Reeves, ed.), pp. 29-97. Kluwer Academic Dordrecht. Dvorak, A. M. (1989). “Human Mast Cells,” Vol. 114 in the series “Advances in Anatomy, Embryology, and Cell Biology” (F. Beck, W. Hild, W. Kriz, R. Ortmann, J. E. Pauly, and T. H. Schiebler, eds). Springer-Verlag, Berlin. Dvorak, A. M. (ed) (1991). “Basophil and Mast Cell Degradation and Recovery,” Vol. 4 in the series “Blood Cell Biochemistry” (J. R. Harris, ed.). Plenum, New York.

218

ANN M. DVORAK

Dvorak, A. M. (1992a). Basophils and mast cells: Piecemeal degranulation in situ and ex vivo: A possible mechanism for cytokine-induced function in disease. In “Granulocyte Responses to Cytokines. Basic and Clinical Research” (R. G. Coffey, ed.), pp. 169-271. Dekker, New York. Dvorak, A. M. (1992b). Human mast cells. Ultrastructural observations of in situ, ex vivo, and in vitro sites, sources, and systems. In “The Mast Cell in Health and Disease” (M. A. Kaliner and D. D. Metcalfe, eds.), Vol. 62 in the series “Lung Biology in Health and Disease” (C. Lenfant, series ed.), pp. 1-90. Dekker, New York. Dvorak, A. M. (1993). Ultrastructural analysis of anaphylactic and piecemeal degranulation of human mast cells and basophils. In “Immunopharmacology of Mast Cells and Basophils” (J. C. Foreman, ed.) in the series “The Handbook of Immunopharmacology” (C. Page, ed.), pp. 89-113. Academic Press, London. Dvorak, A. M. (1994a). Similarities in the ultrastructural morphology and developmental and secretory mechanisms of human basophils and eosinophils. J. Allergy Clin. Immunol. 94, 1103-1134. Dvorak, A. M. (1994b). Recent ultrastructural contributions to the understanding of the cell biology of mast cells and basophils: Identification, homogeneities, heterogeneities, development, secretion, and recovery. In “Basophils, Mast Cells and Airway Epithelial Cells” (G. Marone, E. Pozzi, and S. I. Rennard, eds.), in the series “Meetings on Pathophysiology of Pulmonary Cells” (E. Pozzi, ed.), pp. 1-30. Masson, Milan. Dvorak, A. M. (1994~).Ultrastructural analysis of human basophil and mast cell recovery after secretion. Semin. Clin. Immunol. 8, 5-16. Dvorak, A. M. (1995a). Ultrastuctural analysis of human mast cells and basophils. In “Human Basophils and Mast Cells. Biological Aspects” (G. Marone, ed.), Vol. 61 in the Chemical Immunology series (L. Adorini, K. Arai, J. D. Capra, K. Ishizaka, A.-M. Schmitt-Verhulst, and B. H. Waksman, eds.), pp. 1-33. Karger, Basel. Dvorak, A. M. (1995b). Ultrastructural evidence for vesicular transport in basophil secretion. Fundum. Clin. Immunol. 3, 17-28. Dvorak, A. M. (1996). Human basophil recovery from secretion. A review emphasizing the distribution of Charcot-Leyden crystal protein in cells stained with the electron-dense tracer, cationized ferritin. Histol. Hisfoputhol. 11, 711-728. Dvorak, A. M. (1997). Ultrastructural localization of histamine in human basophils and mast cells; changes associated with anaphylactic degranulation and recovery demonstrated with a diamine oxidase-gold probe. Allergy 52(Suppl. 34), 14-24. Dvorak, A. M., and Ackerman, S. J. (1989). Ultrastructural localization of the Charcot-Leyden crystal protein (lysophospholipase) to granules and intragranular crystals in mature human basophils. Lab. Invest. 60, 557-567. Dvorak, A. M., and Dvorak, H. F. (1979). The basophil. Its morphology, biochemistry, motility, release reactions, recovery, and role in the inflammatory responses of IgE-mediated and cell-mediated origin. Arch. Pufhol. Lab. Med. 103, 551-557. Dvorak, A. M., and Dvorak, H. F. (1993). Cutaneous basophil hypersensitivity-A 20-year perspective, 1970-1990. In “Immunopharmacology of Mast Cells and Basophils” (J. C. Foreman, ed.), in the series “The Handbook of Immunopharmacology” (C. Page, ed.), pp. 153-180. Academic Press, London. Dvorak, A. M., and Ishizaka, T. (1995). Ultrastructural analysis of the development of human basophils and mast cells in vitro. Inf. J. Clin. Lab. Res. 25, 7-24. Dvorak, A. M., and Kissell, S. (1991). Granule changes of human skin mast cells characteristic of piecemeal degranulation and associated with recovery during wound healing in situ. J . Leukocyte Biol. 49, 197-210. Dvorak, A. M., and Monahan, R. A. (1982). Crohn’s disease. Ultrastructural studies showing basophil leukocyte granule changes and lymphocyte parallel tubular arrays in peripheral blood. Arch. Pathol. Lab. Med. 106, 145-149.

CELL BIOLOGY OF THE BASOPHIL

219

Dvorak, A. M., and Monahan, R. A. (1985): Guinea pig bone marrow basophilopoiesis. J. EXP. Pathol. 2, 13-24. Dvorak, A. M., and Monahan-Earley, R. A. (1992). “Diagnostic Ultrastructural Pathology. I. A Text-Atlas of Case Studies Illustrating the Correlative Clinical-Ultrastructural Pathologic Approach to Diagnosis.” CRC Press, Boca Raton, FL. Dvorak, A. M., and Monahan-Earley, R. A. (1995a). Case 20: Thrombocytopenia in a 32year-old woman. Bernard-Soulier syndrome. In “Diagnostic Ultrastructural Pathology. 111. A Text-Atlas of Case Studies Emphasizing Endocrine and Hematopoietic Systems,” pp. 287-314. CRC Press, Boca Raton, FL. Dvorak, A. M., and Monahan-Earley, R. A. (1995b). Case 21: Thrombocytopenia in a 15year-old male. Gray platelet syndrome. In “Diagnostic Ultrastructural Pathology. 111. A Text-Atlas of Case Studies Emphasizing Endocrine and Hematopoietic Systems,” pp. 315366. CRC Press, Boca Raton, FL. Dvorak, A. M., and Monahan-Earley, R. A. (1995~).“Diagnostic Ultrastructural Pathology. 11. A Text-Atlas of Case Studies Emphasizing Respiratory and Nervous Systems.” CRC Press, Boca Raton, FL. Dvorak, A. M., and Monahan-Early, R. A. (1995d). “Diagnostic Ultrastructural Pathology. 111. A Text-Atlas of Case Studies Emphasizing Endocrine and Hematopoietic Systems.” CRC Press, Boca Raton, FL. Dvorak, A. M., and Morgan, E. S. (1996). Ultrastructural detection of histamine in human mast cells developing from cord blood cells cultured with human or murine recombinant c-kit ligands. Int. Arch. Allergy Immunol. 111, 238-244. Dvorak, A. M., and Morgan E. S. (1997). Diamine oxidase-gold enzyme-affinity ultrastructural demonstration that human gut mucosal mast cells secrete histamine by piecemeal degranulation in vivo. J. Allergy Clin. Imrnunol. 99,812-820. Dvorak, A. M., Dvorak, H. F., and Karnovsky, M. J (1972). Uptake of horseradish peroxidase by guinea pig basophilic leukocytes. Lab. Invest. 26, 27-39. Dvorak, A. M., Mihm, M. C., Jr., and Dvorak, H. F. (1976a). Degranulation of basophilic leukocytes in allergic contact dermatitis reactions in man. J. Immunol. 116, 687-695. Dvorak, A. M., Galli, S. J., and Dvorak, H. F. (1980a). Basophilic leukocytes in cell-mediated hypersensitivity: Possible non-anaphylactic mechanisms of mediator release. In “Advances in Allergology and Clinical Immunology” (A. Oehling, I. Glazer, E. Mathov, and C. Arbesman, eds.), pp. 215-222. Pergamon, Oxford. Dvorak, A. M., Newball, H. H., Dvorak, H. F., and Lichtenstein, L. M. (1980b). Antigeninduced IgE-mediated degranulation of human basophils. Lab. Invest. 43, 126-139. Dvorak, A. M., Monahan, R. A,, Osage, J. E., and Dickersin, G. R. (1980~).Crohn’s disease: Transmission electron microscopic studies. 11. Immunologic inflammatory response. Alterations of mast cells, basophils, eosinophils, and the microvasculature. Hum. Pathol. 11, 606-619. Dvorak, A. M., Hammond, M. E., Morgan, E., Orenstein, N. S . , Galli, S. J., and Dvorak, H. F. (1980d). Evidence for a vesicular transport mechanism in guinea pig basophilic leukocytes. Lab. Invest. 42, 263-276. Dvorak, A. M., Galli, S. J., Galli, A. S., Hammond, M. E., and Dvorak, H. F. (1980e). Lymphocyte mediator modulation of basophil motile structures. In “Biochemical Characterization of Lymphokines. Proceedings of the Second International Lymphokine Workshop” (A. L. de Weck, F. Kristensen, and M. Landy, eds.), pp. 205-207. Academic Press, New York. Dvorak, A. M., Monahan, R. A,, and Dickersin, G. R. (1981a). Diagnostic electron microscopy. I. Hematology: Differential diagnosis of acute lymphoblastic and acute myeloblastic leukemia. Use of ultrastructural peroxidase cytochemistry and routine electron microscopic technology. In “Pathology Annual-Part 1” (S. C. Sommers and P. P. Rosen, eds.), Vol. 16, pp. 101-137. Appleton-Century-Crofts, New York.

220

ANN M. DVORAK

Dvorak, A. M., Galli, S. J., Morgan, E., Galli, A. S., Hammond, M. E., and Dvorak, H. F. (1981b). Anaphylactic degranulation of guinea pig basophilic leukocytes. I. Fusion of granule membranes and cytoplasmic vesicles: Formation and resolution of degranulation sacs. Lab. Invest. 44, 174-191. Dvorak, A. M., Lett-Brown, M., Thueson, D., and Grant, J. A. (1981~).Complement-induced degranulation of human basophils. J. Immunol. 126,523-528. Dvorak, A. M., Osage, J. E., Dvorak, H. F., and Galli, S. J. (1981d). Surface membrane alterations in guinea pig basophils undergoing anaphylactic degranulation. A scanning electron microscopic study. Lab. Invest. 45, 58-66. Dvorak, A. M., Nabel, G., Pyne, K., Cantor, H., Dvorak, H. F., and Galli, S. J. (1982a). Ultrastructural identification of the mouse basophil. Blood 59, 1279-1285. Dvorak, A. M., Mihm, M. C., Jr., Osage, J. E., Kwan, T. H., Austen, K. F., and Wintroub, B. U. (1982b). Bullous pemphigoid, an ultrastructual study of the inflammatory response: Eosinophil, basophil and mast cell granule changes in multiple biopsies from one patient. J. Invest. Dermatol. 78, 91-101. Dvorak, A. M., Galli, S. J., Morgan, E., Galli, A. S., Hammond, M. E., and Dvorak, H. F. (1982~).Anaphylactic degranulation of guinea pig basophilic leukocytes. 11. Evidence for regranulation of mature basophils during recovery from degranulation in vitro. Lab. Invest. 46,461-475. Dvorak, A. M., Dvorak, H. F., and Galli, S. J. (1983a). Ultrastructural criteria for identification of mast cells and basophils in humans, guinea pigs, and mice. Am. Rev. Respir. Dis. Suppl. (Comp. Biol. Lung) 128, S49-S52. Dvorak, A. M., Galli, S. J., Schulman, E. S., Lichtenstein, L. M., and Dvorak, H. F. (1983b). Basophil and mast cell degranulation: Ultrastructural analysis of mechanisms of mediator release. Fed. Proc. 42, 2510-2515. Dvorak, A.M., Galli, S. J., Marcum, J. A., Nable, G., Der Simonian, H., Goldin, J., Monahan, R. A,, Pyne, K., Cantor, H., Rosenberg, R. D., and Dvorak, H. F. (1983~).Cloned mouse cells with natural killer function and cloned suppressor T cells express ultrastructural and biochemical features not shared by cloned inducer T cells. J. Exp. Med. 157, 843-861. Dvorak, A. M., Hammel, I., Schulman, E. S., Peters, S. P., MacGlashan, D. W., Jr., Schleimer, R. P., Newball, H. H., Pyne, K., Dvorak, H. F., Lichtenstein, L. M., and Galli, S. J. (1984a). Differences in the behavior of cytoplasmic granules and lipid bodies during human lung mast cell degranulation. J. Cell Biol. 99, 1678-1687. Dvorak, A. M., Lett-Brown, M. A., Thueson, D. O., Pyne, K., Raghuprasad, P. K., Galli, S. J., and Grant, J. A. (1984b). Histamine-releasing activity (HRA). 111. HRA induces human basophil histamine release by provoking noncytotoxic granule exocytosis. Clin. Immunol. Immunopathol. 32, 142-150. Dvorak, A. M., Klebanoff, S. J., Henderson, W. R., Monahan, R. A., Pyne, K., and Galli, S. J. (1985a). Vesicular uptake of eosinophil peroxidase by guinea pig basophils and by cloned mouse mast cells and granule-containing lymphoid cells. Am. J. Pathol. 118,425-438. Dvorak, A. M., Ishizaka, T., and Galli, S. J. (1985b). Ultrastructure of human basophils developing in vitro. Evidence for the acqu on of peroxidase by basophils and for different effects of human and murine growth factors on human basophil and eosinophil maturation. Lab. Invest. 53, 57-71. Dvorak, A. M., Schulman, E. S., Peters, S. P., MacGlashan, D. W., Jr., Newball, H. H., Schleimer, R. P., and Lichtenstein, L. M. (1985~).Immunoglobulin E-mediated degranulation of isolated human lung mast cells. Lab. Invest. 53, 45-56. Dvorak, A. M., Colvin, R. B., and Monahan, R. A. (1985d). Immunoferritin electron microscopic studies with antibasophil serum of guinea pig basophil degranulation and regranulation in vitro. Clin. Immunol. Immunopathol. 37, 63-76. Dvorak, A. M., Monahan-Earley, R. A,, Dvorak, H. F., and Galli, S. J. (1987). Ultrastructural cytochemical and autoradiographic demonstration of nonspecific esterase(s) in guinea pig basophils. J. Histochem. Cytochem. 35, 351-360.

CELL BIOLOGY OF THE BASOPHIL

221

Dvorak, A. M., Letourneau, L., Login, G. R., Weller, P. F., and Ackerman, S. J. (1988). Ultrastructural localization of the Charcot-Leyden crystal protein (lysophospholipase) to a distinct crystalloid-free granule population in mature human eosinophils. Blood 72,150-158. Dvorak, A. M., Monahan-Earley, R. A., Estrella, P., Kissell, S., and Donahue, R. E. (1989a). Ultrastructure of monkey peripheral blood basophils stimulated to develop in vivo by recombinant human interleukin 3. Lab. Invest. 61,677-690. Dvorak, A. M., Saito, H., Estrella, P., Kissell, S., Arai, N., and Ishizaka, T. (1989b). Ultrastructure of eosinophils and basophils stimulated to develop in human cord blood mononuclear cell cultures containing recombinant human interleukin-5 or interleukin-3. Lab. invest. 61, 116-132. Dvorak, A. M., Weller, P. F., Monahan-Earley, R. A., Letourneau, L., and Ackerman, S. J. (1990a). Ultrastructural localization of Charcot-Leyden crystal protein (lysophospholipase) and peroxidase in macrophages, eosinophils and extracellular matrix of the skin in the hypereosinophilic syndrome. Lab. Invest. 62, 590-607. Dvorak, A. M., Letourneau, L., Weller, P. F., and Ackerman, S. J. (1990b). Ultrastructural localization of Charcot-Leyden crystal protein (lysophospholipase) to intracytoplasmic crystals in tumor cells of primary solid and papillary neoplasm of the pancreas. Lab. Invest. 62,608-615. Dvorak, A. M., Warner, J. A., Kissell, S., Lichtenstein, L. M., and MacGlashan, D. W., Jr. (1991a). F-Met peptide-induced degranulation of human basophils. Lab. Invest. 64,234-253. Dvorak, A. M., Furitsu, T., Letourneau, L., Ishizaka, T., and Ackerman, S. J. (1991b). Mature eosinophils stimulated to develop in human cord blood mononuclear cell cultures supplemented with recombinant human interleukin-5. I. Piecemeal degranulation of specific granules and distribution of Charcot-Leyden crystal protein. Am. J. Pathol. 138, 69-82. Dvorak, A. M., Warner, J. A,, Morgan, E., Kissell-Rainville, S., Lichtenstein, L. M., and MacGlashan, D. W., Jr. (1992a). An ultrastructural analysis of tumor-promoting phorbol diester-induced degranulation of human basophils. Am. J. Pathol. 141, 1309-1322. Dvorak, A. M., McLeod, R. S., Onderdonk, A., Monahan-Early, R. A,, Cullen, J. B., Antonioli, D. A,, Morgan, E., Blair, J. E., Estrella, P., Cisneros, R. L., Silen, W., and Cohen, Z. (1992b). Ultrastructural evidence for piecemeal and anaphylactic degranulation of human gut mucosal mast cells in vivo. int. Arch. Allergy Immunol. 99, 74-83. Dvorak, A. M., Ackerman, S. J., Furitsu, T., Estrella, P., Letourneau, L., and Ishizaka, T. (1992~).Mature eosinophils stimulated to develop in human cord blood mononuclear cell cultures supplemented with recombinant human interleukin-5. 11. Vesicular transport of specific granule matrix peroxidase, a mechanism for effecting piecemeal degranulation. Am. J. Pathol. 140,795-807. Dvorak, A. M., Seder, R. A,, Paul, W. E., Kissell-Rainville, S., Plaut, M., and Galli, S. J. (1993a). Ultrastructural characteristics of Fc,R-positive basophils in the spleen and bone marrow of mice immunized with goat anti-mouse IgD antibody. Lab. Invest. 68, 708-715. Dvorak, A. M., Morgan, E. S., Schleimer, R. P., and Lichtenstein, L. M. (1993b). Diamine oxidase-gold labels histamine in human mast-cell granules: A new enzyme-affinity ultrastructural method. J. Histochem. Cytochem. 41,787-800. Dvorak, A. M., Mitsui, H., and Ishizaka, T. (1993~).Ultrastructural morphology of immature mast cells in sequential suspension cultures of human cord blood cells supplemented with c-kit ligand; distinction from mature basophilic leukocytes undergoing secretion in the same cultures. J . Leukocyte Biol. 54,465-485. Dvorak, A. M., Seder, R. A., Paul, W. E., Morgan, E. S., and Galli, S. J. (1994a). Effects of interleukin-3 with or without the c-kit ligand, stem cell factor, on the survival and cytoplasmic granule formation of mouse basophils and mast cells in v i m . Am. J. Pathol. 144,160-170. Dvorak, A. M., Morgan, E. S., Lichtenstein, L. M., and MacGlashan, D. W., Jr. (1994b). Activated human basophils contain histamine in cytoplasmic vesicles. Int. Arch. Allergy Immunol. 105,s-11.

222

ANN M. DVORAK

Dvorak, A. M., Ishizaka, T., Letourneau, L., Albee, E. A,, Mitsui, H., and Ackerman, S. J. (1994~).Charcot-Leyden crystal protein distribution in basophils and its absence in mast cells that differentiate from human umbilical cord blood precursor cells cultured in murine fibroblast culture supernatants or in recombinant human c-kit ligand. J. Histochern. Cytochem. 42,251-263. Dvorak, A. M., Furitsu, T., Estrella, P., Letourneau, L., Ishizaka, T., and Ackerman, S . J. (1994d). Ultrastructural localization of major basic protein in the human eosinophil lineage in vitro. J. Histochem. Cytochem. 42, 1443-1451. Dvorak, A. M., Tepper, R. I., Weller, P. F., Morgan, E. S., Estrella, P., Monahan-Earley, R. A., and Galli, S. J. (1994e). Piecemeal degranulation of mast cells in the inflammatory eyelid lesions of interleukin-4 transgenic mice. Evidence of mast cell histamine release in vivo by diamine oxidase-gold enzyme-affinity ultrastructural cytochemistry. Blood 83,36003612. Dvorak, A. M., MacGlashan, D. W., Jr., Morgan, E. S . , and Lichtenstein, L. M. (1995a). Histamine distribution in human basophil secretory granules undergoing FMLP-stimulated secretion and recovery. Blood 86, 3560-3566. Dvorak, A. M., Morgan, E. S., Monahan-Earley, R. A., Estrella, P., Schleimer, R. P., Weller, P. F., Tepper, R. I., Lichtenstein, L. M., and Galli, S. J. (1995b). Analysis of mast cell activation using diamine oxidase-gold enzyme-affinity ultrastructural cytochemistry. Int. Arch. Allergy Irnmunol. 107, 87-89. Dvorak, A. M., Ackerman, S. J., Letourneau, L., Morgan, E. S . , Lichtenstein, L. M., and MacGlashan, D. W., Jr. (1996a). Vesicular transport of Charcot-Leyden crystal protein in tumor-promoting phorbol diester-stimulated human basophils. Lab. Invest. 74, 967-974. Dvorak, A. M., Warner, J. A,, Fox, P., Lichtenstein, L. M., and MacGlashan, D. W. (199613). Recovery of human basophils after FMLP-stimulated secretion. Clin. Exp. Allergy 26, 281-294. Dvorak, A. M., Morgan, E. S., Schleimer, R. P., and Lichtenstein, L. M. (1996~).Diamine oxidase-gold ultrastructural localization of histamine in isolated human lung mast cells stimulated to undergo anaphylactic degranulation and recovery in vitro. J. Leukocyte Bid. 59, 824-834. Dvorak, A. M., Kohn, S . , Morgan, E. S., Fox, P., Nagy, J. A,, and Dvorak, H. F. (1996d). The vesiculo-vacuolar organelle (VVO): A distinct endothelial cell structure that provides a transcellular pathway for macromolecular extravasation. J. Leukocyte Bid. 59, 100-115. Dvorak, A. M., Schroeder, J. T., MacGlashan, D. W., Jr., Byran, K. P., Morgan, E. S . , Lichtenstein, L. M., and MacDonald, S. M. (1996e). Comparative ultrastructural morphology of human basophils stimulated to release histamine by anti-IgE, recombinant IgE-dependent histamine-releasing factor, or monocyte chemotactic protein-1. J. Allergy Clin. Irnmunol. 98,355-370. Dvorak, A. M., MacGlashan, D. W., Jr., Morgan, E. S., and Lichtenstein, L. M. (1996f). Vesicular transport of histamine in stimulated human basophils. Blood 88, 4090-4101. Dvorak, A. M., MacGlashan, D. W., Jr., Warner, J. A,, Letourneau, L., Morgan, E. S., Lichtenstein, L. M., and Ackerman, S. J. (1997a). Localization of Charcot-Leyden crystal protein in individual morphologic phenotypes of human basophils stimulated by f-Met peptide. Clin. Exp. Allergy 27, 452-474. Dvorak, A. M., MacGlashan, D. W., Jr., Warner, J. A., Letourneau, L., Morgan, E. S., Lichtenstein, L. M., and Ackerrnan, S. J. (1997b). Vesicular transport of Charcot-Leyden crystal protein in f-Met peptide-stimulated human basophils. Int. Arch. Allergy Irnrnunol. 113,465-477. Dvorak, H. F., and Dvorak, A. M. (1972). Basophils, mast cells, and cellular immunity in animals and man. Hum. Pathol. 3, 454-456. Dvorak, H. F., and Dvorak, A. M. (1973). Basophilic leukocytes in delayed-type hypersensitivity reactions in experimental animals and man. In “Microenvironmental Aspects of Immu-

CELL BIOLOGY OF THE BASOPHIL

223

nity,” Vol. 29 in the series “Advances in Experimental Medicine and Biology” (B. D. Jankovic and K. Isakovic, eds.), pp. 573-579. Plenum. New York. Dvorak, H. F., and Dvorak, A. M. (1974). Cutaneous basophil hypersensitivity. In “Progress in Immunology 11,” Vol. 3, “Biological Aspects 11” (L. Brent and J. Holborow, eds.), pp. 171-181. North-Holland, Amsterdam. Dvorak, H. F., and Dvorak, A. M. (1975). Basophilic leucocytes: Structure, function and role in disease. In “Clincis in Haematology,” Vol. 4, No. 3, “Granulocyte and Monocyte Abnormalities” (M. A. Lichtman, ed.), pp. 651-683. Saunders, London. Dvorak, H. F., Dvorak, A. M., Simpson, B. A., Richerson, H. B., Leskowitz, S., and Karnovsky, M. J. (1970). Cutaneous basophil hypersensitivity. 11. A light and electron microscopic description. J . Exp. Med. 132, 558-582. Dvorak, H. F., Dvorak, A. M., and Churchill, W. F. (1973). Immunologic rejection of diethylnitrosamine-induced hepatomas in strain 2 guinea pigs. Participation of basophilic leukocytes and macrophage aggregates. J. Exp. Med. 137,751-775. Dvorak, H. F., Dvorak, A. M., and Mihm, M. C., Jr. (1974a). Morphologic studies in cellular hypersensitivity in guinea pigs and man. In “Monographs in Allergy,” Vol. 8, “Contact Hypersensitivity in Experimental Animals’’ (D. Parker and J. L. Turk, eds.), pp. 54-65. Karger, Basel. Dvorak, H. F., Mihm, M. C., Jr., Dvorak, A. M., Johnson, R. A,, Manseau, E. J., Morgan, E., and Colvin, R. B. (1974b). Morphology of delayed type hypersensitivity reactions in man. I. Quantitative description of the inflammatory response. Lab. Invest 31, 111-130. Dvorak, H. F., Selvaggio, S. S., Dvorak, A. M., Colvin, R. B., Lean, D. B., and Rypysc, J. (1974~).Purification of basophilic leukocytes from guinea pig blood and bone marrow. J. Immunol. 113,1694-1702. Dvorak, H. F., Mihm, M. C., Jr., and Dvorak, A. M. (1976b). Morphology of delayed-type hypersensitivity reactions in man. J. Invest. Dermatol. 67, 391-401. Dvorak, H. F., Orenstein, N. S., Galli, S. J., and Dvorak, A. M. (1979a). Plasminogen activator of guinea pig basophils: A protease probably localized to the cell surface. In “Monographs in Allergy” (P. Kallos, ed.), Vol. 14, pp. 249-252. Karger, Basel. Dvorak, H. F., Orenstein, N. S., Galli, S. J., and Dvorak, A. M. (1979b). Cutaneous basophil hypersensitivity. In “The Mast Cell. Its Role in Health and Disease” (J. Pepys and A. M. Edwards, eds.), pp. 76-82. Pitman, Kent, UK. Dvorak, H. F., Mihm, M. C., Jr., Dvorak, A. M., Barnes, B. A., Manseau, E. J., and Galli, S. J. (1979~).Rejection of first-set skin allografts in man. The microvasculature is the critical target of the immune response. J. Exp. Med. 150,322-337. Dvorak, H. F., Galli, S. J., and Dvorak, A. M. (1980f). Expression of cell-mediated hypersensitivity in vivo-Recent advances. In “International Review of Experimental Pathology” (G. W. Richter and M. A. Epstein, eds.), Vol. 21, pp. 119-194. Academic Press, New York. Dvorak, H. F., Mihm, M. C., Jr., Dvorak, A. M., Barnes, B. A,, and Galli, S. J. (1980g). The microvasculature is the critical target of the immune response in vascularized skin allgoraft rejection. J. Invest. Dermutol. 74, 280-284. Dvorak, H. F., Galli, S. J., and Dvorak, A. M. (1986). Cellular and vascular manifestations of cell-mediated immunity. Hum. Pathof. 17, 122-137. Dvorak, H. F., Brown, L. F., Detmar, M., and Dvorak, A. M. (1995~).Vascular permeability factorlvascular endothelial cell growth factor, microvascular hyperpermeability, and angiogenesis. Am. J. Pathof. 146, 1029-1039. Eguchi, M. (1991). Comparative electron microscopy of basophils and mast cells, in vivo and in vitro. Electron Microsc. Rev. 4, 293-318. Eguchi, M., Sakakibara, H., Iwama, Y., Ochiai, R., Furukawa, T., Yamaguchi, K., Suda, T., and Suda, J. (1985). Ultrastructural characterization of cells with basophilic granules in hemopoietic colonies. J. Clin. Electron Microsc. 18, 5-6.

224

ANN M. DVORAK

Eguchi, M., Suda, J., Sugita, K., Suda, T., and Furukawa, T. (1989). Cultured human basophils with ultrastructural and ultracytochemical features of mast cells. Eur. J. Haematof.42,81-89. Ehrich, W. E. (1953). Histamine in mast cells. Science 118, 603. Ehrlich, P. (1877). Beitrage zur kenntnis der anilinfarbungen und ihrer verwendung in der mikroskopischen technik. Arch. Mikrosk. Anat. 13,263-277. Ehrlich, P. (1879). Beitrage zur kenntnis der granuliertan bindegewebszellen und der eosinophilen leukocyten. Arch. Anat. Physiol. 3, 166-169. Espersen, F., Jarlov, J. O., Jensen, C., Skov, P. S., and Norn, S . (1984). Staphylococcus aureus peptidoglycan induces histamine release from basophil human leukocytes in vitro. Infect. Immun. 46,710-714. Farnam, J., Grant, J. A,, Lett-Brown, M. A., Hunt, C., Thueson, D. O., and Giclas, P. C. (1985). Complement- and IgE-mediated release of histamine from basophils in v i m . J. Immunol. 134,541-547. Fedorko, M. E., and Hirsch, J. G. (1965). Crystalloid structure in granules of guinea pig basophils and human mast cells. J. Cell Biol. 26, 973-976. Feng, D., Nagy, J. A,, Hipp, J., Dvorak, H. F., and Dvorak, A. M. (1996). Vesiculo-vacuolar organelles and the regulation of venule permeability to macromolecules by vascular permeability factor, histamine and serotonin. J. Exp. Med. 183, 1981-1986. Fernandez, J. M., Villalon, M., and Verdugo, P. (1991). Reversible condensation of mast cell secretory products in vitro. Biophys. J. 59, 1022-1027. Ferro-Novick, S., and Jahn, R. (1994). Vesicle fusion from yeast to man. Nature 370,191-193. Findlay, S . R., Lichtenstein, L. M., and Grant, J. A. (1980). Generation of slow reacting substance by human leukocytes. 11. Comparison of stimulation by antigen, anti-IgE, calcium ionophore, and C5-peptide. J. Immunol. 124,238-242. Findlay, S. R., Dvorak, A. M., Kagey-Sobotka, A., and Lichtenstein, L. M. (1981). Hyperosmolar triggering of histamine release from human basophils. J. Clin. Invest. 67, 1604-1613. Fine, R. E. (1989). Vesicles without clathrin: Intermediates in bulk flow exocytosis. Cell 58,609-610. Fishman, P., Djaldetti, M., Hart, J., and Sredni, B. (1985). Growth of human basophil lines derived from chronic myelocytic leukaemia cells in v i m : Ultrastructure and x-ray microanalysis studies. Immunology 55, 105-113. Fox, B., Bull, T. B., and Guz, A. (1981). Mast cells in the human alveolar wall: An electronmicroscopic study. J. Clin. Pathol. 34, 1333-1342. Fox, C. C., Dvorak, A. M., MacGlashan, D. W., Jr., and Lichtenstein, L. M. (1984). Histaminecontaining cells in human peritoneal fluid. J. Immunol. 132,2177-2179. Frens, G. (1973). Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions. Nature Phys. Sci. 241, 20-22. Frigeri, L. G., Zuberi, R. I., and Liu, F.-T. (1993). EBP, a P-galactoside-binding animal lectin, recognizes IgE receptor (FccRI) and activates mast cells. Biochemistry 32,7644-7649. Furitsu, T., Saito, H., Dvorak, A. M., Schwartz, L. B., Irani, A.-M. A,, Burdick, J. F., Ishizaka, K., and Ishizaka, T. (1989). Development of human mast cells in vitro. Proc. Natl. Acad. Sci. USA 86, 10039-10043. Furutani, Y., Nomura, H., Notake, M., Oyamada, Y., Fukui, T., Yamada, M., Larsen, C. G., Oppenheim, J. J., and Matsushima, K. (1989). Cloning and sequencing of the cDNA for human monocyte chemotactic and activating factor (MCAF). Biochem. Biophys. Res. Commun. 159,249-255. Galli, S. J., and Dvorak, A. M. (1995). Production, biochemistry, and function of basophils and mast cells. I n “Williams Hematology” (E. Beutler, M. A. Lichtman, B. S. Coller, and T. J. Kipps, eds.), 5th Ed., pp. 805-810. McGraw-Hill, New York. Galli, S. J., Orenstein, N. S., Gill, P. J., Silbert, J. E., Dvorak, A. M., and Dvorak, H. F. (1979). Sulphated glycosaminoglycans synthesised by basophil-enriched human leukaemic

CELL BIOLOGY OF THE BASOPHIL

225

granulocytes. In “The Mast Cell. Its Role in Health and Disease” (J. Pepys and A. M. Edwards, eds.), p. 842. Pitman, Kent, UK. Galli, S. J., Dvorak, A. M., Hammond, M. E., Morgan, E., Galli, A. S., and Dvorak, H. F. (1981). Guinea pig basophil morphology in virro. I. Ultrastructure of uropod-bearing (motile) basophils and modulation of motile structures by serum and substrate effects. J. Immunol. 126 1066-1074. Galli, S. J., Dvorak, A. M., and Dvorak, H. F. (1983a). Mouse mast cells and other granulated leukocyte clones. In “Monographs in Allergy” (M. Ricci and G. Marone, eds.), Vol. 18, pp. 129-137. Karger, Basel. Galli, S. J., Dvorak, A. M., and Dvorak, H. F. (1983b). Morphology, biochemistry, and function of basophils. In “Hematology” (W. J. Williams, E. Beutler, A. J. Erslev, and M. A. Lichtman, eds.), 3rd Ed., pp. 820-825. McGraw-Hill, New York. Galli, S. J., Dvorak, A. M., and Dvorak, H. F. (1984). Basophils and mast cells: Morphologic insights into their biology, secretory patterns, and function. In “Progress in Allergy,” Vol. 34, “Mast Cell Activation and Mediator Release” (K. Ishizaka, ed.), pp. 1-141. Karger, Basel. Galli, S. J., Arizono, N., Murakami, T., Dvorak, A. M., and Fox, J. G. (1987). Development of large numbers of mast cells at sites of idiopathic chronic dermatitis in genetically mast cell-deficient WBB6F1-W W mice. Blood 69, 1661-1666. Galli, S. J., Gordon, J. R., and Wershil, B. K. (1991). Cytokine production by mast cells and basophils. Curr. Opin. Immunol. 3, 865-872. Glasser, L., Corrigan, J. J., Jr., and Payne, C. (1976). Basophilic meningitis secondary to lymphoma. Neurology 26,899-902. Glovsky, M. M., Hugli, T. E., Ishizaka, T., Lichtenstein, L. M., and Erickson, B. W. (1979). Anaphylatoxin induced histamine release with human leukocytes: Studies of C3a leukocyte binding and histamine release. J. Clin. Invesr. 64,804-811. Goldstein, J. L., Anderson, R. G. W., and Brown, M. S. (1979). Coated pits, coated vesicles, and receptor-mediated endocytosis. Nature 279, 679-685. Graham, H. T., Lowry, 0. H., Wheelwright, F., Lenz, M. A,, and Parish, H. H., Jr. (1955). Distribution of histamine among leukocytes and platelets. Blood 10, 467-481. Grant, J. A., Dupree, E., Goldman, A. S., Schultz, D. R., and Jackson, A. L. (1975). Complement-mediated release of histamine from human leukocytes. J . Immunol. 114,11011106. Grant, J. A., Settle, L., Whorton, E. B., and Dupree, E. (1976). Complement-mediated release of histamine from human basophils. 11. Biochemical characterization of the reaction. J. Immunol. 117,450-456. Grant, J. A., Dupree, E., and Thueson, D. 0. (1977). Complement-mediated release of histamine from human basophils. 111. Possible regulatory role of microtubules and microfilaments. J. Allergy Clin. Immunol. 60, 306-311. Grant, J. A., Alam, R., and Lett-Brown, M. A. (1991). Histamine-releasing factors and inhibitors: Historical perspectives and possible implications in human illness. J. Allergy Clin. Immunol. 88,683-693. Haak-Frendscho, M., Arai, N., Arai, K.-I., Baeza, M. L., Finn, A., and Kaplan, A. P. (1988a). Human recombinant granulocyte-macrophage colony-stimulating factor and interleukin 3 cause basophil histamine release. J. Clin. Invesr. 82, 17-20. Haak-Frendscho, M., Dinarello, C., and Kaplan, A. P. (1988b). Recombinant human interleukin-1 beta causes histamine release from human basophils. J . Allergy Clin. Immunol. 82,218-223. Hammel, I., Dvorak, A. M., and Galli, S. J. (1987). Defective cytoplasmic granule formation. I. Abnormalities affecting tissue mast cells and pancreatic acinar cells of beige mice. Lab. Invesr. 56, 321-328. Handagama, P., Scarborough, R. M., Shuman, M. A., and Bainton, D. F. (1993). Endocytosis of fibrinogen into megakaryocyte and platelet @-granulesis mediated by (glycoprotein IIb-IIIa) [published erratum appears in Blood 82, 2936, 19931. Blood 82, 135-138.

226

ANN M. DVORAK

Handagama, P. J., George, J. N., Shuman, M. A., McEver, R. P., and Bainton, D. F. (1987). Incorporation of a circulating protein into megakaryocyte and platelet granules. Proc. Nutl. Acad. Sci. USA 84,861-865. Handagama, P. J., Shuman, M. A., and Bainton, D. F. (1989). Incorporation of intravenously injected albumin, immunoglobulin G, and fibrinogen in guinea pig megakaryocyte granules. J. Clin. Invest. 84, 73-82. Hardin, J. H., and Spicer, S. S. (1971). Ultrastructural localization of dialyzed iron-reactive mucosubstance in rabbit heterophils, basophils, and eosinophils. J. Cell Biol. 48,368-386. Harrison, P., Savidge, G. F., and Cramer, E. M. (1990). The origin and physiological relevance of a-granule adhesive proteins. Br. J. Huematol. 74, 125-130. Hastie, R. (1974). A study of the ultrastructure of human basophil leukocytes. Lab Invest. 31,223-231. Hastie, R. (1990). Ultrastructure of human basophil leukocytes studied after spray freezing and freeze-substitution. Lab. Invest. 62, 119-130. Hastie, R., Heroy, J. H., 111, and Levy, D. A. (1979). Basophil leukocytes and mast cells in human nasal secretions and scrapings studied by light microscopy. Lab. Invest. 40,554-561. Hebert, H., and Lindberg, M. (1982). Granules in basophil leukocytes from guinea pig dermis and granules isolated from basophil leukocytes in guinea pig blood and bone marrow: A structural study by electron microscopy and optical diffraction. J. Ultrastruct. Res. 78, 215-225. Hermine, 0..Mayeux, P., Titeux, M., Mitjavila, M.-T., Casadevall, N., Guichard, J., Komatsu, N., Suda, T., Miura, Y., Vainchenker, W., and Breton-Gorius, J. (1992). Granulocytemacrophage colony-stimulating factor and erythropoietin act competitively to induce two different programs of differentiation in the human pluripotent cell line UT-7. Blood 80,30603069. Hernandez-Asensio, M., Hooks, J. J., Ida, S., Siraganian, R. P., and Notkins, A. L. (1979). Interferon-induced enhancement of IgE-mediated histamine release from human basophils requires RNA synthesis. J. Immunol. 122, 1601-1603. Hill, S. A., Shaughnessy, S. G., Joshua, P., Ribau, J., Austin, R. C., and Podor, T. J. (1996). Differential mechanisms targeting type 1 plasminogen activator inhibitor and vitronectin into the storage granules of a human megakaryocytic cell line. Blood 87, 5061-5073. Hirai, K., Morita, Y., Misaki, Y., Ohta, K., Takaishi, T., Suzuki, S., Motoyoshi, K., and Miyamoto, T. (1988). Modulation of human basophil histamine release by hemopoietic growth factors. J. Immunol. 141, 3958-3964. Hirai, K., Yamaguchi, M., Misaki, Y., Takaishi, T., Ohta, K., Morita, Y., Ito, K., and Miyamoto, T. (1990). Enhancement of human basophil histamine release by interleukin 5. J. Exp. Med. 172, 1525-1528. Hoenig, E. M., and Levine, S. (1974). Three localized forms of experimental allergic encephalomyelitis: An ultrastructural comparison. J. Neuroputhol. Exp. Neurol. 33, 251-259. Hook, W. A,, and Siraganian, R. P. (1977). Complement-induced histamine release from human basophils. 111. Effect of pharmacologic agents. J. Immunol. 118, 679-684. Hook, W. A,, Siraganian, R. P., and Wahl, S. M. (1975). Complement-induced histamine release from human basophils. I. Generation of activity in human serum. J. Immunol. 114, 1185-1190. Hook, W. A., Schiffmann, E., Aswanikumar, S., and Siraganian, R. P. (1976). Histamine release by chemotactic, formyl methionine-containing peptides. J. Immunol. 117, 594-596. Horn, R. G., and Spicer, S. S. (1964). Sulfated mucopolysaccharide and basic protein in certain granules of rabbit leukocytes. Lab. Invest. 13, 1-15. Ida, S., Hooks, J. J., Siraganian, R. P., and Notkins, A. L. (1977). Enhancement of IgEmediated histamine release from human basophils by viruses: Role of interferon. J. Exp. Med. 145, 892-906.

CELL BIOLOGY OF THE BASOPHIL

227

Ida, S., Hooks, J. J., Siraganian, R. P., and Notkins, A. L. (1980). Enhancement of IgEmediated histamine release from human basophils by immune-specific lymphokines. Clin. Exp. Immunol. 41,380-387. Iliopoulos, O., Baroody, F. M., Naclerio, R. M., Bochner, B. S., Kagey-Sobotka, A., and Lichtenstein, L. M. (1992). Histamine-containing cells obtained from the nose hours after antigen challenge have functional and phenotypic characteristics of basophils. J. Immunol. 148,2223-2228. Irani, A,-M. A,, Bradford, T. R., Kepley, C. L., Schechter, N. M., and Schwartz, L. B. (1989). of human mast cells by immunohistochemistry using Detection of MCT and M C T types ~ new monoclonal anti-tryptase and anti-chymase antibodies. J. Histochem. Cytochem. 37, 1509-1515. Ishizaka, K., Tomioka, H., and Ishizaka, T. (1970). Mechanisms of passive sensitization. I. Presence of IgE and IgG molecules on human leukocytes. 1.Immunol. 105, 1459-1467. Ishizaka, T., and Ishizaka, K. (1975). Cell surface IgE on human basophilic granulocytes. Ann. NY Acad. Sci. 254,462-415. Ishizaka, T., Sterk, A. R., and Ishizaka, K. (1979). Demonstration of Fcy receptors on human basophil granulocytes. J. Immunol. 123, 578-583. Ishizaka, T., Dvorak, A. M., Conrad, D. H., Niebyl, J. R., Marquette, J. P., and Ishizaka, K. (1985a). Morphologic and immunologic characterization of human basophils developed in cultures of cord blood mononuclear cells. J. Immunol. 134,532-540. Ishizaka, T., Conrad, D. H., Huff, T. F., Metcalfe, D. D., Stevens, R. L., and Lewis, R. A. (1985b). Unique features of human basophilic granulocytes developed in in vitro culture. In?. Arch. Allergy Appl. Immunol. 77, 137-143. Ishizaka, T., Saito, H., Furitsu, T., and Dvorak, A. M. (1989a). Growth of human basophils and mast cells in vitro. In “Mast Cell and Basophil Differentiation and Function in Health and Disease” (S. J. Galli and K. F. Austen, eds.), pp. 39-47. Raven Press, New York. Ishizaka, T., Saito, H., Hatake, K., Dvorak, A. M., Leiferman, K. M., Arai, N., and Ishizaka, K. (1989b). Preferential differentiation of inflammatory cells by recombinant human interleukins. Int. Arch. Allergy Appl. Immunol. 88, 46-49. Jamieson, J. D., and Palade, G. E. (1971). Synthesis, intracellular transport, and discharge of secretory proteins in stimulated pancreatic exocrine cells. J. CeN Biol. 50, 135-158. Javid, J. (1978). Human haptoglobins. Curr. Top. Hematol. 1, 151-192. Jolly, M. J. (1900). Recherches sur la division indirecte des cellules lymphatiques granuleuses de la moelle des 0s. Arch. Anat. Microsc. 3 (Fasc. 11-III), 168-228. Kagey-Sobotka, A., Dembo, M., Goldstein, B., Metzger, H., and Lichtenstein, L. M. (1981). Qualitative characteristics of histamine release from human basophils by covalently crosslinked IgE. J. Immunol. 127,2285-2291. Kaplan, A. P., Reddigari, S., Baeza, M., and Kuna, P. (1991). Histamine releasing factors and cytokine-dependent activation of basophils and mast cells. Adv. Immunol. 50, 237-260. Karnovsky, M. J. (1967). The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. J. Cell Biol. 35, 213-236. Kelly, R. B. (1985). Pathways of protein secretion in eukaryotes. Science 230, 25-32. Kepley, C . , Craig, S., and Schwartz, L. (1994). Purification of human basophils by density and size alone. J. Immunol. Methods 175, 1-9. Kepley, C. L., Craig, S. S., and Schwartz, L. B. (1995). Identification and partial characterization of a unique marker for human basophils. J. Immunol. 154, 6548-6555. Kobayasi, T., Midtgsrd, K., and Asboe-Hansen, G. (1968). Ultrastructure of human mast-cell granules. J. Ultrastruct. Res. 23, 153-165. Kohn, S., Nagy, J. A., Dvorak, H. F., and Dvorak, A. M. (1992). Pathways of macromolecular tracer transport across venules and small veins. Structural basis for the hyperpermeability of tumor blood vessels. Lab. Invest. 67, 596-607.

228

ANN M. DVORAK

Komiyama, A., and Spicer, S. S. (1974). Ultrastructural localization of a characteristic acid phosphatase in granules of rabbit basophils. J. Histochem. Cytochem. 22, 1092-1104. Krieger, M., Brunner, T., Bischoff, S. C., von Tscharner, V., Walz, A., Moser, B., Baggiolini, M., and Dahinden, C . A. (1992). Activation of human basophils through the IL-8 receptor. J. Immunol. 149,2662-2667. Kuna,P., Reddigari, S. R., Rucinski, D., Oppenheim, J. J., and Kaplan, A. P. (1992a). Monocyte chemotactic and activating factor is a potent histamine-releasing factor for human basophils. J . Exp. Med. 175,489-493. Kuna, P., Reddigari, S. R., Schall, T. J., Rucinski, D., Viksman, M. Y., and Kaplan, A. P. (1992b). RANTES, a monocyte and T lymphocyte chemotactic cytokine releases histamine from human basophils. J. Immunol. 149,636-642. Kurimoto, Y., de Weck, A. L.. and Dahinden, C. A. (1989). Interleukin 3-dependent mediator release in basophils triggered by C5a. J. Exp. Med. 170, 467-479. Lacy, P. E. (1975). Endocrine secretory mechanisms. A review. Am. J. Pathol. 79, 170-187. Lagunoff, D., Phillips, M., and Benditt, E. P. (1961). The histochemical demonstration of histamine in mast cells. J . Histochem. Cytochem. 9, 534-541. Le Gros, G., Ben-Sasson, S. Z., Conrad, D. H., Clark-Lewis, I., Finkelman, F. D., Plaut, M., and Paul, W. E. (1990). IL-3 promotes production of IL-4 by splenic non-B, non-T cells in response to Fc receptor cross-linkage. J. Immunol. 145,2500-2506. Leiferman, K. M., Gleich, G. J., Kephart, G. M., Haugen, H. S., Hisamatsu, K.-I., Proud, D., Lichtenstein, L. M., and Ackerman, S. J. (1986). Differences between basophils and mast cells: Failure to detect Charcot-Leyden crystal protein (lysophospholipase) and eosinophil granule major basic protein in human mast cells. J. Immunol. 136,852-855. Leonidas, D. D., Elbert, B. L., Zhou, Z., Leffler, H., Ackerman, S. J., and Acharya, K. R. (1995). Crystal structure of human Charcot-Leyden crystal protein, an eosinophil lysophospholipase, identifies it as a new member of the carbohydrate-binding family of galectins. Structure 3, 1379-1393. Li, H., Sim, T. C., and Alam, R. (1996). IL-13 released by and localized in human basophils. J. Irnmunol. 156,4833-4838. Lichtenstein, L. M. (1971). The immediate allergic response: In vitro separation of antigen activation, decay and histamine release. J. Immunol. 107, 1122-1130. Lichtenstein, L. M. (1975). The mechanism of basophil histamine release induced by antigen and by the calcium ionophore A23187. J. Immunol. 114, 1692-1699. Lichtenstein, L. M., and Bochner, B. S. (1991). The role of basophils in asthma. Ann. NY Acud. Sci. 629, 48-61. Lichtenstein, L. M., and Gillespie, E. (1973). Inhibition of histamine release by histamine controlled by HZreceptor. Nature 244,287-288. Lichtenstein, L. M., and Osler, A. G. (1964). Studies on the mechanisms of hypersensitivity phenomena. IX. Histamine release from human leukocytes by ragweed pollen antigen. J. Exp. Med. 120,507530. Lichtenstein, L. M., Marone, G., Thomas, L. L., and Malveaux, F. J. (1978). The role of basophils in inflammatory reactions. J . Invest. Dermatol. 71, 65-69. Lichtenstein, L. M., Schleimer, R. P., Peters, S. P., Kagey-Sobotka, A., Adkinson, N. F., Jr., Adams, G. K., 111, Schulman, E. S., and MacGlashan, D. W., Jr. (1983). Studies with purified human basophils and mast cells. Monogr. Allergy 18, 259-264. Liu, M. C . ,Proud, D., Lichtenstein, L. M., MacGlashan, D. W., Jr., Schleimer, R. P., Adkinson, N. F., Jr., Kagey-Sobotka, A., Schulman, E. S., and Plaut, M. (1986). Human lung macrophage-derived histamine-releasing activity is due to IgE-dependent factors. J . Immunol. 136,2588-2595. Login, G. R., Galli, S. J., Morgan, E., Arizono, N., Schwartz, L. B., and Dvorak, A. M. (1987). Rapid microwave fixation of rat mast cells. I. Localization of granule chymase with an ultrastructural postembedding immunogold technique. Lab. Invest. 57, 592-599.

CELL BIOLOGY OF THE BASOPHIL

229

Login, G . R., Quissell, D. O., Bryan, K. P., and Dvorak, A. M. (1994). A role for cytoplasmic vesicles in amylase release from secretory granules in isolated rat parotid gland acinar cells. Int. Congress Electron Microsc. 13, 83-84. Login, G. R., Yang, J., Bryan, K. P., Digenis, E. C., McBride, J., Elovic, A., Quissell, D. O., Dvorak, A. M., and Wong, D. T. W. (1997). Characterization of the synthesis and storage of TGF-a in rat parotid acinar and intercalated duct cells. Gmtrointest. Liver Physiol. 35, G553-GS62. Lopez, A. F., Eglinton, J. M., Lyons, A. B., Tapley, P. M., To, L. B., Park, L. S., Clark, S. C., and Vadas, M. A. (1990). Human interleukin-3 inhibits the binding of granulocyte-macrophage colony-stimulating factor and interleukin-5 to basophils and strongly enhances their functional activity. J. Cell. Physiol. 145, 69-77. MacDonald, S. M. (1993). Histamine releasing factors and IgE heterogeneity. In “AllergyPrinciples and Practice” (E. Middleton, C. E. Reed, E. F. Ellis, N. F. Adkinson, J. W. Yunginger, and W. W. Busse, eds.), 4th Ed., pp. 1-11. Mosby-Year Book, St. Louis. MacDonald, S. M., Lichtenstein, L. M., Proud, D., Plaut, M., Naclerio, R. M., MacGlashan, D. W., and Kagey-Sobotka, A. (1987). Studies of IgE-dependent histamine releasing factors: Heterogeneity of IgE. J. Immunol. 139,506-512. MacDonald, S. M., Schleimer, R. P., Kagey-Sobotka, A,, Gillis, S., and Lichtenstein, L. M. (1989). Recombinant IL-3 induces histamine release from human basophils. J. Irnmunol. 142, 3527-3532. MacDonald, S. M., Rafnar,T., Langdon, J., and Lichtenstein, L. M. (1995). Molecular identification of an IgE-dependent histamine-releasing factor. Science 269, 688-690. MacGlashan, D., Jr., and Guo, C. B. (1991). Oscillations in free cytosolic calcium during IgEmediated stimulation distinguish human basophils from human mast cells. J. Immunol. 147,2259-2269. MacGlashan, D., Jr., and Warner, J. (1991). Stimulus-dependent leukotriene release from human basophils: A comparative study of C5a and Fmet-leu-phe. J. Leukocyte Biol. 49, 29-40. MacGlashan, D., Jr., White, J. M., Huang, S. K., Ono, S. J., Schroeder, J. T., and Lichtenstein, L. M. (1994a). Secretion of IL-4 from human basophils. The relationship between IL-4 mRNA and protein in resting and stimulated basophils. J. Immunol. 152,3006-3016. MacGlashan, D. W., Jr. (1995). Graded changes in the response of individual human basophils to stimulation: Distributional behavior of events temporally coincident with degranulation. J. Leukocyte Biol. 58, 177-188. MacGlashan, D. W., Jr., and Hubbard, W. C. (1993). IL-3 alters free arachidonic acid generation in C5a-stimulated human basophils. J. Immunol. 151, 6358-6369. MacGlashan, D. W., Jr., and Lichtenstein, L. M. (1980). The purification of human basophils. J. Immunol. 124,2519-2521. MacGlashan, D. W., Jr., Lichtenstein, L. M., Galli, S. J., Dvorak, A. M., and Dvorak, H. F. (1982a). Purification of basophilic leukocytes from guinea pig and human blood and from guinea pig bone marrow. In “Cell Separation: Methods and Selected Applications” (T. G. Pretlow, 11, and T. P. Pretlow, eds.), Vol. 1, pp. 301-320. Academic Press, New York. MacGlashan, D. W., Jr., Schleimer, R. P., Peters, S. P., Schulman, E. S., Adams, G. K., 111, Newball, H. H., and Lichtenstein, L. M. (1982b). Generation of leukotrienes by purified human lung mast cells. J. Clin. Invest. 70, 747-751. MacGlashan, D. W., Jr., Schleimer, R. P., Peters, S. P., Schulman, E. S., Adams, G . K., Sobotka, A. K., Newball, H. H., and Lichtenstein, L. M. (1983). Comparative studies of human basophils and mast cells. Fed. Proc. 42,2504-2509. MacGlashan, D. W., Jr., Bochner, B. S., and Warner, J. A. (1994b). Graded changes in the response of individual human basophils to stimulation: Distributional behavior of early activation events. J . Leukocyte Biol. 55, 13-23.

230

ANN

M. DVORAK

Malhotra, V., Serafini, T., Orci, L., Shepherd, J. C., and Rothman, J. E. (1989). Purification of a novel class of coated vesicles mediating biosynthetic protein transport through the Golgi stack. Cell 58, 329-336. Maloney, W. C., and Lange, R. D. (19.54). Cytologic and biochemical studies on the granulocytes in early leukemia among atomic bomb survivors. Tex. Rep. Biol. Med. 12, 887-897. Marone, G., Poto, S., Petracca, R., Triggiani, M., de Lutio di Castelguidone, E., and Condorelli, M. (1982). Activation of human basophils by staphylococcal protein A. I. The role of cyclic AMP, arachidonic acid metabolites, microtubules and microfilaments. Clin. Exp. Immunol. 50,661-668.

Martin, R. R., and White, A. (1969). The in witro release of leukocyte histamine by staphylococcal antigens. J. Immunol. 102,437-441. Massey, W. A., Randall, T. C., Kagey-Sobotka, A,, Warner, J. A,, MacDonald, S. M., Gillis, S., Allison, A. C., and Lichtenstein, L. M. (1989). Recombinant human IL-la and -1p potentiate IgE-mediated histamine release from human basophils. J. Immunol. 143, 18751880.

Matsushima, K., Larsen, C. G., DuBois, G . C., and Oppenheim, J. J. (1989). Purification and characterization of a novel monocyte chemotactic and activating factor produced by a human myelomonocytic cell line. J. Exp. Med. 169, 1485-1490. Matsuuchi, L., and Kelly, R. B. (1991). Constitutive and basal secretion from the endocrine cell line. AtT-20. J. Cell Biol. 112, 843-852. Maximow, A. (1913). Untersuchungen iiber blut und bindegewebe. VI. Uber blutmastzellen. Arch. Mikrosk. Anat. Ver. Exp. Hisrol. Entwicklungsgesch. 83 (Abt. I, Heft 3), 247-289. Mayer, P., Valent, P., Schmidt, G., Liehl, E., and Bettelheim, P. (1989). The in wiwo effects of recombinant human interleukin-3: Demonstration of basophil differentiation factor, histamine-producing activity, and priming of GM-CSF-responsive progenitors in nonhuman primates. Blood 74, 613-621. McBride, P., Bradley, D., and Kaliner, M. (1988). Evaluation of a radioimrnunoassay for histamine measurement in biologic fluids. J. Allergy Clin. Immunol. 82, 638-646. Metcalfe, D. D., Lewis, R. A., Silbert, J. E., Rosenberg, R. D., Wasserman, S. I., and Austen, K. F. (1979). Isolation and characterization of heparin from human lung. J . Clin. Invesr. 64, 1537-1543.

Metcalfe, D. D., Wasserman, S. I., and Austen, K. F. (1980). Isolation and characterization of sulphated mucopolysaccharides from rat leukaemic (RBL-1) basophils. Biochem. J . 185,367-372.

Metcalfe, D. D., Bland, C. E., and Wasserman, S. I. (1984). Biochemical and functional characterization of proteoglycans isolated from basophils of patients with chronic myelogenous leukemia. J . Imrnunol. 132, 1943-1950. Michaelis, L. (1902). Uber mastzellen. Munch. Med. Wochensch. 49 (Pt. I, No. 6), 225-226. Mitchell, E. B., Crow, J., Chapman, M. D., Jouhal, S. S., Pope, F. M., and Platts-Mills, T. A. E. (1982). Basophils in allergen-induced patch test sites in atopic dermatitis. Lancer i (8264), 127-130. Mitsui, H., Furitsu, T., Dvorak, A. M., Irani, A.-M. A., Schwartz, L. B., Inagaki, N., Takei, M., Ishizaka, K., Zsebo, K. M., Gillis, S., and Ishizaka, T. (1993). Development of human mast cells from umbilical cord blood cells by recombinant human and murine c-kit ligand. Proc. Narl. Acad. Sci. USA 90, 735-739. Monahan, R. A., and Dvorak, A. M. (1985). One-micron plastic sections of human peripheral blood and bone marrow specimens and peroxidase cytochemistry: Use in the differential diagnosis of acute leukemias. In “Pathology Annual-Part 1 (S. C. Sornrners, P. P. Rosen, and R. E. Fechner, eds.), Vol. 20, pp. 383-401. Appleton-Century-Crofts, Norwalk, CT. Morita, Y., Goto, M., and Miyamoto, T. (1987). Effect of interleukin 2 on basophil histamine release. Allergy 42, 104-108.

CELL BIOLOGY OF THE BASOPHIL

231

Mueller, R., Heusser, C. H., Rihs, S., Brunner, T., Bullock, G. R., and Dahinden, C. A. (1994). Immunolocalization of intracellular interleukin-4 in normal human peripheral blood basophils. Eur. J. Zmmunol. 24, 2935-2940. Murata, F., and Spicer, S. S. (1974). Ultrastructural comparison of basophilic leukocytes and mast cells in the guinea pig. Am. J. Anaf. 139, 335-352. Naclerio, R. M., Proud, D., Togias, A. G., Adkinson, N. F., Jr., Meyers, D. A., Kagey-Sobotka, A., Plaut, M., Norman, P. S., and Lichtenstein, L. M. (1985). Inflammatory mediators in late antigen-induced rhinitis. N . Engl. J. Med. 313, 65-70. Nakagawa, T., and de Weck, A. L. (1983). Membrane receptors for the IgG4 subclass on human basophils and mast cells. Clin. Rev. Allergy 1, 197-206. Nanavati, C., and Fernandez, J. M. (1993). The secretory granule matrix: A fast-acting smart polymer. Science 259, 963-965. Nichols, B. A., and Bainton, D. F. (1973). Differentiation of human monocytes in bone marrow and blood. Sequential formation of two granule populations. Lab. Invest. 29, 27-40. Norn, S., Jensen, C., Dahl, B. T., Stahl Skov, P., Baek, L., Permin, H., Jarlov, J. O., and Sorensen, H. (1986). Endotoxins release histamine by complement activation and potentiate bacteria-induced histamine release. Agents Actions 18, 149-152. Ochensberger, B., Rihs, S., Brunner, T., and Dahinden, C. A. (1995). IgE-independent interleukin-4 expression and induction of a late phase of leukotriene C4 formation in human blood basophils. Blood 86, 4039-4049. O’Connor, V., Augustine, G . J., and Betz, H. (1994). Synaptic vesicle exocytosis: Molecules and models. Cell 76,785-787. Ogawa, M., Nakahata, T., Leary, A. G., Sterk, A. R., Ishizaka, K., and Ishizaka, T. (1983). Suspension culture of human mast cellsbasophils from umbilical cord blood mononuclear cells. Proc. Natl. Acad. Sci. USA 80, 4494-4498. Okayama, Y., and Church, M. K. (1992). IL-3 primes and evokes histamine release from human basophils but not mast cells. Int. Arch. Allergy Immunol. 99, 343-345. Olsen, U. B., Selmer, J., and Kahl, J. U. (1988). Complement C5a receptor antagonism by protamine and poly-L-Arg on human leukocytes. Complement 5, 153-162. Orchard, M. A., Kagey-Sobotka, A., Proud, D., and Lichtenstein, L. M. (1986). Basophil histamine release induced by a substance from stimulated human platelets. J. Zmmunol. 136,2240-2244. Orci, L., Malaisse-Lagae, F., Ravazzola, M., Amherdt, M., and Renold, A. E. (1973). Exocytosis-endocytosis coupling in the pancreatic beta cell. Science 181,561-562. Orenstein, N. S., Galli, S. J., Dvorak, A. M., Silbert, J. E., and Dvorak, H. F. (1978). Sulfated glycosaminoglycans of guinea pig basophilic leukocytes. J. Immunol. 121, 586-592. Orenstein, N. S., Galli, S. J., Dvorak, A. M., and Dvorak, H. F. (1981). Glycosaminoglycans and proteases of guinea pig basophilic leukocytes. In “Biochemistry of the Acute Allergic Reactions: Fourth International Symposium” (E. L. Becker, A. S. Simon, and K. F. Austen, eds.), pp. 123-143. A. R. Liss, New York. Orr, T. S. C. (1977). Fine structure of the mast cells with special reference to human cells. Scand. J. Respir. Dis.Suppl. 98, 1-7. Ostermann, J., Orci, L., Tani, K., Amherdt, M., Ravazzola, M., Elazar, Z., and Rothman, J. E. (1993). Stepwise assembly of functionally active transport vesicles. Cell 75, 1015-1025. Palade, G. (1975). Intracellular aspects of the process of protein synthesis. Science 189,347-358. Palade, G. E. (1955). Studies on the endoplasmic reticulum. 11. Simple dispositions in cells in situ. J. Biophys. Biochem. Cyrol. 1,567-582. Parmley, R. T., Ogawa, M., Spicer, S. S., and Wright, N. J. (1976). Ultrastructure and cytochemistry of bone marrow granulocytes in culture. Exp. Hematol. 4, 75-89. Patterson, R., Ts’ao, C.-h., and Suszko, I. M. (1980). Heterogeneity of bronchial lumen mast cells which are homogeneous by electron microscopy. J. Allergy Clin. Immunol. 65,278-284.

232

ANN M. DVORAK

Paul, W. E., Seder, R. A., and Plaut, M. (1993). Lymphokine and cytokine production by Fc&RI+cells. Adv. Immunol. 53, 1-29. Pearce, F. L., Behrendt, H., Blum, U., Poblete-Freundt, G., Pult, P., Stang-Voss, C. H., and Schmitzler, W. (1977). Isolation and study of functional mast cells from lung and mesentery of guinea pig. Agent Actions 7, 45-56. Pedersen, M., Permin, H., Jensen, C., Stahl Skov, P., Norn, S., and Faber, V. (1987). Histamine release from basophil leukocytes induced by microbial antigen preparations in patients with AIDS. Allergy 42, 291-297. Peters, S. P., MacGlashan, D. W., Jr., Schulman, E. S., Schleimer, R. P., Hayes, E. C . , Rokach, J., Adkinson, N. F., Jr., and Lichtenstein, L. M. (1984). Arachidonic acid metabolism in purified human lung mast cells. J . Immunol. 132, 1972-1979. Proost, P., Wuyts, A., and Van Damme, J. (1996). Human monocyte chemotactic proteins-2 and -3: Structural and functional comparison with MCP-1. J. Leukocyte Biol. 59, 67-74. Pruzansky, J. J., and Patterson, R. (1967). Subcellular distribution of histamine in human leukocytes. Proc. SOC.Exp. Biol. Med. l24, 56-59. Qu-Hong, Nagy, J. A., Senger, D. R., Dvorak, H. F., and Dvorak, A. M. (1995). Ultrastructural localization of vascular permeability factor/vascular endothelial growth factor (VPFNEGF) to the abluminal plasma membrane and vesiculovacuolar organelles of tumor microvascular endothelium. J. Histochem. Cytochem. 43,381-389. Reshef, A,, and MacGlashan, D. W. (1987). Immunogold probe for the light-microscopic phenotyping of human mast cells and basophils. J. Immunol. Methods 99,213-219. Riley, J. F., and West, G. B. (1953). The presence of histamine in tissue mast cells. J. Physiol. (London) 120,528-537. Rimmer, E. F., and Horton, M. A. (1986). In-vitro culture of basophils from human bone marrow. Leukocyte Res. 10,1241-1248. Rothman, J. E. (1994). Mechanisms of intracellular protein transport. Nature 372, 55-63. Rothman, J. E., and Wieland, F. T. (1996). Protein sorting by transport vesicles. Science 272,227-234. Saito, H., Hatake, K., Dvorak, A. M., Leiferman, K. M., Donnenberg, A. D., Arai, N., Ishizaka, K., and Ishizaka, T. (1988). Selective differentiation and proliferation of hematopoietic cells induced by recombinant human interleukins. Proc. Nail. Acad. Sci. USA 85,2288-2292. Sampson, H. A., Broadbent, K. R., and Bernhisel-Broadbent, J. (1989). Spontaneous release of histamine from basophils and histamine-releasing factor in patients with atopic dermatitis and food hypersensitivity. N. Engl. J. Med. 321,228-232. Sarmiento, E. U., Espiritu, B. R., Gleich, G. J., and Thomas, L. L. (1995). IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor potentiate basophil mediator release stimulated by eosinophil granule major basic protein. J. Immunol. 155, 221 1-2221. Schatz, G., and Dobberstein, B. (1996). Common principles of protein translocation across membranes. Science 271, 1519-1526. Schayer, R. W. (1959). Catabolism of physiological quantities of histamine in vivo. Physiol. Rev. 39, 116-126. Schekman, R., and Orci, L. (1996). Coat proteins and vesicle budding. Science 271,1526-1533. Schleimer, R. P., Gillespie, E., and Lichtenstein, L. M. (1981). Release of histamine from human leukocytes stimulated with the tumor-promoting phorbol diesters. I. Characterization of the response. J. Immunol. 126,570-574. Schleimer, R. P., Gillespie, E., Daiuta, R., and Lichtenstein, L. M. (1982). Release of histamine from human leukocytes stimulated with the tumor-promoting phorbol diesters. 11. Interaction with other stimuli. J. Immunol. 128, 136-140. Schleimer, R. P., Derse, C . P., Friedman, B., Gillis, S., Plaut, M., Lichtenstein, L. M., and MacGlashan, D. W., Jr. (1989). Regulation of human basophil mediator release by cytokines I. Interaction with antiinflammatory steroids. J. Immunol. 143, 1310-1317.

CELL BIOLOGY OF THE BASOPHIL

233

Schmid, S. L., and Damke, H. (1995). Coated vesicles: A diversity of form and function. FASEB J. 9, 1445-1453. Schnitzer, J. E., Oh, P., Jacobson, B. S . , and Dvorak, A. M. (1995a). Caveolae from luminal plasmalemma of rat lung endothelium: Microdomains enriched in caveolin, Ca"-ATPase, and inositol triphosphate receptor. Proc. Natl. Acad. Sci. USA 92, 1759-1763. Schnitzer, J. E., McIntosh, D. P., Dvorak, A. M., Liu, J., and Oh, P. (1995b). Separation of caveolae from associated microdomains of GP1-anchored proteins. Science 269,1435-1439. Schroeder, J. T., and Hanrahan, L. R., Jr. (1990). Purification of human basophils using mouse monoclonal IgE. J. Immunol. Methods 133,269-277. Schroeder, J. T., MacGlashan, D. W., Jr., Kagey-Sobotka, A,, White, J. M., and Lichtenstein, L. M. (1994). IgE-dependent IL-4 secretion by human basophils. The relationship between cytokine production and histamine release in mixed leukocyte cultures. J. Immunol. 153, 1808-1817. Schroeder, J. T., Lichtenstein, L. M., and MacDonald, S . M. (1996). An immunoglobulin Edependent recombinant histamine-releasing factor induces interleukin-4 secretion from human basophils. J. Exp. Med. 183, 1265-1270. Schulman, E. S., McGettigan, M. C., Post, T. J., Vigderman, R. J., and Shapiro, S. S. (1988). Human monocytes generate basophil histamine-releasing activities. J . Immunol. 140,23692375. Schwartz, L. B., Irani, A,-M. A,, Roller, K., Castells, M. C., and Schechter, N. M. (1987). Quantitation of histamine, tryptase, and chymase in dispersed human T and TC mast cells. J. Immunol. 138,2611-2615. Seder, R. A,, Paul, W. E., Dvorak, A. M., Sharkis, S. J., Kagey-Sobotka, A., Niv, Y., Finkelman, F. D., Barbieri, S. A,, Galli, S . J., and Plaut, M. (1991a). Mouse splenic and bone marrow cell populations that express high-affinity Fc, receptors and produce interleukin 4 are highly enriched in basophils. Proc. Natl. Acad. Sci. USA 88, 2835-2839. Seder, R. A,, Plaut, M., Barbieri, S., Urban, J., Jr., Finkelman, F. D., and Paul, W. E. (1991b). Purified FcsR' bone marrow and splenic non-B, non-T cells are highly enriched in the capacity to produce IL-4 in response to immobilized IgE, IgG2a, or ionomycin. J . Immunol. 147, 903-909. Seldin, D. C., Caulfield, J. P., Hein, A., Osathanondh, R., Nabel, G., Schlossman, S. F., Stevens, R. L., and Austen, K. F. (1986). Biochemical and phenotypic characterization of human basophilic cells derived from dispersed fetal liver with murine T cell factors. J. Immunol. 136,2222-2230. Siraganian, P. A., and Siraganian, R. P. (1974). Basophil activation by concanavalin A: Characteristics of the reaction. J. Immunol. 112, 2117-2125. Siraganian, R. P., and Hook, W. A. (1976). Complement-induced histamine release from human basophils. 11. Mechanism of the histamine release reaction. J. Immunol. 116,639-646. Siraganian, R. P., and Hook, W. A. (1977). Mechanism of histamine release by formyl methionine-containing peptides. J. Immunol. 119, 2078-2083. Siraganian, R. P., and Siraganian, P. A. (1975). Mechanism of action of concanavalin A on human basophils. J. Immunol. 114, 886-893. Sorensen, L. S., Mullarkey, M. F., Bean, M. A., Mochizuki, D. Y., Chi, E. Y., and Henderson, W. R. (1988). Propagation and characterization of human blood basophils. Int. Arch. Allergy Appl. Immunol. 86,267-280. Sperr, W. R., Agis, H., Czerwenka, K., Klepetko, W., Kubista, E., Boltz-Nitulescu, G., Lechner, K., and Valent, P. (1992). Differential expression of cell surface integrins on human mast cells and human basophils. Ann. Hematol. 65, 10-16. Stain, C., Stockinger, H., Scharf, M., Jager, U., Gossinger, H., Lechner, K., and Bettelheim, P. (1987). Human blood basophils display a unique phenotype including activation linked membrane structures. Blood 70, 1872-1879.

234

ANN M. DVORAK

Stock, E. L., Hill, R. A,, Boyle-Vavra, S . , and Roth, S . I. (1989). Eosinophils and mast cell homogeneity of the guinea pig eyelid skin, conjunctiva, and ileum. Am. J. Anal. 186,359-368. Stockinger, H., Valent, P., Majdic, O., Bettelheim, P., and Knapp, W. (1990). Human blood basophils synthesize interleukin-2 binding sites. Blood 75, 1820-1826. Subramanian, N., and Bray, M. A. (1987). Interleukin 1 release histamine from human basophils and mast cells in vitro. J . Immunol. 138, 271-275. Suda, T., Suda, J., Miura, Y., Hayashi, Y., Eguchi, M., Tadokoro, K., and Saito, M. (1985). Clonal analysis of basophil differentiation in bone marrow cultures from a Down's syndrome patient with megakaryoblastic leukemia. Blood 66, 1278-1283. Sudhof, T. C. (1995). The synaptic vesicle cycle: A cascade of protein-protein interactions. Nature 375, 645-653. Sudhof, T. C., De Camilli, P., Niemann, H., and Jahn, R. (1993). Membrane fusion machinery: Insights from synaptic proteins. Cell 75, 1-4. Sue, T. K., and Jaques, L. B. (1974). Sulfated mucopolysaccharides and basophilic leucocytes in rabbits on a high cholesterol/oil diet. Proc. Soc. Exp. Bid. Med. 146, 1006-1013. Sullivan, A. L., Grimley, P. M., and Metzger, H. (1971). Electron microscopic localization of immunoglobulin E on the surface membrane of human basophils. J. Exp. Med. 134, 14031416. Sztul, E., Kaplin, A., Saucan, L., and Palade, G. (1991). Protein traffic between distinct plasma membrane domains: Isolation and characterization of vesicular carriers involved in transcytosis. Cell 64,8149. Taichman, N. S . (1970). Ultrastructure of guinea pig mast cells. J. Ulrrastruct. Res. 32,284-292. Taichman, N. S. (1971). Ultrastructural alterations in guinea pig mast cells during anaphylaxis. Int. Arch. Allergy Appl. Immunol. 40, 934-942. Takizawa, P. A., and Malhotra, V. (1993). Coatomers and SNARES in promoting membrane traffic. Cell 75, 593-596. Tanno, Y., Bienenstock, J., Richardson, M., Lee, T. D. G., Befus, A. D., and Denburg, J. A. (1987). Reciprocal regulation of human basophil and eosinophil differentiation by separate T-cell-derived factors. Exp. Hemafol. 15, 24-33. Tedeschi, A,, Palella, M., Milazzo, N., Lorini, M., and Miadonna, A. (1995). IL-3-induced histamine release from human basophils. Dissociation from cationic dye binding and downregulation by Na' and K". J. Immunol. 155,2652-2660. Terry, R. W., Bainton, D. F., and Farquhar, M. G. (1996). Formation and structure of specific granules in basophilic leukocytes of the guinea pig. Lab. Invest. 21, 65-76. Terstappen, L. W. M. M., Hollander, Z., Meiners, H., and Loken, M. R. (1990). Quantitative comparison of myeloid antigens on five lineages of mature peripheral blood cells. J. Leukocyte B i d . 48, 138-148. Thueson, D. O., Speck, L. S., Lett-Brown, M. A,, and Grant, J. A. (1979a). Histamine-releasing activity (HRA). I. Production by mitogen- or antigen-stimulated human mononuclear cells. J. Immunol. 123,626-632. Thueson, D. O.,Speck, L. S . , Lett-Brown, M. A,, and Grant, J. A. (1979b). Histamine-releasing activity (HRA). 11. Interaction with basophils and physicochemical characterization. J . Immunol. 123,633-639. Truong, M.-J., Gruart, V., Liu, F. T., Prin, L., Capron, A,, and Capron, M. (1993). IgE-binding molecules (Mac-2/sBP) expressed by human eosinophils. Implication in IgE-dependent eosinophil cytotoxicity. Eur. J. Immunol. 23, 3230-3235. Ts'ao, C.-h., Metzger, W. J., Patterson, R., and Suszko, I. M. (1976). Histamine-containing cells in bronchial lavage fluid. I. Ultrastructural characterization and comparison with mast cells in three types of tissues of Rhesus monkeys. Inf. Arch. Allergy Appl. Zmmunol. 52, 315-324. Ts'ao, C.-h., Patterson, R., McKenna, J. M., and Suszko, I. M. (1977). Ultrastructural identification of mast cells obtained from human bronchial lumens. J. Allergy Clin. Immunol. 59, 320-326.

CELL BIOLOGY

OF THE BASOPHIL

235

Valent, P., and Bettelheim, P. (1992). Cell surface structures on human basophils and mast cells: Biochemical and functional characterization. Adv. Immunol. 52, 333-423. Valent, P., Besemer, J., Muhm, M., Majdic, O., Lechner, K., and Bettelheim, P. (1989). Interleukin 3 activates human blood basophils via high-affinity binding sites. Proc. Narl. Acad. Sci. USA 86,5542-5546. Valent, P., Majdic, O., Maurer, D., Bodger, M., Muhm, M., and Bettelheim, P. (1990a). Further characterization of surface membrane structures expressed on human basophils and mast cells. Int. Arch. Allergy Appl. Immunol. 91, 198-203. Valent, P., Besemer, J., Kishi, K., Di Padova, F., Geissler, K., Lechner, K., and Bettelheim, P. (1990b). Human basophils express interleukin-4 receptors. Blood 76, 1734-1738. van Gils, F. C. J. M., van Teeffelen, M. E. J. M., Neelis, K. J., Hendrikx, J., Burger, H., van Leen, R. W., Knol, E., Wagemaker, G., and Wognum, A. W. (1995). Interleukin-3 treatment of Rhesus monkeys leads to increased production of histamine-releasing cells that express interleukin-3 receptors at high levels. Blood 86, 592-597. Volc-Platzer, B., Valent, P., Radaszkiewicz, T., Mayer, P., Bettelheim, P., and Wolff, K. (1991). Recombinant human interleukin 3 induces proliferation of inflammatory cells and keratinocytes in vivo. Lab. Invest. 64, 557-566. Wagemaker, G . ,van Gils, F. C. J. M., Burger, H., Dorssers, L. C. J., van Leen, R. W., Persoon, N. L. M., Wielenga, J. J., Heeney, J. L., and Knol, E. (1990). Highly increased production of bone marrow-derived blood cells by administration of homologous interleukin-3 to Rhesus monkeys. Blood 76, 2235-2241. Wagner, L., Gessl, A., Baumgartner Parzer, S., Base, W., Waldhausl, W., and Pasternack, M. S. (1996). Haptoglobin phenotyping by newly developed monoclonal antibodies. Demonstration of haptoglobin uptake into peripheral blood neutrophils and rn0nocytes.J. Immunol. 156, 1989-1996. Warner, J. A,, Pienkowski, M. M., Plaut, M., Norman, P. S., and Lichtenstein, L. M. (1986). Identification of histamine releasing factor(s) in the late phase of cutaneous IgE-mediated reactions. J. Immunol. 136, 2583-2587. Warner, J. A., Freeland, H. S., MacGlashan, D. W., Jr., Lichtenstein, L. M., and Peters, S. P. (1987).Purified human basophils do not generate LTB4.Biochem. Pharmacol. 36,3195-3199. Warner, J. A,, Peters, S. P., Lichtenstein, L. M., Hubbard, W., Yancey, K. B., Stevenson, H. C., Miller, P. J., and MacGlashan, D. W., Jr. (1989). Differential release of mediators from human basophils: Differences in arachidonic acid metabolism following activation by unrelated stimuli. J. Leukocyte Biol. 45, 558-571. Watanabe, Y. (1954). An electron microscopic study of the leukocytes in bone marrow of guinea pig. J. Electron Microsc. (Tokyo) 2, 34-39. Weber, M., Uguccioni, M., Ochensberger, B., Baggiolini, M., Clark-Lewis, I., and Dahinden, C. A. (1995). Monocyte chemotactic protein MCP-2 activates human basophil and eosinophil leukocytes similar to MCP-3. J. Immunol. 154, 4166-4172. Weber, M., Uguccioni, M., Baggiolini, M., Clark-Lewis, I., and Dahinden, C. A. (1996). Deletion of the NHz-terminal residue converts monocyte chemotactic protein 1 from an activator of basophil mediator release to an eosinophil chemoattractant. J . Exp. Med. 183, 681-685. Weller, P. F., Bach, D. S., and Austen, K. F. (1984). Biochemical characterization of human eosinophil Charcot-Leyden crystal protein (lysophospholipase). J. Biol. Chem. 259,15100151005. Wetzel, B. K. (1970). The comparative fine structure of normal and diseased mammalian granulocytes. In “Regulation of Hematopoiesis,” Vol. 2, “White Cell and Platelet Production” (A. S. Gordon, ed.), pp. 819-872. Appleton-Century-Crofts, New York. Wetzel, B. K., Horn, R. G., and Spicer, S. S. (1967). Fine structural studies on the development of heterophil, eosinophil, and basophil granulocytes in rabbits. Lab. Invest. 16, 349-382.

236

ANN M. DVORAK

White, M. V., and Kaliner, M. A. (1987). Neutrophils and mast cells. I Human neutrophilderived histamine-releasing activity. J . Immunol. 139, 1624-1630. White, M. V., Kaplan, A. P., Haak-Frendscho, M., and Kaliner, M. (1988). Neutrophils and mast cells. Comparison of neutrophil-derived histamine-releasing activity with other histamine-releasing factors. J. Immunol. 141, 3575-3583. White, M. V., Igarashi, Y., Emery, B. E., Lotze, M. T., and Kaliner, M. A. (1992). Effects of in vivo administration of interleukin-2 (IL-2) and IL-4, alone and in combination, on ex vivo human basophil histamine release. Blood 79, 1491-1495. Winqvist, G. (1960). The ultrastructure of the granules of the basophil granulocyte. 2. Zellforsch. Mikrosk. Anat. 52, 475-481. Winqvist, G. (1963). Electron microscopy of the basophilic granulocyte. Ann. NY Acad. Sci. 103,352-375. Wognum, A. W., Visser, T. P., de Jong, M. O., Egeland, T., and Wagemaker, G. (1995). Differential expression of receptors for interleukin-3 on subsets of CD34-expressing hematopoietic cells of Rhesus monkeys. Blood 86,581-591. Yoshimura, T., Robinson, E. A,, Tanaka, S., Appella, E., and Leonard, E. J. (1989). Purification and amino acid analysis of two human monocyte chemoattractants produced by phytohemagglutinin-stimulated human blood mononuclear leukocytes. J . Immunol. 142, 1956-1962. Zachariae, C. O., Anderson, A. O., Thompson, H. L., Appella, E., Mantovani, A,, Oppenheim, J. J., and Matsushima, K. (1990). Properties of monocyte chemotactic and activating factor (MCAF) purified from a human fibrosarcoma cell line. J . Exp. Med. 171,2177-2182. Zeller, E. A. (1965). Identity of histaminase and diamine oxidase. Fed. Proc. 24, 766-768. Zhou, Z.-Q., Tenen, D. G., Dvorak, A. M., and Ackerman, S. J. (1992). The gene for human eosinophil Charcot-Leyden crystal protein directs expression of lysophospholipase activity and spontaneous crystallization in transiently transfected COS cells. J. Leukocyte Biol. 52,588-595. Zimmermann, A. (1908). Uber das vorkommen der mastzellen beim meerschweinchen. Arch. Mikrosk. Anat. Entwicklungsgesch. 72, 662-670. Zucker-Franklin, D. (1967). Electron microscopic study of human basophils. Blood 29, 878-890.

Membrane Receptors for Endocytosis in the Renal Proximal Tubule Erik llss Christensen,* Henrik Birn,* Pierre Verroust,t and W e n K. Moestrupt * Department of Cell Biology, Institute of Anatomy, and 4 Department of Medical Biochemistry, University of Aarhus, DK-8000 Aarhus C, Denmark; and INSERM U64, Hbpital Tenon, Paris, France

The renal proximal tubule exhibits a very extensive apical endocytic apparatus consisting of an elaborate network of coated pits and small coated and noncoated endosomes. In addition, the cells contain a large number of late endosomesl prelysosomes, lysosomes, and so-called dense apical tubules involved in receptor recycling from the endosomes to the apical plasma membrane. This endocytic apparatus is involved in the reabsorption of molecules filtered in the glomeruli. The process is very effective as demonstrated by the fact that although several grams of protein are filtered daily in the human glomeruli, human urine is virtually devoid of proteins under physiological conditions. Several key receptors appear to be involved in this function, which selves not only to conserve protein as such for the organism but also to reabsorb vital substances such as different vitamins in complex with their binding proteins. Recent research has established megalin, a 600-kDa protein belonging to the LDL receptor family, as probably the most important receptor in this process in the proximal tubule mediating endocytosis of a large variety of ligands and therefore classifying it as a scavenger receptor. More specific receptors like the folate receptor, IGF-IIIMan-6-P receptor, and gp280/1FR, identical to the intrinsic factor receptor, are also functioning in the apical endocytic pathway of renal proximal tubules. A better understandingof these receptors will give us new insight into these very important processes for the organism. KEY WORDS: Endocytosis receptors, Renal proximal tubule, gp330/megalin, gp280/ intrinsic factor receptor, IGF-II/Man-6-Preceptor, Folate receptor.

1. Introduction The renal proximal tubule cell is one of the most effective mammalian cells involved in endocytosis. This process serves several functions. It has been Inrrrnorional Review of Cyrology, Vol. 180

0074-7696/98 $ZS.OO

237

Copyright 0 1998 by Academic Press. All rights of reproduction in any form reserved.

238

ERIK lLS0 CHRISTENSEN E f AL.

known for decades that intensive reabsorption of protein filtered in the renal glomeruli takes place in the renal proximal tubule (Maack et al., 1985; Christensen and Nielsen, 1991; Maunsbach and Christensen, 1992). In the adult human kidney, several grams of protein are filtered daily, but under physiological conditions, the urine is almost devoid of protein, illustrating the importance of the reabsorption process by which protein is saved for the organism. Recent studies carried out in our and other laboratories demonstrate that endocytosis in the proximal tubule via well-defined receptors serves yet another key role by conserving vital substances such as vitamins. Receptor-mediated endocytosis is also involved in the uptake of different drugs and may thereby constitute the first step in the development of nephrotoxicity. In addition, the endocytic process has been implicated in other functions for which its role may not be clearly demonstrated. It has been suggested that another reason for this proximal reabsorption could be to avoid having active low-molecular-weight peptide hormones in more distal parts of the nephron. However, most known receptors for these hormones are localized at the basolateral membranes of the tubule cells and would therefore probably never meet the filtered hormones. A final role could be to keep the urine sterile. Indeed, along with glucose and amino acids, which are efficiently removed in the renal proximal tubule by apical plasma membrane transporters, proteins are also excellent bacterial substrates. Their removal may therefore be crucial in preventing infections in the human urinary tract. This suggestion is supported by the fact that patients with proteinuria of different origins have an increased rate of urinary infections. The molecular mechanisms behind proximal tubular reabsorption have been largely unknown although several theories have been put forward, including the electrical charge of proteins (Just and Habermann, 1977; Just et al., 1977; Christensen et al., 1981, 1983; Cojocel et al., 1981; Sumpio and Maack, 1982; Baumann et al., 1983; Park and Maack, 1984; Ottosen et al., 1985; Christensen and Bjerke, 1986). The intracellular destiny of proteins reabsorbed by endocytosis into proximal tubule cells has also been intensively discussed over the years. It was suggested that a large fraction of reabsorbed protein was transported intact through the proximal tubule cells by transcytosis (Maack and Kinter, 1969; Maack et al., 1971). However, several later studies (Ottosen and Maunsbach, 1973; Just et al., 1975; Ottosen, 1978; Ottosen et al., 1979; Nielsen et al., 1985, 1986, 1987; Cui et al., 1993b) demonstrated that transcytosis in these cells is quantitatively unimportant and that virtually all protein taken up by endocytosis is targeted to lysosomes for degradation, as shown first by Straus (1959, 1962, 1964) and Novikoff (1960, 1963). Luminal endocytosis in the proximal tubule cells takes place probably exclusively via clathrin-coated pits. Protein ligands dissociate from their

RENAL PROXIMAL TUBULE ENDOCYTOSIS MEMBRANE RECEPTORS

239

receptors in early and late endosomes and are targeted to lysosomes for degradation, whereas the receptors are sorted back to the apical plasma membrane via dense apical tubules. Basolateral endocytic uptake in the proximal tubule is insignificant for most proteins (Christensen and Nielsen, 1991; Maunsbach and Christensen, 1992). However, for certain hormones and growth factors for which receptors may be localized at basolateral membranes, it can be of quantitative importance. Thus, for example, the basolateral uptake of insulin constitutes about 15% of total tubular uptake (Nielsen et al., 1987). We recently reviewed proximal tubular handling of hormones and growth factors (Christensen and Birn, 1997). In this review we shall briefly describe the general ultrastructure of the apical endocytic apparatus of renal proximal tubule cells followed by a detailed description of important receptors involved in apical endocytosis.

II. Ultrastructure of the Endocytic Apparatus in the Proximal Tubule The mammalian proximal tubule (Fig. l ) , starting at the glomerulus and ending abruptly at the thin descending limb of Henle, can be subdivided into three segments as has been thoroughly done for the rat proximal tubule (Maunsbach, 1966b) of which the first two segments comprise most of the pars convoluta (Fig. 2) and the third segment the major part of the pars recta (for an extensive review of the ultrastructure of the mammalian proximal tubule see Maunsbach and Christensen, 1992). The endocytic apparatus in the proximal tubule consists of the following components: (1) apical clathrin-coated pits/endocytic invaginations (Fig. 3); (2) small, <0.5-pm, coated and noncoated early endosomes; (3) large, >0.5-pm, noncoated endosomes (Fig. 4) which can be subdivided into endosomes with (early) and without (late) an internal coat (Cui and Christensen, 1993); (4) dense apical tubules (DAT), 0.07-0.09 pm, free in the apical cytoplasm or connected to endosomes; ( 5 ) the lysosomes; and finally (6) small tubulovesicular structures (<0.05 p m in diameter) smaller and different from DAT, so far only described in rabbit proximal tubules (Cui et al., 1993b) and probably representing a vehicle for transcytosis. The clathrin-coated pits are located at the base of the very well developed brush border (Fig. 2) and are much more numerous in the first two segments than in segment 3. They are not simple pits but are often extensively branched (Fig. 3) (Cui and Christensen, 1993). In addition to the cytoplasmic clathrin coat they have a thick extracellular coat and several proteins have been localized to this coated plasma membrane as discussed later. Coated pits are only

240

ERIK lLS0 CHRlSTENSEN ET AL.

FIG.1 Electron micrograph of a cross-sectioned rat renal proximal tubule, late segment 1, fixed by microperfusion through the tubule lumen. The cells have a well-developed brush border (BB), microvilli being about 2 p m in height. The apical cytoplasm is rich in small and large endosomes (arrows), and numerous electron-lucent lysosomes (L) are seen deeper in the cytoplasm. Surrounding the tubules, capillaries (CAP) are seen filled with erythrocytes due to the fixation mode. Original magnification, X2200.

occasionally seen at the basolateral membrane. The small endosomes/endocytic vesicles are numerous in the apical cytoplasm, especially in segments 1 and 2 (Fig. 2). Several of the apparently early endosomes (Fig. 3) are in fact cross-sectioned coated pits (from 30 to 50%) as demonstrated by serial sectioning (Cui and Christensen, 1993) and by ruthenium red staining of the plasma membrane of fixed cells (Birn er al., 1993a). The large endosomes, often up to 1 p m or more in diameter, can be subdivided into at least two types (Cui and Christensen, 1993) based initially exclusively on

RENAL PROXIMAL TUBULE ENDOCYTOSIS MEMBRANE RECEPTORS

24 1

FIG. 2 Proximal tubule from late segment 1 as in Fig. 1. The micrograph illustrates the extensive apical endocytic apparatus, including coated pits (arrows) and small and large endosomes (E). Deeper in the cytoplasm, electron-lucent lysosomes (L) together with perpendicular-oriented mitochondria (M) are noted. BB, brush border, N, nucleus. Original magnification, X9000.

morphological characteristics: the early type has an internal coat similar to the coat seen in small endosomes and the extracellular coat seen in coated pits. This coat is not or only in part present in these later vacuoles, which also contain small amounts of electron-dense material (Fig. 4). These two types of large vacuoles probably represent different stages of late endosomes since megalin is only expressed in the membrane of the early type as described below and shown in Fig. 9. Very few large endosomes are present in segment 3 of the proximal tubule. Purified endosomal fractions have been obtained from rat renal cortex and fusion events studied in v i m (Hammond et al., 1994) as has also been done in other systems (Yamamoto et al., 1984; Gruenberg and Howell, 1986; Braell, 1987). The DAT, which represent the recycling vehicle for membrane proteins (Christensen, 1982), are often seen connected to both the small and the large endosomes (Fig. 5 ) and, in fact, based on serial sections we calculated, that 80% of the recycling DAT originates from the small endosomes (Cui and Christensen,

242

ERIK lLS0 CHRISTENSEN ET AL.

RENAL PROXIMAL TUBULE ENDOCYTOSIS MEMBRANE RECEPTORS

243

1993). We also estimated that an amount of membrane equal to the surface density of the dense apical tubules is being recycled within 92 s; in other words, this is an extremely rapid process (Birn et al., 1993a). At the ultrastructural level, the lysosomes in the proximal tubule have been identified by enzymatic cytochemical methods as well as by immunocytochemistry, locating cathepsins B, H, and L to the lysosomes (Yokota et al., 1986,1988; Yokota and Kato, 1988). The ultrastructure of the lysosomes varies significantly between different segments of the proximal tubule, probably reflecting the fact that the most intensive endocytic reabsorption takes place in the early parts of the proximal tubule in which the lysosomes are relatively electron lucent (Fig. 4) (Maunsbach, 1966b), whereas in later parts they become more electron dense. In segment 3 the lysosomes are generally much smaller. The ultrastructure of lysosomes also varies between species (Maunsbach and Christensen, 1992). The small tubulovesicular structures have only been described in rabbit proximal tubules. Using cationized ferritin as tracer (Cui et al., 1993b) they were initially located in the apical cytoplasm, either free in the cytoplasm or connected to endosomes like DAT. However, whereas D A T are confined to the apical cytoplasm, the small tubulovesicular structures containing ferritin at later stages could be found in the basal part of the proximal tubule cells simultaneous with the appearance of small amounts of ferritin in the intercellular spaces. As mentioned above, this transcytotic process in the proximal tubule is insignificant, probably amounting to less than 1% of the protein taken up (Cui et al., 1993b).

111. Megalin Megalin is a high-molecular-weight membrane receptor mediating endocytosis of proteins in the glomerular ultrafiltrate and first described in glomerular epithelial cells and proximal tubule cells by Kerjaschki and Farquhar (1982). The receptor constitutes a significant amount (probably several

FIG. 3 Apical cytoplasm of proximal cells from six consecutive ultrathin sections, a-f, illustrating the very complicated network of coated pits and small endosomes. (a) Three apparent small coated vesicles (1-3) and one invagination (4) are seen. (b) The small coated vesicle (1) is connected to the small coated vesicle (2). (c) Furthermore, 1 is seen connected to invagination (4) and another small coated vesicle (5) appears but can also be seen faintly in b. ( d ) The connection between 3 and 5 is shown. (e and f ) Connections between 5 and 3 with invagination (4) are shown. Original magnification, X48,OOO. (Reproduced from Cui and Christensen, 1993, by permission from Karger, Basel.)

244

ERIK ILS0 CHRISTENSEN ET AL.

FIG. 4 Electron micrographs from proximal tubule cells showing fusions between different types of vacuoles of the endocytic apparatus with and without inner coats (arrows). (a) Fusion between a small endosome <0.5 p m in diameter (SEV) and a prelysosome without an inner coat (PL). Original magnification, X54,OOO. (b) Fusion between two large endosomes >O.5 p n in diameter (LEV), both with an inner coat. Original magnification, X30,OOO. (c) Fusion between a PL and an LEV. Original magnification X23,OOO. ( d ) Fusion between an LEV and a lysosome (LY). Original magnification, X18,OOO. (e) Fusion between a lysosome and a prelysosome. Original magnification, X27.000. (f ) Fusion between two lysosomes. Original magnification, X 13,500. (Reproduced from Cui and Christensen, 1993, by permission from Karger, Basel.)

percent) of the total protein in the proximal tubule membranes. The kidney has the highest concentration of the receptor of all tissues but expression of megalin has also been reported in the parathyroids (Lundgren er al., 1994) and several specialized epithelia including the epididymis epithelium, type I1 pneumocytes, visceral yolk sac (Doxsey et al., 1984; Chatelet et al.,

RENAL PROXIMAL TUBULE ENDOCYTOSIS MEMBRANE RECEPTORS

245

FIG. 5 Electron micrographs of large endosomes with inner coats from proximal tubule cells demonstrating connections to dense apical tubule (DAT; arrows). a and b show one DAT connected to an LEV. Original magnifications, X58,OoO and X48,OOO. c shows two DAT connected. Original magnification, X36,OOO. d shows one DAT connected and a DAT apparently connecting a small vesicle and the large vacuole. Original magnification, X62,OOO.

1986b; Leung et al., 1987; Sahali et al., 1988; Le Panse et al., 1997a), ependyma and ciliary epithelium of eye (Zheng et al., 1994), inner ear epithelium (Kounnas et al., 1994), and trophectodermic cells (Gueth-Hallonet et al.,

246

ERIK lLS0 CHRISTENSEN ET AL.

1994) (Table I). Furthermore, the protein has also been found in rat yolk sac carcinoma cell lines (Lundstrom et al., 1993; Orlando and Farquhar, 1993; Moestrup et al., 1994; Le Panse et al., 1995, 1997b), which have been the basis for several functional analyses difficult to perform in vivo.

A. Structure The 600-kDa megalin protein is, as the name indicates, the largest known membrane receptor. Previously, the protein was designated “gp330” or Heymann nephritis antigen (Kerjaschki and Farquhar, 1982). The primary sequence of the rat (Saito et al., 1994) and human receptor (Hjalm et al., 1996) disclosed that the receptor belongs to the low-density lipoprotein (LDL) receptor family. Figure 6 displays the close structural relationship between megalin, the LDL receptor (Yamamoto et al., 1984), the VLDL receptor (Takahashi et al., 1992), the a2-macroglobulin receptor called LDL receptor-related protein (LRP) (Herz et al., 1988), and a megalin homologue cloned in the nematode Cuenorhabditis elegans (Yochem and Greenwald, 1993). The high structural homology between the C. elegans receptor and

TABLE I Expression of Megalin and gp28011FR

Kidney Proximal tubule Glomerulus-Podocytes Lung-Pneumocytes

type I1

Epididymis Endometrium Oviduct Yolk sac Parathyroid Ependyma Ileal enterocytes Placenta-Syncytiotrophoblast Eye-Ciliary epithelium Trophectoderm

gp280/IFR

++++ + ++ +++

++++ -

+++

Thyroid

Inner ear-Epithelial

Megalin

cells

++ +++ ++ +++ ++ + +

-

++++ ? ? -

++ ?

++

?

++

?

++

Note. Data for megalin were modified from Zheng et al. (1994).

247

RENAL PROXIMAL TUBULE ENDOCYTOSIS MEMBRANE RECEPTORS I11

N’ ,

IV

c

Megalin

>

C Elegans protein LRP VLDL

I]

lhgand binding repeat (class A motif) EGF repeat (class 6.1 motif) growth factor repeat (class 6.2 motif) YWTD spacer region (xcysteine)

I 1

a f n k e d sugar domain

,

main

:

i

’

’

j

LDLR

(ONPXI..NPXY-like) R-X-WR-R sequence

FIG. 6 The structure of five closely related receptors.

the mammalian homologues suggests a fundamental function of the receptor in a substantial time span of its evolution. The extracellular domain is characterized by four main types of extracellular modules, the LDL receptor class A repeats, the E G F repeats, the growth factor (EGF-like) repeats, and the YWTD repeats. Both the class A repeats and the EGF-like repeats contain six cysteines in a 40-aminoacid stretch. The 36 class A repeats are rich in acidic residues and arranged in four different clusters with 7-11 repeats. The repeats have also been designated “ligand-binding repeats” based on studies on the LDL receptor showing the class A repeat cluster as the ligand-binding region (Russell et al., 1989). The clusters are flanked by the less acidic EGF-like growth factor repeats. The E G F repeats are located close to the transmembrane domain. The spaces between the clusters of cysteine-rich repeats are encoded by the YWTD repeats (also called “spacer repeats”). The 209-amino-acid cytoplasmic domain of the human receptor (Hjalm et al., 1996) which is very similar to the corresponding rat sequence shows little overall homology with the other members of the LDL receptor family except for the two copies of the consensus sequence FXNPXY important for coated pit internalization (Chen et al., 1990). The intracellular domain also contains regions that might be involved in signal transduction, e.g., three conserved Scr-homology binding regions, three protein kinase C phosphorylation sites, and seven casein kinase I1 sites. Rat megalin, but not human megalin, contains as LRP a furin cleavage site. This may explain the findings of soluble forms of megalin (Bachinsky et al., 1993).

6 . Expression

The expression of megalin in the renal proximal tubule cells was initially found to be restricted to the clathrin-coated pits at the base of the microvilli

248

ERIK lLS0 CHRISTENSEN ET AL.

and in apical endocytic vacuoles (Fig. 7), at distinct variance with brush border proteins such as maltase (Kerjaschki et al., 1984). However, megalin was later also found in DAT (Fig. 8) (Biemesderfer et al., 1992; Christensen et al., 1992, 1995), strongly suggesting recycling of megalin via DAT. As shown in Fig. 9, it is not all apparent endosomes which express megalin in their limiting membrane. Probably, as also discussed above, the large vacuoles which do not have a continuous inner coat and do not express megalin (Fig. 9) correspond to late endosomes/prelysosomes. Immunocytochemistry

FIG. 7 Immunocytochemical localization of megalin on an ultrathin cryosection from the apical part of a rat proximal tubule cell, late segment 1, as visualized by incubation with a primary sheep anti-megalin antibody and a secondary antibody coupled to 10 nm colloidal gold. Megalin is seen on the microvillar (MV) and coated pit (CP) membrane. Original magnification, X56,OOO.

RENAL PROXIMAL TUBULE ENDOCYTOSIS MEMBRANE RECEPTORS

249

FIG.8 Immunocytochemical localization for megalin as in Fig. 7, illustrating localization in coated endosomes (arrow), dense apical tubules (arrowheads), and on the membrane of a large endosome (LEV). Original magnification, X46,OOO.

has also localized megalin to the lysosomes of especially segments 1 and 2 (Fig. 9) (Christensen et al., 1995). The immunocytochemical reaction was mainly due to low-molecular-weight peptides, probably lysosomal degradation products of megalin, and most likely representing a certain “spill-over” of the receptor during the endocytic process. Also, megalin is not restricted to the apical endocytic apparatus of the proximal tubule cells. Several immunocytochemical studies using monoclonal and polyclonal antibodies have shown a variable degree of brush border labeling (Fig. 7) (Chatelet et al., 1986a; Abbate et al., 1993; Bachinsky et al., 1993), and we recently demonstrated a distinct segmental variation (Christensen et al., 1995) between the three different segments of the proximal tubule in three different species, rat, rabbit, and man, using several monoclonal and polyclonal antibodies. No labeling of the brush border is observed in the initial part of segment 1; however, in the remaining part of segment 1 and in segment 2 there is a distinct labeling of the microvilli (Fig. 7) and, in segment 3, groups of microvilli are distinctly labeled from bottom to tip whereas

250

ERIK lLS0 CHRISTENSEN ET AL.

Immunocytochemical localization for megalin as in Fig. 7, demonstrating membrane labeling of coated pits (CP) and small and large endosomes (LEV). A prelysosome (PL) without an inner coat is virtually not labeled. In the bottom right corner a lysosome (L) with labeling of the matrix is seen. Original magnification, X25,OOO. FIG. 9

neighboring regions are unlabeled (Fig. 10). The expression of megalin in the endocytic apparatus is much smaller in segment 3 compared to that in segments 1 and 2. In contrast to other members of the LDL receptor family, the expression of megalin is always apical in polarized epithelial cells. During development, megalin is intracellularly detectable in the trophectodermic cells as early as in the Day 4 preimplantation embryo and on their plasma membrane immediately after compaction (Sahali et al., 1993; Gueth-Hallonet et al., 1994). Subsequently, megalin is detectable in the first endodermal cells that arise from the inner cell mass. In the kidney, megalin is first detectable in the mesonephros and in the nephronic vesicle as well as in the ureteric bud (Sahali et al., 1993). In the S-shaped body, megalin is localized in areas that will give rise to the glomerulus, the proximal tubule, and the distal tubule, and only later during development do the areas of expression narrow down to the glomerulus and the proximal tubule, being progressively concentrated in clathrin-coated pits simultane-

RENAL PROXIMAL TUBULE ENDOCYTOSIS MEMBRANE RECEPTORS

251

FIG. 10 Irnrnunocytochemical labeling for megalin in segment 3, pars recta of rat. The labeling is confined to a cluster of microvilli; neighboring parts of the brush border are negative. Arrow points to a tight junction between two cells. Dense apical tubules demonstrate strong labeling (arrowheads). Original magnification, X27,OOO. (Reproduced from Christensen et al., 1995, by permission from Wissenschaftliche Verlagsgesellschaft, Stuttgart.)

ously with the initiation of glomerular filtration (Sahali et al., 1993). These results agree well with the observation of a close relationship between the ability of the proximal tubule to reabsorb protein and the expression of megalin in coated pits (Biemesderfer et al., 1992). Under different experimental conditions, megalin is redistributed in the proximal tubules. After treatment of rats with sodium maleate, which significantly reduces protein reabsorption and induces an extensive apical vacuolization in proximal tubule cells (Christensen and Maunsbach, 1980), megalin was concentrated in newly formed apical vacuoles (Rodman et al.,

252

ERIK lLS0 CHRISTENSEN ET AL.

1986;Bergeron et af., 1996). In rats treated with colchicine, vesicles containing megalin were found dispersed throughout the entire cell cytoplasm, indicating that microtubules are involved in the apical accumulation of megalin containing vacuoles (Gutmann et al., 1989; Elkjar et al., 1995). Besides the expression in the proximal tubule, megalin is also expressed in the glomerular podocytes (Kerjaschki and Farquhar, 1983). Interestingly, the glomerular expression which has been confirmed by several laboratories has only been observed in rats and only in some strains. In humans, for example, it has not been possible to detect glomerular staining (Kerjaschki et aL, 1987). C. Function

A function of megalin as an endocytic receptor was early suggested from studies showing its localization in coated pits (Kerjaschki et al., 1984) and partial sequencing (Raychowdhury et al., 1989) showing its relationship to the LDL receptor. Later, several functional studies established its role as an endocytic receptor for a wide spectrum of ligands (for references see Table 11). The ligands reported to bind to megalin (Table 11) represent substances of different classes including calcium, lipoprotein particles, proteinases and proteinase-inhibitor complexes, drugs, albumin, vitamincarrier complexes, and receptor-associated protein (RAP). Binding of all the ligands is dependent on calcium. The megalin ligands have with few exceptions (e.g., clusterin/apolipoprotein J and transcobalamin-vitamin B12 (TC-B,,)) also been reported as ligands for the a2-macroglobulin receptor. Most relevant for the function of megalin as an endocytic receptor in the proximal tubule epithelium seems to be the small ligands which are filtered in the glomeruli. Several functional studies on megalin in the proximal tubule have shown that the megalin-mediated uptake of ligands follows the classic description of receptor-mediated endocytosis (Brown and Goldstein, 1979, 1986): the ligand-receptor complex formed in the coated pits is transported to endosomes followed by segregation of the receptor-ligand complex and recycling of the receptors back to the plasma membrane via DAT while the ligandcontaining endosomes fuse with lysosomes (Fig. 11). It is not entirely clear how so many ligands of completely different natures can bind to megalin, but one common feature among many of the ligands is the presence of basic patches on the surface of molecules. This is evident from studies (Christensen et a[., 1992; Moestrup et a/., 1993b, 1996) with heparin, which binds to TC-BIZ, plasminogen activator-inhibitor complexes, apolipoproteins, lipoprotein lipase, and RAP. Heparin strongly interferes with the binding of several of the ligands to megalin (Christensen

RENAL PROXIMAL TUBULE ENDOCYTOSIS MEMBRANE RECEPTORS

253

TABLE I1 Substances Reported to Bind to Megalin

Enzymes and enzyme inhibitors PAI-1 PAI-1-urokinase PAI-1-tPA Prourokinase Lipoprotein lipase Aprotinin

(Stefansson ef al., 1995b) (Moestrup et al., 1993b) (Willnow et al., 1992; Moestrup et al., 1993b) (Stefansson et al., 1995) (Kounnas et al., 1993) (Moestrup et al., 1995)

Vitamin-binding protein Transcobalamin-vitamin B

(Moestrup et al., 1996)

Apolipoproteins Apolipoprotein B Apolipoprotein E Apolipoprotein Jlclusterin

(Stefansson et al., 1995a) (Willnow et al., 1992) (Kounnas et al., 1995)

Polybasic drugs Aminoglycosides Polymyxin B Aprotinin

(Moestrup et al., 1995) (Moestrup et al., 1995) (Moestrup et aL, 1995)

Other Plasminogen Albumin Lactoferrin RAP Ca2’

(Kanalas and Makker, 1991) (Cui et al., 1996) (Willnow et al., 1992) (Christensen et al., 1992; Orlando et al., 1992; Kounnas et al., 1992; Willnow et al., 1992) (Christensen et al., 1992)

et al., 1992; Moestrup et al., 1993b, 1996). Furthermore, a recent study (Moestrup et al., 1995) of the role of megalin for the renal clearance and uptake of polybasic drugs, including the proteinase inhibitor aprotinin and aminoglycoside antibiotics, has demonstrated the capability of the receptor to mediate uptake of filtered basic molecules. Aprotinin is a 6-kDa protein readily filtered in the glomeruli. Site-directed mutagenesis analysis (Moestrup et al., 1995) showed that the mutation of certain basic residues causes decreased affinity for megalin and increased excretion of aprotinin after intravenous injection in rats. It is, however, interesting that megalin also mediates endocytosis of anionic proteins. We have previously suggested that it may not be the isoelectric point of a protein but rather the distribution of surface charges of the molecule which determines the efficiency of reabsorption in the proximal tubule (Christenen et al., 1983). Serum albumin has attracted much attention with respect to renal handling. Proximal tubular

254

ERIK lLS0 CHRISTENSEN ET AL.

Ligand

y Megalin

nJ FIG. 11 Diagram illustrating the function of megalin in the endocytic process in renal proximal tubule cells. Ligands bind to megalin in apical clathrin-coated pits (CP) which pinch off to form clathrin-coated vesicles. After loosing the clathrin coat, ligands start to dissociate in small uncoated early acidic endosomes (SEV), a process which continues in the large endosomes (LEV). From both types of endosomes, but mainly from the small endosomes, megalin is returned to the apical plasma membrane via dense apical tubules (DAT). The ligands arc transferred to late endosomes/prelysosomes (PL) together with small amounts of megalin by fusion processes and finally accumulated in lysosomes (LYS) for degradation and further processing.

uptake of homologous albumin was demonstrated at the ultrastructural level by micropuncture experiments in rat (Maunsbach, 1966a) and we recently demonstrated that although albumin is an anionic protein (PI 4.6), its tubular uptake is at least in part mediated by megalin (Figs. 12 and 13) (Cui et aL, 1996).

RENAL PROXIMAL TUBULE ENDOCYTOSIS MEMBRANE RECEPTORS

255

FIG. 12 Proximal tubule microinfused with colloidal gold particles coated with bovine serum albumin and fixed 7 min later. A few gold particles are seen in LEV; however, the prelysosomes demonstrate heavy accumulation. Original magnification, X30,OOO.

In rats with Heymann nephritis, there is a loss of megalin in the apical endocytic pathway simultaneous with a reduced uptake of dextran (Gutmann et al., 1991), which indicates a general perturbation in the endocytic process since dextran is known to be taken up by fluid-phase endocytosis in the proximal tubule (Christensen and Maunsbach, 1979). Similarly, the endocytic uptake of horseradish peroxidase in proximal tubules is significantly reduced in rats with Heymann nephritis, not only in the early stages but also in later stages when the brush border is in part regenerated (Kitazawa et al., 1986). The physiological importance of megalin-mediated uptake of ligands in the proximal tubule is only partially clarified and awaits further investigations such as specific knockout of the megalin gene in the kidney. A recent report on knockout of megalin in mice (Willnow et al., 1996a) has shown

256

ERIK lLS0 CHRISTENSEN ET AL.

FIG. 13 Proximal tubule prepared as in Fig. 12 except that RAP, 2.5 X 10-6M, was added to the perfusate. RAP significantly reduced uptake and in addition the albumin-gold particles were found nearly exclusively in LEV. A quantitative analysis (Cui et al., 1996) revealed a reduction in uptake of about 83%. Original magnification, X30,OOO.

that complete knockout of megalin in all tissues is incompatible with normal life. The newborn megalin knockout mice have serious malformations in several tissues including brain and lung and most die within few hours, apparently from respiratory failure. Microscopic examination of the kidneys of the knockout mice showed almost normal morphology of the proximal tubules except for a decreased number of endocytic vesicles in the cells. One tempting hypothesis for the main physiological role of megalin is that it preserves nutrients, which would be lost in the urine in the absence

RENAL PROXIMAL TUBULE ENDOCYTOSIS MEMBRANE RECEPTORS

257

of a tubular uptake mechanism. A recent study provides evidence for a role of megalin in the preservation of the pool of vitamin BIZin the organism by an efficient megalin-mediated uptake of filtered TC-BIZ(Fig. 14) (Moestrup et al., 1996). The amount of the TC-facilitated BIZuptake has been estimated to approximately 1.5 p g per day, which equals the daily intestinal uptake. Future studies will show if other vitamins are taken up by means of megalin. Another interesting nutrient is Ca2+.Analysis of rabbit and rat kidney disclosed megalin as the major calcium-binding protein of the renal cortex (Christensen etal., 1992). Studies of the similar LRP molecule (Moestrup et al., 1990, 1993a) suggest multiple Ca2+-bindingsites in negatively charged class A repeats. In view of the high expression of megalin in tubules, megalin might account for a large part of the nonregulated calcium uptake in the proximal tubules. Ca2+ uptake via megalin may also be a part of a calcium-sensing mechanism, as indicated by studies on placenta and the parathyroid gland (Lundgren et al., 1994).

FIG. 14 Semithin, 0.8-pm cryosection, incubated with 1251-TC-B12 and prepared for light microscope autoradiography. Labeling is observed in the apical part of the proximal tubules similar to the immunohistochemical localization of megalin. No other nephron segments show any significant labeling. Original magnification, X 1000.

258

ERIK ILS0 CHRISTENSEN ET AL.

0 . Megalin and RAP

One of the most interesting megalin ligands is RAP, which has been the subject of intensive research in recent years, but this molecule still has a glory of mysticism. RAP is a 40-kDa protein present in the endoplasmic reticulum. It has been cloned as a heparin-binding protein in mouse (Furukawa et af., 1990) and later from rat (Pietromonaco et af., 1990) and man (Strickland et af., 1991). Rat RAP was originally designated “Heymann nephritis target antigen” because it was thought to be part of megalin (Pietromonaco et aL, 1990). The human version was originally identified as a protein binding to the ectodomain of LRP (Jensen et af., 1989; Ashcom et af., 1990; Moestrup and Gliemann, 1991). The sequence of RAP shows an extraordinary high percentage of charged residues and alignment studies indicate that the molecule has a three-domain structure with internal homology (Ellgaard et af., 1997). Apparently, all the domains have binding sites for the LDLreceptor-related protein. The strongest affinity is seen in the N- and Cterminal domains (Warshawsky et af., 1993).The C-terminal sequence has a signal sequence for retention in the endoplasmic reticulum (Bu and Rennke, 1996; Strickland et af., 1991). RA P has a very high affinity for megalin (Christensen et al., 1992; Orlando et af., 1992) and RAP-affinity chromatography of renal cortex is an efficient method for purification of megalin (Kounnas et af., 1993; Moestrup et al., 1993b). Furthermore, RAP has the unique quality of inhibiting the binding of all known megalin ligands (Moestrup, 1994). It is not known whether this is an allosteric effect or a direct competition on the binding sites. Surface plasmon resonance analysis has demonstrated at least two binding sites in megalin (Moestrup et af., 1996). However, this estimate is a minimum value and RA P may bind to all the class A repeat clusters. Although experiments have shown that megalin mediates internalization of RAP, leading to its lysosomal degradation (Christensen et al., 1992), the intracellular localization of RAP in the endoplasmic reticulum contradicts a role for RAP as an ordinary ligand. This localization seems predominant, if not exclusive, as shown by Abbate et af. (1993), demonstrating that the previously reported brush border localization of R A P was artifactually induced during tissue processing. A clue to its function has come from recent analyses including analysis of RAP knockout mice (Willnow et af., 1995, 1996b; Bu and Rennke, 1996) indicating that RAP is important for the early processing of LDL receptor family proteins. Willnow et af. (1995, 1996b) showed that much less LRP is expressed in the absence of RAP. Apparently, RAP functions as an escort protein binding to the receptors, thereby preventing aggregation and inactivation of the receptors. Bu and Rennke (1996) suggested that RAP segregates from the receptor in acidic

RENAL PROXIMAL TUBULE ENDOCYlOSlS MEMBRANE RECEPTORS

259

compartments before release to the cell surface. Alternatively, RAP might be transported to the surface in complex with a receptor as a “quality control protein” which would be degraded after the first round of internalization, while the receptor recycles in ligand-binding active form to the surface (Moestrup et al., 1994). In these studies, the relation between RAP and LRP has been used as a model for RAP-receptor interaction, but in view of the similarity between megalin and LRP and the fact that RAP has a high expression in the proximal tubules, it would seem reasonable to suggest similar results if the RAP-megalin interaction had been analyzed.

E. Role of Megalin in Renal Proximal Tubule Disease Megalin seems to be involved in pathogenic mechanisms of both glomerular and proximal tubule disease. Heymann nephritis, a rat model of human membranous glomerulonephritis is caused by autoantibodies against antigenic epitopes in rat megalin leading to antibody recognition of megalin expressed in the glomerular podocytes of rats. The binding causes shedding of antibodies and destruction of the basement membrane leading to the syndrome of membranous glomerulonephritis. The role of megalin in this disease has recently been reviewed by Farquhar et al. (1995). Study on the binding and megalin-mediated uptake of polybasic drugs (Moestrup et al., 1995) indicates a central role of megalin in some renal proximal tubule diseases caused by receptor-mediated uptake of toxic substances. The study indicates that megalin is important for aminoglycoside-induced destruction of the proximal tubule leading to a Fanconi-like syndrome characterized by increased urinary excretion of glucose, amino acids, phosphate, bicarbonate, and low-molecular-weight proteins. Aminoglycosides, which bind to megalin, constitute a serious problem in antibiotic therapy because of their accumulation in lysosomes and subsequent damaging effect on the proximal tubule epithelium. Other nephrotoxic drugs or toxic constituents might also be taken up in the proximal tubules via megalin.

IV. gp280/lntrinsic Factor Receptor (IFR] gp280/IFR constitutes another, large endocytic membrane receptor localized to the kidney proximal tubule. This protein was initially identified during studies on megalin through the use of monoclonal antibodies raised against rat renal brush border (Sahali et aL, 1988). Table I compares the organ expression of megalin and gp2801IFR. An initial survey of various organs indicated that expression was limited to the kidney and yolk sac,

260

ERIK lLS0 CHRISTENSEN ET AL.

predominantly on the visceral but also on the parietal layer (Sahali et al., 1988). Recent studies have established that gp280/IFR is identical to the intrinsic factor cobalamin receptor (Birn et al., 1997b; Seetharam et al., 1997) mediating endocytosis of intrinsic factor-vitamin B12 in the terminal ileum. The highest concentrations of gp280/IFR are found in organs which are characterized by a high level of endocytosis of proteins facing their apical pole followed by degradation of the internalized proteins and recovery of the degraded amino acids. The ability to internalize and degrade large amounts of protein is a, if not the, key property of yolk sac visceral epithelial cells in rodents (Jollie, 1990; Lloyd, 1990). It provides the only source of amino acids used for embryonic development between Day 7 and Day 11 of gestation, i.e., at a time when the yolk sac is the only foeto maternal interface. Embryo cultures, surrounded by the visceral layer of the yolk sac, can be maintained ex vivo under conditions which maintain the full degree of epithelial differentiation. This model as well as that of yolk sac epithelial cell lines has been used for several of the functional studies of gp280IIFR presented in this review.

A. Structure The molecular mass of gp280/IFR was first estimated at 280 kDa (Sahali et al., 1988) by reference to megalin which was initially ascribed a mass of 330 kDa. Cloning of the latter protein (Saito et al., 1994; Hjalm et al., 1996) revealed its molecular weight to be close to 600 kDa. This observation and the use of other reference proteins including LRP indicate that the molecular weight of gp280/IFR is approximately 460 kDa (Birn eta!., 1997b). The slower migration on SDS-PAGE of gp280/IFR under reducing conditions indicates the likely presence of intrachain disulfide bridges. There is at present little information on the structure of gp280/IFR. Proteins similar to gp280/IFR in terms of mass and tissue distribution have been detected in several species including rabbit (Birn et al., 1997b), mouse (Verroust, unpublished observations), and man (Sahali et al., 1992). Comparative twodimensional maps of CNBr peptides derived from rat, human, and rabbit proteins (Sahali ef at., 1992; Moestrup, unpublished observations) suggest that its structure, and in particular a 100-kDa peptide which appears to be specific for gp280/IFR (Sahali et aL., 1993), is conserved across species (Sahali et al., 1992). The amino-terminal and CNBr peptide sequences which have been obtained indicate that gp280/IFR is a yet undescribed protein (Birn et al., 1997b). Active cloning studies using cDNAs derived from both yolk sac epithelial cells and renal cortex are being undertaken.

RENAL PROXIMAL TUBULE ENDOCYTOSIS MEMBRANE RECEPTORS

261

B. Expression Within the kidney, gp2801IFR is only detectable in proximal tubule cells, in contrast to megalin, which is also expressed by rat glomerular epithelial cells. At the ultrastructural level the localization of gp280/IFR in proximal tubule resembles that of megalin (Sahali et al., 1988). On the apical membrane, it is concentrated in the intermicrovillar areas but also extends to the microvillar domain. gp280/IFR is detectable along the intracellular compartments of the endocytic pathway (Fig. 15) including coated and noncoated endosomes, large endocytic vacuoles, DAT, and some lysosomes, mainly in segment S2 (Seetharam etal., 1997). Compared to megalin, expression seems relatively predominant on the lysosomal membrane (Fig. 16) (Seetharam et al., 1997). Finally, synthesis of gp2801IFR by proximal tubule cells may be evidenced by the presence of immunoreactive protein in the biosynthetic compartments (Sahali et al., 1993). During embryonic development, gp280/IFR along with megalin is detectable in the first endodermal cells that arise from the inner cell mass. Renal expression of gp2801IFR resembles that of megalin. gp2801IFR is detectable on S-shaped bodies in the presumptive areas of the proximal tubule and glomerulus and initially assumes a diffuse pattern of membrane expression which becomes restricted to the proximal tubule and concentrated in the intermicrovillar domain approximately at the time glomerular filtration begins (Sahali et al., 1993).

C. lntracellular Traffic of gp280/1FR 1. Internalization and Recycling

Studies based on biotin labeling of surface membrane protein and quantitative immunocytochemistry on yolk sac cells incubated with gold-labeled monoclonal anti-gp280hFR antibodies as well as Fab antibody fragments (Le Panse et al., 1997a,b) have clearly shown that gp280/IFR is internalized and very efficiently recycled as indicated by the accumulation of gold particles in DAT. Transfer to the lysosomes is minimal even after long (30 min) incubation times. Gold particles tracing megalin indicated that this protein was also efficiently recycled although the lysosomal accumulation was measurably higher. Whether internalization is constitutive or induced by ligand binding is unknown, in particular since our knowledge of the latter is still limited. 2. Biosynthesis

In yolk sac epithelial cells (BN/MSV) expressing both gp280/IFR and megalin in vitro, both receptors are independently synthesized and rapidly associ-

FIG. 15 Ultrathin cryosection from the apical part of a proximal tubule, incubated with rabbit anti-gp28O/IFR and subsequently with 10 nm colloidal gold particles labeled with secondary antibodies. Labeling is seen on microvilli (MV), along the membrane of coated pits (arrows), in endosomes (E), and in dense apical tubules (arrowheads). Original magnification, X63.000.

RENAL PROXIMAL TUBULE ENDOCMOSIS MEMBRANE RECEPTORS

263

FIG. 16 Ultrathin cryosection from proximal tubule, incubated as in Fig. 15. The lysosomal membrane is intensely labeled for gp280/IFR (arrowheads) and, in addition, some labeling is seen in the lysosomal matrix. Original magnification, X63,OOO.

ate to form homodimers and homopolymers of very high molecular mass, in part at least linked by disulfide bonds (Baricault et al., 1995). The observations on megalin confirmed results obtained with kidney slices (Biemesderfer et al., 1993), suggesting that conclusions derived from BNMSV cells are valid for tubular cells. After long incubation times (24 h), gp280/IFR and megalin are released in the cell supernatant. Given the delays involved, it is most likely that the soluble forms of megalin and gp280/IFR are derived from proteolytic cleavage rather than being the product of an alternate splicing. This observation may be of significance since megalin (Kounnas et al., 1993) and IFR activity are detectable in the urine (see below). Pulse biosynthetic labeling in combination with biotin labeling of membrane proteins (Baricault et al., 1995) showed that maturation of gp280/IFR was very slow since endo H-sensitive forms were still detectable after 24 h of chase. By comparison, maturation of megalin, although slow compared to

264

ERIK lLS0 CHRISTENSEN ET AL.

most proteins, required only 4 h. Furthermore, the targeting pathways of newly synthesized megalin and gp280/IFR were distinct. Whereas the bulk of megalin inserted in the plasma membrane was in a mature, fully glycosylated form, gp280/IFR was inserted in the plasma membrane in an endo Hsensitive form, indicating that it had not been processed through the Golgi. With increasing times of chase, membrane gp280/IFR acquired complex glycosylation although it was still incomplete after 6 h. These findings were confirmed by the demonstration of a large intracellular pool of unprocessed gp280/IFR in the steady state. The results indicate that the intracellular trafficking of gp280/IFR is highly complex. After internalization, as described for numerous receptors, gp280/IFR is recycled from the endocytic compartments to the plasma membrane via the efficient recycling system provided by DAT (Le Panse et al., 1997a,b). In addition, after biosynthesis, gp280/IFR is recycled from the plasma membrane to the early compartments of the Golgi apparatus to undergo complex glycosylation (Baricault et al., 1995). It is possible that the two recycling pathways intersect at some point, but this remains to be established.

D. Function The expression of gp280/IFR in association with clathrin suggested that it could be involved in the endocytic process and the first ligand was recently identified as the gastric intrinsic factor-vitamin B12 ( IF-BI2) complex (Seetharam et al., 1997). These studies were prompted by the similar immunocytochemical distribution of the protein identified as gp280 (Sahali et al., 1988) and the protein isolated from solubilized renal cortex via its binding properties for the IF-BIZcomplex, referred to as the intrinsic factor cobalamin receptor (IFR) (Seetharam et al., 1988). Antibodies independently raised against gp280 or IFR recognized the same protein in the brush border (Seetharam et al., 1997). Furthermore, immunoprecipitation with anti-gp280 antibodies completely depleted a pulse-labeled BN cell lysate of the protein reactive with the anti-IFR antibodies and vice versa. These experiments demonstrate that gp280 and IFR are indeed the same protein. In addition, binding of IF-B12to gp280 was demonstrated by immunoadsorption. Sepharose 4B linked to a monoclonal anti-gp280 antibody retained the entire IFBIZ-bindingcapacity applied on the resin. The single protein eluted, migrated with the expected molecular weight, and bound IF-BIZwith high affinity. Only holo-IF-BIZbinds to gp280/IFR (Fig. 17a), whereas apo-IF does not bind to the receptor (Birn et al., 1997b). Micropuncture studies show that gp280/IFR is indeed an endocytic receptor mediating uptake of IF-BIZin the proximal tubule by endocytosis followed by translocation of the endocytosed B12to lysosomes (Fig. 17b) (Birn et al., 1997b). Opossum

RENAL PROXIMAL TUBULE ENDOCYTOSIS MEMBRANE RECEPTORS

265

kidney cells have been reported to express IF-Blz-bindingsites in vitro and to mediate transcytosis of BIZbut the protein involved has not been clearly identified (Ramanujam et al., 1992, 1994). The physiological significance of gp280/IFR in the kidney proximal tubule and yolk sac epithelial cells is, however, unknown, mainly because I F is present in very low amounts in the circulation and only filtered in small amounts because of its size. Nevertheless, IF-like activity has been described associated with the proximal convoluted tubular brush border and in the urine (Wahlstedt and Grasbeck, 1985; Ramanujam et al., 1990) and I F is definitely filtered to some extent (Birn et al., 1997b). It had been reported 30 years ago that yolk sac membranes bound radiolabeled BIZ,binding being considerably increased in the presence of crude preparations of IF (Padykula et aL, 1966). We have recently shown (Verroust et al., 1997) that the receptor could effect transcytosis as described in intestinal cells or renal cells. Whether IF-BI2is the only ligand of gp280/IFR is at present unknown but several observations suggest otherwise. A first line of evidence derives from observations made in dogs who have an inherited deficiency of the IFR (Fyfe et al., 1989, 1991a,b). The defect is clinically characterized by megaloblastic anemia and proteinuria which persists after intracellular deficiency of BI2and megaloblastic anemia have been corrected by parenteral administration of BIZ.Biochemical studies indicate that both the intestinal and the renal proteins are abnormal (Fyfe et al., 1991b), suggesting (i) that they are indeed the product of the same gene and (ii) that the renal receptor may have the ability to bind other proteins. Similar observations have been made in humans in inherited malabsorption of BIZ(Imerslund-Grasbeck syndrome) (Grasbeck et al., 1960; Imerslund, 1960), which, at least in some instances, is related to abnormal receptor synthesis and/or expression (Gueant et al., 1995). A second line of evidence derives from the inhibition of the endocytosis of peroxidase and the disruption of the intracellular endocytic apparatus by anti-gp280/IFR antibodies (Le Panse et al., 1995). However, this abnormality could be related to an antibody-induced trafficking abnormality rather than to the blocking of a multibinding capacity. Third, recent results that gp280/IFR binds RAP are an indication of at least one additional potential ligand (Birn et al., 1997b), although R AP is essentially an intracellular protein acting as a novel kind of chaperone, as discussed above. This observation is also valuable as it points to another functional similarity with megalin and LRP, the two RAP-binding multiligand receptors.

V. IGF-II/Man-6-P Receptor The insulin-like growth factor II/mannose-6-phosphate receptor (IGF-111 M-6-P receptor) represents another receptor, which has been localized to

266

ERIK lLS0 CHRISTENSEN ET AL.

RENAL PROXIMAL TUBULE ENDOCYTOSIS MEMBRANE RECEPTORS

267

the rat renal proximal tubule (Cui et al., 1993a). The receptor, which is identical to the cation-independent M-6-P receptor (Macdonald et al., 1988), is expressed in apical coated pits, endocytic vacuoles, and DAT (Cui et al., 1993a), as are megalin and gp280/IFR. In addition, the receptor is present in cytoplasmic bodies, probably prelysosomes, which is not surprising considering its function as an M-6-P receptor. However, conflicting results are reported on the renal segmental and subcellular localization of the receptor. Studies on renal cortical membrane fractions indicated that the receptor was distributed either equally, 50% in apical and 50% in basolateral membranes of the proximal tubule (Hammerman and Rogers, 1987), or severalfold higher in basolateral membrane fractions (Jacobsen et al., 1995), and the receptor was also basolaterally located in isolated proximal tubular segments (Mellas et al., 1986). In situ hybridization experiments (Chin and Bondy, 1992) revealed very little expression of the IGF-II/Man-6-P receptor in the proximal tubule. Human kidney sections incubated with labeled IGFI1 showed binding to the inner medulla and glomeruli (Chin et al., 1994), and specificbinding sites were also observed in isolated rat kidney glomeruli using labeled IGF-I1 (Haskell et al., 1988). A recent immunohistochemical study (Evan etal., 1995) showed labeling for the IGF-II/Man-6-P receptor in principal cells of cortical collecting ducts, and in all cells of inner medullary collecting ducts and loops of Henle. The apical localization in the endocytic pathway probably to some extent reflects its function as a receptor mediating endocytosis of IGF-11. As visualized by ligand binding on cryosections from renal cortex followed by autoradiography, the receptor bound IGF-I1 very strongly, IGF-I to a lesser extent, but not insulin (Cui et al., 1993a). Thus, contrary to megalin, this receptor seems to be rather specific in mediating endocytosis of IGF-11. Since the proximal tubule cell has a very extensive endocytic apparatus, including a high amount of lysosomes when compared to that in all other nephron segments, including glomerular cells, as well as compared to collecting duct cells, it appears reasonable to expect that the M-6-P receptor, mediating transport of lysosomal enzymes, is highly expressed in these cells. However, in view of the conflicting results described above, and also in

FIG. 17 (a) Semithin, 0.8-pm cryosection, incubated with IF-S7Co-BIZand prepared for light microscope autoradiography. Labeling is observed in the apical part of the proximal tubules (P) similar to the immunohistochemical localization of gp280iIFR. in particular, concentrated at the base of the brush border. No labeling of glomerulus (G) or distal nephron (D) is seen. Original magnification, X 1000. (b) Electron microscope autoradiography of rat proximal tubule microinfused with IF-57Co-B,,showing endocytosis. S7Co-BIZ can be localized as autoradiographic grains at the brush border (BB) and in endocytic vesicles (V). Original magnification, X 18,000.

268

ERIK ILS0 CHRISTENSEN ET AL.

view of its function as a receptor for IGF-11, further studies are needed to clarify the localization of this receptor in the kidney.

VI. Folate Receptor The vitamin folate (in this context, folate represents all physiological folate derivatives) is absorbed in the gastrointestinal tract and is essential to DNA synthesis. Folate deficiency is implicated in both hematological and fetal abnormalities. Several folate analogues are used for both therapeutic and diagnostic purposes in a wide variety of diseases. Most folates in serum are present as 5-methyltetrahydrofolate (5-THF) (Scott and Gregory, 1996), and the concentration of folates in human serum is in the order of 10 ng/ ml (Branda, 1981). As plasma protein binding of folates is limited (Goresky et al., 1963; Williams and Huang, 1982; Selhub et al., 1987a), the filtered fraction of folates largely exceeds the minimum daily dose necessary to maintain normal plasma folate, thus indicating the need for efficient renal tubular reabsorption. This reabsorption occurs within the proximal tubule (Williams and Huang, 1982; Selhub et al., 1987b; Hjelle et al., 1991; Birn et al., 1997a) and involves the folate receptor (FR) (Selhub et al., 1987a; Hjelle et al., 1991; Birn et al., 1993b).

A. Structure and Expression Folate-binding proteins (FBPs) were originally identified as a soluble folatebinding factor in milk (Ghitis, 1967). Later, its ubiquitous presence was established in both serum and tissues (Henderson, 1990; Antony, 1992, 1996; Selhub, 1994) as a 28- to 40-kDa protein with a high affinity for physiological folates existing both in a soluble form and as a membranebound protein. The term folate receptor (Antony et al., 1981) has later been applied to indicate its function in cellular folate uptake. Three different isoforms of human FR, designated FR-a, FR-P, and FR-y (Elwood, 1989; Lacey et al., 1989; Sadasivan and Rothenberg, 1989; Ratnam er al., 1989; Sadasivan et al., 1994; Shen et al., 1995), have been cloned. The genes are localized to chromosome l l q 1 3 (Ragoussis et al., 1992). FR-y probably represents a secretory form mainly restricted to hematopoietic cells (Shen et al., 1995), whereas FR-a and FR-/3 are glycosyl-phosphatidylinositol (GP1)-linked proteins (Lacey et al., 1989; Lee et al., 1992;Verma et al., 1992) expressing different affinities for different stereo-specific folate analogues (Wang et al., 1992). Binding of folates to FR induces conformational changes in the binding protein (Kaarsholm et al., 1993). The relationship between

RENAL PROXIMAL TUBULE ENDOCYTOSIS MEMBRANE RECEPTORS

269

free and membrane-bound FR has not been fully established. It is possible that free FR may result from the enzymatic release of membrane-bound FR (Antony et al., 1989; Lacey et al., 1989; Elwood et al., 1991; Lee et al., 1992). FRs are expressed in many tissues and cell lines, both normal and malignant, e.g., hematopoietic cells, placenta, ovary, choroid plexus, salivary glands, colon, bronchial cells, pancreas, mammary glands, and several carcinomas, as well as being found in plasma and cerebrospinal fluid (see reviews). The relationship between different isoforms varies among tissues (Ross et al., 1994). The expression of FR is regulated by the extracellular folate concentration in several cell systems (Henderson et al., 1988; Kane et al., 1988; Jansen et al., 1989) and the expression of FR may inversely affect cell proliferation (Sun et al., 1995); however, such regulation has not been established in the kidney. Membrane-bound FR has been located to the kidney (Kamen and Caston, 1975; Selhub and Rosenberg, 1978; Selhub and Franklin, 1984) and FR has also been found in human urine (Hansen et al., 1989). Based on mRNA analysis, adult human kidney expresses both FR-a and FR-P (Ross et al., 1994). Immunocytochemical studies have localized the FR to the proximal tubule of human (Holm et al., 1992) and rat kidney (Selhub and Franklin, 1984; Corrocher et al., 1985), which is confirmed by electron microscopy showing localization to the brush border, endocytic invaginations, including coated pits, vacuoles, and DA T (Hjelle et al., 1991; Birn et al., 1993b, 1997a), and the FR has thus been localized to all the components of the endocytic apparatus (Fig. 18). 6. Function

Several studies have suggested that the proximal tubule FR is involved in renal folate reabsorption (Selhub et al., 1987a,b; McMartin et al., 1992). Selhub ef al. (1987a) originally showed that a similar specificity for different folate analogues could be observed between tubular reabsorption rate and binding to FBP. Furthermore, micropuncture and autoradiographic studies have shown that folate is reabsorbed by endocytosis in the proximal tubule (Selhub el al., 1987b; Hjelle et al., 1991; Birn et al., 1997a). Micropuncture in combination with immunocytochemistry has suggested that FR is endocytosed and recycled through DAT (Birn et al., 1993b). Binding sites are regenerated with t112i= 29 s (Selhub et al., 1987b). Studies on a monkey kidney cell line, MA104, and other cell lines have suggested that FRmediated folate uptake involves caveolae rather than the clathrin-coated pit pathway (Rothberg et al., 1990; Anderson et al., 1992; Chang et al., 1992; Ritter et al., 1995; Smart et al., 1995, 1996; Lee et al., 1996). In this process the folate-FR complex is clustered into caveolae followed by acidification

270

ERIK lLS0 CHRISTENSEN ET AL.

FIG. 18 Lowicryl section from the apical part of a rat proximal tubule incubated with rabbit anti-FR and subsequently 10nm colloidal gold particles conjugated to goat anti-rabbit antibodies. Labeling is confined to the brush border (BB), imaginations (EI), vesicles (V), and dense apical tubules (arrowhead). Original magnification, X52,OOO.

and dissociation of the ligand (Lee et al., 1996). The folates are transported to the cytoplasm, possibly through a carrier protein (Kamen et al., 1991; Prasad et al., 1994),whereas FR returns to the plasma membrane by reopening of the caveolae (Rothberg ef al., 1990). As caveolae are almost never observed in kidney proximal tubules (Breton et al., 1996), it is unclear

RENAL PROXIMAL TUBULE ENDOCYTOSIS MEMBRANE RECEPTORS

271

whether this mechanism operates in these cells. Endocytosed folate-gold particles can be localized to coated pits and coated vesicles (Birn et al., 1997a) and FR is localized to the compartments of the classical endocytic pathway (Fig. 18) (Hjelle et al., 1991; Birn et al., 1993b), suggesting that coated pits are involved in folate internalization followed by transport to vesicles, dissociation of the ligand, and recycling of FR to the luminal membrane through dense apical tubules. Furthermore, other studies have challenged the caveolae hypothesis by showing that GPI-linked receptors concentrate in caveolae by crosslinking with antibodies, but not with folate (Mayor et al., 1994), and that in KB cells most internalized folate bypasses the caveolae (Rijnboutt et al., 1996). A third mechanism of folate transport involving FR has been suggested based on studies of perfused human placentas in which the FR serves a concentrative role by binding folates, resulting in a threefold increase in local concentration facilitating passive flux (Henderson et al., 1995). Such a function has not yet been suggested in other systems. Another system for cellular uptake of folates involves the reduced folate carrier. This low-affinity, high-capacity system has different affinity than FR for folates and antifolates (Westerhof et al., 1991, 1995; Spinella et al., 1995; Henderson, 1990; Antony, 1992). This carrier has been located in human kidney by immunocytochemistry (Matherly et aL, 1994) as well as in mouse kidney by Northern blotting (Said et al., 1996). However, the function of this low-affinity system in kidney folate reabsorption has not been established and may be minor considering the relatively low concentration of folates in the glomerular ultrafiltrate. Recently, it was suggested that renal reabsorption of 5-THF does not involve the FR since folic acid, which has high affinity for the FR, did not inhibit reabsorption of 5-THF in the isolated perfused kidney (Muldoon et al., 1996). In order for folate to return to the circulation, proximal tubules must be capable of transcellular transport of folate. Studies on isolated perfused proximal tubules have shown limited capacity for apical to basolateral transport of folic acid (Birn et al., 1997a), confirming previous studies suggesting a saturable process (Johns et al., 1961; Goresky et al., 1963; Williams and Huang, 1982; Selhub et al., 1987b). Microperfusion studies suggested rapid luminal uptake followed by slow cellular transport (Selhub et aL, 1987b). Similarly, an apical to basal transport can be observed with proximal tubule cells in vitro (McMartin et al., 1993; Morshed and McMartin, 1996) in addition to a quantitatively smaller basal to apical transport (Williams and Huang, 1982; Morshed and McMartin, 1996). The cellular handling of internalized folates is unknown although studies on the isolated perfused kidney have suggested metabolism of some of the internalized folates (Muldoon et al., 1992). In particular, it is not known how folates are translocated from an intracellular compartment to the basolateral surface.

272

ERIK lLS0 CHRISTENSEN ET AL.

Conditions associated with increased urinary folates and decreased serum folates include acute and subacute ethanol intoxication (McMartin, 1984; McMartin et al., 1985, 1986a,b; Muldoon and McMartin, 1994). The increased urinary folates appear to result from a direct effect of ethanol on the renal proximal tubule (Muldoon and McMartin, 1994) although the mechanism is unknown.

VII. Concluding Remarks In the present paper we have described the extensive endocytosis taking place in the renal proximal tubule and, in addition, reviewed our present knowledge of four of the receptors involved in receptor-mediated endocytosis in these cells. It appears to us that megalin plays a very central role in the removal of proteins and other molecules from the tubular fluid. A number of ligands have been listed for this receptor; however, there is no doubt that this list will be significantly extended in the coming years. Thus, the development of knockout for megalin in mice (Willnow et al., 1996a) and future models of knockouts such as organ-specific knockouts will significantly enhance our knowledge of the physiological role of megalinmediated endocytosis in the kidney as well as that in other organs where this protein is expressed. Similar models will also be developed for gp280/ IFR and thereby help to clarify the function of this protein especially in the kidney, since the magnitude of expression of this receptor in the proximal tubule still remains a mystery. Future studies will also focus in more detail on the role of the folate receptor in capturing the folate by endocytosis and clarify the mechanisms for intracellular folate transport and release at the basolateral membranes.

References Abbate, M., Bachinsky, D., Zheng, G., Stamenkovic, I., McLaughlin, M., Niles. J. L., McCluskey, R. T., and Brown, D. (1993). Location of gp330/a2-m receptor-associated protein (a2-MRAP)and its binding sites in kidney: Distribution of endogenousa2-MRAP is modified I . Cell Biol. 61, 139-149. by tissue processing. Ear. . Anderson, R. G., Kamen, B. A., Rothberg, K. G., and Lacey, S. W. (1992). Potocytosis: Sequestration and transport of small molecules by caveolae. Science 255, 410-41 1. Antony. A. C. (1992). The biological chemistry of folate receptors. Blood 79, 2807-2820. Antony, A. C . (1996). Folate receptors. Annu. Rev. Nutr. 16, 501-521. Antony, A. C., Utley, C., Van Horne, K. C., and Kolhouse, J. F. (1981). Isolation and characterization of a folate receptor from human placenta. J . B i d Chem. 256, 9684-9692.

RENAL PROXIMAL TUBULE ENDOCYTOSIS MEMBRANE RECEPTORS

273

Antony, A. C., Verma, R. S., Unune, A. R., and LaRosa, J. A. (1989). Identification of a Mg*+-dependent protease in human placenta which cleaves hydrophobic folate-binding proteins to hydrophilic forms. J. B i d . Chem. 264, 1911-1914. Ashcom, J. D., Tiller, S. E., Dickerson, K., Cravens, J. L., Argraves, W. S., and Strickland, D. K. (1990). The human alpha 2-macroglobin receptor: identification of a 420-kD cell surface glycoprotein specific for the activated conformation of alpha 2-macroglobulin. J . Cell Biol. 110, 1041-1048. Bachinsky, D. R., Zheng, G., Niles, J. L., McLaughlin, M., Abbate, M., Andres, G., Brown, D., and McCluskey, R. T. (1993). Detection of two forms of GP330: Their role in Heymann nephritis. Am. J. Pathol. 143, 598-611. Baricault, L., Galceran, M., Ronco, P. M., Trugnan, G., and Verroust, P. J. (1995). Unusual processing of GP280,a protein associated with the intermicrovillar areas of yolk sac epithelial cells: Plasma membrane delivery of immature protein. Biochem. Biophys. Res. Commun. 212,353-359. Baumann, K., Cojocel, C., and Pape, W. (1983). Effect of molecular charge of protein on its reabsorption in microperfused proximal renal tubule. I n “The Pathogenicity of Cationic Proteins” (P. P. Lambert, P. Bergmann, and R. Beauvens, eds.), pp. 249-260. Raven Press, New York. Bergeron, M., Mayers, P., and Brown D. (1996). Specific effect of maleate on an apical membrane glycoprotein (gp330) in proximal tubule of rat kidneys. Am. J. Physiol. (Renal Fluid Electrolyte Physiol. 40) 271, F908-F916. Biemesderfer, D., Dekan, G., Aronson, P. S., and Farquhar, M. G. (1992). Assembly of disfinctive coated pit and microvillar microdomains in the renal brush border. Am. J. Physiol. 262, F55-F67. Biemesderfer, D., Dekan, G., Aronson, P. S., and Farquhar, M. G. (1993). Biosynthesis of the gp330/44-kDa Heymann nephritis antigenic complex: Assembly takes place in the ER. Am. J. Physiol. 264, FlOl1-F1020. Birn, H., Christensen, E. I., and Nielsen, S. (1993a). Kinetics of endocytosis in renal proximal tubule studied with ruthenium red as membrane marker. Am. J. Physiol. (Renal Fluid Electrolyte Physiol.) 264, F239-F250. Birn, H., Selhub, J., and Christensen, E. I. (1993b). Internalization and intracellular transport of folate-binding protein in rat kidney proximal tubule. Am. J. Physiol. 264, C302-C310. Birn, H., Nielsen, S., and Christensen, E. I. (1997a). Internalization and apical to basolateral transport of folate in rat kidney proximal tubule. Am. J. Physiol. 272, F70-F78. Birn, H., Verroust, P., Nex0, E., Hager, H.. Jacobsen, C., Christensen, E. I., and Moestrup, S. K. (1997b). Characterization of an epithelial -460 kDa protein that facilitates endocytosis of intrinsic factor-vitamin BI2 and binds receptor associated protein. J. Biol. Chem. 272, 26497-26504. Braell, W. A. (1987). Fusion between endocytic vesicles in a cell-free system. Proc. Natl. Acud. Sci. USA 84, 1137-1141. Branda, R. F. (1981). Transport of 5-methyltetrahydrofolic acid in erythrocytes from various mammalian species. J. Nutr. 111,618-623. Breton, S., Lisanti, M. P., and Brown, D. (1996). Caveolin-1 is not associated with H+-ATPase recycling vesicles in proximal tubules (PT) and collecting duct (CD) intercalated cells (IC). Am. Soc. Nephrol. 7(9), 1308. [Abstract] Brown, M. S., and Goldstein, J. L. (1979). Receptor-mediated endocytosis: Insights from the lipoprotein receptor system. Proc. Natl. Acad. Sci. USA 76,3330-3337. Brown, M. S., and Goldstein J. L. (1986). A receptor-mediated pathway for cholesterol homeostasis. Science 232, 34-47. Bu, G., and Rennke, S. (1996). Receptor-associated protein is a folding chaperone for low density lipoprotein receptor-related protein. J. Biol. Chem. 271, 22218-22224.

274

ERIK lLS0 CHRISTENSEN ET AL.

Chang, W. J., Rothberg, K. G., Kamen, B. A,, and Anderson, R. G. (1992). Lowering the cholesterol content of MA104 cells inhibits receptor-mediated transport of folate. J. Cell. Biol. 118, 63-69. Chatelet, F., Brianti, E., Ronco, P., Roland, J., and Verroust, P. (1986a). Ultrastructural localization by monoclonal antibodies of brush border antigens expressed by glomeruli. I. Renal distribution. Am. J. Pathol. 122, 500-511. Chatelet, F., Brianti, E., Ronco, P., Roland, J., and Verroust, P. (1986b). Ultrastructural localization by monoclonal antibodies of brush border antigens expressed by glomeruli. 11. Extrarenal distribution. Am. J. Pathol. 122, 512-519. Chen, W. J., Goldstein, J. L., and Brown, M. S. (1990). NPXY, a sequence often found in cytoplasmic tails, is required for coated pit-mediated internalization of the low density lipoprotein receptor. J. Biol. Chem. 265, 3116-3123. Chin, E., and Bondy, C. (1992). Insulin-like growth factor system gene expression in the human kidney. J. Clin. Endocrinol. Metab. 75, 962-968. Chin, E., Michels, K., and Bondy, C. A. (1994). Partition of insulin-like growth factor (1GF)binding sites between the IGF-I and IGF-I1 receptors and IGF-binding proteins in the human kidney. J. CIin Endocrinol. Metab. 78, 156-164. Christensen, E. I. (1982). Rapid membrane recycling in renal proximal tubule cells. Eur. J. Cell Biol. 29, 43-49. Christensen, E. I., and Birn, H. (1997). Hormone, growth factor, and vitamin handling by proximal tubule cells. Curr. Opin. Nephrol. Hypertens. 6 , 20-27. Christensen, E. I., and Bjerke, T. (1986). Renal tubular uptake of protein: Effect of pH. Renal Physiol. 9, 160-166. Christensen, E. I., and Maunsbach, A. B. (1979). Effects of dextran on lysosomal ultrastructure and protein digestion in renal proximal tubule. Kidney Znt. 16, 301-311. Christensen, E. I., and Maunsbach, A. B. (1980). Proteinuria induced by sodium maleate in rats. Effects on ultrastructure and protein handling in renal proximal tubule. Kidney Znt. 17,771-787. Christensen, E. I., and Nielsen, S. (1991). Structural and functional features of protein handling in the kidney proximal tubule. Semin. Nephrol. 11,414-439. Christensen, E. I., Carone, F. A., and Rennke, H. G. (1981). Effect of molecular charge on endocytic uptake of ferritin in renal proximal tubule cells. Lab. Invest. 44, 351-358. Christensen, E. I., Rennke, H. G., and Carone, F. A. (1983). Renal tubular uptake of protein: Effect of molecular charge. Am. J. Physiol. (Renal Fluid Electrolyte Physiol. 13) 244, F436F441. Christensen, E. I., Gliemann, J., and Moestrup, S. K. (1992). Renal tubule gp330 is a calciumbinding receptor for endocytic uptake of protein. J. Hisrochem. Cytocyem. 40, 1481-1490. Christensen, E. I., Nielsen, S., Moestrup, S. K., Borre, C., Maunsbach, A. B., De Heer, E., Ronco, P., Hammond, T. G., and Verroust, P. (1995). Segmental distribution of the endocytosis receptor gp330 in renal proximal tubules. Eur. J. Cell Biol. 66, 349-364. Cojocel, C., Franzen-Sieveking, M., Beckmann, G., and Baumann, K. (1981). Inhibition of renal accumulation of lysozyme (basic low molecular weight protein) by basic proteins and other basic substances. Pjiigers Arch. 390,211-215. Corrocher, R., Abramson, R. G., King, V. F., Schreiber, C., Dikman, S., and Waxman, S. (1985). Differential binding of folates by rat renal cortex brush border and basolateral membrane preparations. Proc. SOC.Exp. Biol. Med. 178,73-84. Cui, S., and Christensen, E. I. (1993). Three-dimensional organization of the vacuolar apparatus involved in endocytosis and membrane recycling of rat kidney proximal tubule cells. An electron-microscopic study of serial sections. Exp. Nephrol. 1, 175-184. Cui, S., Flyvbjerg, A., Nielsen, S., Kiess, W., and Christensen, E. I. (1993a). IGF-IIIMan-6-P receptors in rat kidney: Apical localization in proximal tubule cells. Kidney Znr. 43,796-807.

RENAL PROXIMAL TUBULE ENDOCYTOSIS MEMBRANE RECEPTORS

275

Cui, S., Nielsen, S., Bjerke, T., and Christensen, E. I. (1993b). Transcellular transport of ferritin in rabbit renal proximal tubules. Exp. Nephrol. 1,309-318. Cui, S., Verroust, P., Moestrup, S. K., and Christensen, E. I. (1996). Megalinlgp330 mediates uptake of albumin in renal proximal tubule. Am. J. Physiol. (Renal Fluid Electrolyte Physiol. 40) 271, F900-F907. Doxsey, S., Kerjaschki, D., and Farquhar, M. G. (1984). A large glycoprotein (gp330) is a resident of coated pits of several absorptive epithelia. J. Cell Biol. 98, 178a. [Abstract] Elkjzr, M. L., Birn, H., Agre, P., Christensen, E. I., and Nielsen, S. (1995). Effects of microtubule disruption on endocytosis, membrane recycling and polarized distribution of Aquaporin-1 and gp330 in proximal tubule cells. Eur. J. Cell Biol. 67, 57-72. Ellgaard, L., Holtet. T. L., Nielsen, P. R., Etzerodt, M., Gliemann, J., and Thflgersen, H. C. (1997). Dissection of the domain architecture of RAP, the alpha2-macroglobin receptor associated protein. Eur. J. Biochem. 244, 544-551. Elwood, P. C. (1989). Molecular cloning and characterization of the human folate-binding protein cDNA from placenta and malignant tissue culture (KB) cells. J. Biol. Chem. 264, 14893-14901. Elwood, P. C., Deutsch, J. C., and Kolhouse, J. F. (1991). The conversion of the human membrane-associated folate binding protein (folate receptor) to the soluble folate binding protein by a membrane-associated metalloprotease. J. Biol. Chem. 266, 2346-2353. Evan, A. P., Henry, D. P., Connors, B. A,, Summerlin, P., and Lee, W. H. (1995). Analysis of insulin-like growth factors (1GF)-I, and -11, type I1 IGF receptor and IGF-binding protein-2 mRNA and peptide levels in normal and nephrectomized rat kidney. Kidney Int. 48,1517-1529. Farquhar, M. G., Saito, A., Kerjaschki, D., and Orlando, R. A. (1995). The Heymann nephritis antigenic complex: Megalin (gp330) and RAP. J. Am. SOC.Nephrol. 6, 35-47. [Editorial] Furukawa, T., Ozawa, M., Huang, R. P., and Muramatsu, T. (1990). A heparin binding protein whose expression increases during differentiation of embryonal carcinoma cells to parietal endoderm cells: cDNA cloning and sequence analysis. J. Biochem. (Tokyo) 108,297-302. Fyfe, J. C., Jezyk, P. F., Giger, U., and Patterson, D. F. (1989). Inherited selective malabsorption of vitamin B12 in giant schnauzers. J. Am. Anim. Hosp. Assoc. 25, 533-539. Fyfe, J. C., Giger, U., Hall, C. A., Jezyk, P. F., Klumpp, S. A., Levine, J. S., and Patterson, D. F. (1991a). Inherited selective intestinal cobalamin malabsorption and cobalamin deficiency in dogs. Pediatr. Res. 29,24-31. Fyfe, J. C., Ramanujam, K. S., Ramaswamy, K., Patterson, D. F., and Seetharam, B. (1991b). Defective brush-border expression of intrinsic factor-cobalamin receptor in canine inherited intestinal cobalamin malabsorption. J. Biol. Chem. 266,4489-4494. Ghitis, J. (1967). The folate binding in milk. Am. J. Clin. Nutr. 20, 1-4. Goresky, C. A,, Watanabe, H., and Johns, D. G. (1963). The renal excretion of folic acid. J. Clin. Invest. 42, 1841-1849. Grasbeck, R., Gordin, R., Kantero, I., and Kuhlback, B. (1960). Selective vitamin B12 malabsorption and proteinuria in young people. Acta Med. Scand. 167, 289-296. Gruenberg, J. E., and Howell, K. E. (1986). Reconstitution of vesicle fusions occurring in endocytosis with a cell-free system. E M 3 0 J. 5,3091-3101. Gueant, J. L., Saunier, M., Gastin, I., Safi, A., Lamireau, T., Duclos, B., Bigard, M. A., and Grasbeck, R. (1995). Decreased activity of intestinal and urinary factor in GrasbeckImerslund disease. Gastroenterology 108, 1622-1628. Gueth-Hallonet, C., Santa Maria, A,, Verroust, P., and Maro, B. (1994). Gp330 is specifically expressed in outer cells during epithelial differentiation in the preimplantation mouse embryo. Development 120,3289-3299. Gutmann, E. J., Niles, J. L., McCluskey, R. T., and Brown, D. (1989). Colchicine-induced redistribution of an apical membrane glycoprotein (gp330) in proximal tubules. Am. J. Physiol. (Cell Physiol. 26) 257, C397-C407.

276

ERIK lLS0 CHRISTENSEN ET AL.

Gutmann, E. J.. Niles, J. L., McCluskey, R. T., and Brown, D. (1991). Loss of antigens associated with the apical endocytotic pathway in proximal tubules from rats with heymann nephritis. Am. J. Pathol. 138, 1243-1255. Haminerman, M. R., and Rogers, S. (1987). Distribution of IGF receptors in the plasma membrane of proximal tubular cells. A m . J. Physiol. (Renal Fluid Electrolyte Physiol. 22) 253, F841-F847. Hammond, T. G., Majewski, R. R., Muse, K. E., Oberley, T. D., Morrissey. L. W., and Amendt Raduege, A. M. (1994). Energy transfer assays of rat renal cortical endosomal fusion: Evidence for superfusion. A m . J. Physiol. 267, F1021-F1033. Hansen, S. I., Holm, J., and Hf~ierMadsen, M. (1989). A high-affinity folate binding protein in human urine. Radioligand binding characteristics, immunological properties and molecular size. Biosci. Rep. 9, 93-97. Haskell, J. F., Pillion. D. J., and Meezan. E. (1988). Specific, high affinity receptors for insulinlike growth factor I1 in the rat kidney glomerulus. Endocrinology 123, 774-780. Henderson, G. B. (1990). Folate-binding proteins. Anntt. Rev. Nurr. 10, 319-335. Henderson, G. B., Tsuji, J. M., and Kumar, H. P. (1988). Mediated uptake of folate by a high-affinity binding protein in sublines of L1210 cells adapted to nanomolar concentrations of folate. J . Membr. Bid. 101, 247-258. Henderson, G. I., Perez, T., Schenker, S., Mackins, J., and Antony, A. C. (1995). Maternalto-fetal transfer of 5-methyltetrahydrofolate by the perfused human placental cotyledon: Evidence for a concentrative role by placental folate receptors in fetal folate delivery. J . Lab. Clin. Med. 126, 184-203. Herz, J., Hamann, U., Rogne, S., Myklebost, 0..Gausepohl, H., and Stanley K. K. (1988). Surface location and high affinity for calcium of a 500-kd liver membrane protein closely related to the LDL-receptor suggests a physiological role as lipoprotein receptor. EMBO J . 7,4119-4127. Hjalm, G., Murray, E., Crumley, G., Harazim, W., Lundgren, S., Onyango. I., Ek. B., Larsson, M., Juhlin, C., Hellman, P., Davis, H., Akerstrom, G., Rask. L., and Morse, B. (1996). Cloning and sequencing of human gp330, a Ca(2+)-binding receptor with potential intracelMa r signaling properties. Eur. J . Biochem. 239, 132-137. Hjelle, J. T., Christensen, E. I., Carone, F. A., and Selhub, J. (1991). Cell fractionation and electron microscope studies of kidney folate-binding protein. Am. J. Physiol. 260, C338C346. Holm, J., Hansen, S. I.. H f ~ ie rMadsen, M., and Bostad, L. (1992). A high-affinity folate binding protein in proximal tubule cells of human kidney. Kidney Inr. 41, 50-55. Imerslund, 0. (1960). Idiopathic chronic megaloblastic anemia in children. Acta Paediatr. Scand. 49(Suppl. 119), 1-115. Jacobsen, C., Jessen, H., and Flyvbjerg, A. (1995). IGF-I1 receptors in luminal and basolateral membranes isolated from pars convoluta and pars recta of rabbit proximal tubule. Biochim. Biophys. Acta 1235, 85-92. Jansen, G., Kathmann, I., Rademaker, B. C., Braakhuis, 6. J., Westerhof, G. R., Rijksen, G., and Schornagel, J. H. (1989). Expression of a folate binding protein in L1210 cells grown in low folate medium. Cancer Res. 49, 1959-1963. Jensen, P. H., Moestrup, S. K., and Gliemann, J. (1989). Purification of the human placental alpha2-macroglobulin receptor. FEBS Left. 255,275-280. Johns, D. G., Sperti, S., and Burgen, A. S. V. (1961). The metabolism of tritiated folk acid in man. J . Clin. Invest 40, 1684-1695. Jollie, W. P. (1990). Development, morphology, and function of the yolk-sac placenta of laboratory rodents. Teratology 41, 361-381. Just, M., and Habermann, E. (1977). The renal handling of polybasic drugs. 2. In vitro studies with brush border and lysosomal preparations. Naunyn-Schrniedeberg's Arch. Pharmacol. 300,67-76.

RENAL PROXIMAL TUBULE ENDOCYTOSIS MEMBRANE RECEPTORS

277

Just, M., Rockel, A., Stanjek, A.. and Bode, F. (1975). Is there any transtubular reabsorption of filtered proteins in rat kidney? Naunyri-Schmiedeberg’s Arch. Pharmacol. 289, 229-236. Just, M., Erdmann, G., and Habermann, E. (1977). The renal handling of polybasic drugs. 1. Gentamicin and aprotinin in intact animals. 2. In vitro studies with brush border and lysosomal preparations. Naunyn-Schnziedeberg’s Arch. Pharmucol. 300, 57-76. Kaarsholm, N. C., Kolstrup, A. M., Danielsen, S. E., Holm, J., and Hansen, S. I. (1993). Ligand induced conformation change in folate binding protein. Adv. Exp. Med. Biol. 338,753-756. Kamen, B. A., and Caston, J. D. (1975). Identification of a folate binder in hog kidney. J . Biol. Chem. 250, 2203-2205. Kamen, B. A., Smith, A. K., and Anderson, R. G. (1991). The folate receptor works in tandem with a probenecid-sensitive carrier in MA104 cells in vitro. J . Clin Invesr. 87, 1442-1449. Kanalas, J J., and Makker, S. P. (1991). Identification of the rat Heymann nephritis autoantigen (GP330) as a receptor site for plasminogen. J. B i d . Chem. 266, 10825-10829. Kane, M. A., Elwood, P. C., Portillo, R. M., Antony, A. C., Najfeld, V., Finley, A., Waxrnan, S., and Kolhouse, J. F. (1988). Influence on immunoreactive folate-binding proteins of extracellular folate concentration in cultured human cells. J . Clin. Invest. 81, 1398-1406. Kerjaschki, D., and Farquhar, M. Ci. (1982). The pathogenic antigen of Heymann nephritis is a membrane glycoprotein of the renal proximal tubule brush border. Proc. Natl. Acad. Sci. USA 79, 5557-5561. Kerjaschki, D., and Farquhar, M. G. (1983). Immunocytochemical localization of the Heymann nephritis antigen (GP330) in glomerular epithelial cells of normal Lewis rats. J. Exp. Med. 157,667-686. Kerjaschki, D., Noronha-Blob, L., Sacktor, B., and Farquhar, M. G. (1984). Microdomains of distinctive glycoprotein composition in the kidney proximal tubule brush border. J . Cell Biol. 98, 1505-1513. Kerjaschki, D., Horvat, R., Binder, S., Susani, M., Dekan, G., Ohja, P. P., Hillemanns, P., and Ulrich, W. (1987). Identification of a 400-kd protein in the brush border of human kidney tubules that is similar to gp330. the nephritogenic antigen of rat Heymann nephritis. Am. J. Parhol. 129, 183-191. Kitazawa, K., Gorfien, S., Brentjens, J. R., Andres, G., and Noble, B. (1986). Reabsorption of horseradish peroxidase by proximal tubules in rats with Heymann nephritis. Am. J. Kidney Dis.7, 58-68. Kounnas, M. Z., Argraves, W. S., and Strickland, D. K. (1992). The 39-kDa receptor-associated protein interacts with two members of the low density lipoprotein receptor family, alpha 2-macroglobulin receptor and glycoprotein 330. J. Biol. Chem. 267, 21162-21166. Kounnas, M. Z . , Chappell, D. A,, Strickland, D. K., and Argraves, W. S. (1993). Glycoprotein 330, a member of the low density lipoprotein receptor family, binds lipoprotein lipase in vitro. J. Biol. Chem. 268, 14176-14181. Kounnas, M. Z., Haudenschild, C. C., Strickland, D. K., and Argraves, W. S. (1994). Immunological localization of glycoprotein 330, low density lipoprotein receptor related protein and 39 kDa receptor associated protein in embryonic mouse tissues. In Vivo 8, 343-351. Kounnas, M. Z., Loukinova, E. B., Stefansson, S., Harmony, J. A., Brewer, B. H., Strickland, D. K., and Argraves, W. S. (1995). Identification of glycoprotein 330 as an endocytic receptor for apolipoprotein Jklusterin. J. Biol. Chem. 270, 13070-13075. Lacey, S. W., Sanders, J. M., Rothberg, K. G., Anderson, R. G., and Kamen, B. A. (1989). Complementary DNA for the folate binding protein correctly predicts anchoring to the membrane by glycosyl-phosphatidylinositol.J. Clin. Invest. 84, 715-720. Lee, H. C., Shoda, R., Krall, J. A,, Foster, J. D., Selhub, J., and Rosenberry, T. L. (1992). Folate binding protein from kidney brush border membranes contains components characteristic of a glycoinositol phospholipid anchor. Biochemistry 31, 3236-3243. Lee, R. J., Wang, S., and Low, P. S. (1996). Measurement of endosome pH following folate receptor-mediated endocytosis. Biochim. Biophys. Acru 1312,237-242.

278

ERIK ILS0 CHRISTENSEN ET AL.

Le Panse, S., Galceran, M., Pontillon, F., Lelongt, B., van de Putte, M., Ronco, P. M., and Verroust, P. J. (1995). Immunofunctional properties of a yolk sac epithelial cell line expressing two proteins gp280 and gp330 of the intermicrovillar area of proximal tubule cells: Inhibition of endocytosis by the specific antibodies. Eur. J. Cell Biol. 67, 120-129. Le Panse, S., Ayani, E., Nielsen, S., Ronco, P., Verroust, P., and Christensen, E. I. (1997a). Internalization and recycling of glycoprotein 280 in epithelial cells of yolk sac. Eur. J. Cell Biol. 72,257-267. Le Panse, S., Verroust, P., and Christensen, E. I. (1997b). Internalization and recycling of glycoprotein 280 in BNlMSV yolk sac epithelial cells: A model system of relevance to receptor-mediated endocytosis in the renal proximal tubule. Exp. Nephrol. 5, 375-384. Leung, C. C., Cheewatrakoolpong, B., O’Mara, T., and Black, M. (1987). Passive Heymann nephritis induced by rabbit antiserum to membrane antigens isolated from rat visceral yolksac microvilli. Am. J. Anat. 179, 169-174. Lloyd, J. B. (1990). Cell physiology of the rat visceral yolk sac: A study of pinocytosis and lysosome function. Teratology 41, 383-393. Lundgren, S., Hjalm, G., Hellman, P., Ek, B., Juhlin, C., Rastad, J., Klareskog, L., Akerstrom, G., and Rask, L. (1994). A protein involved in calcium sensing of the human parathyroid and placental cytotrophoblast cells belongs to the LDL-receptor superfamily. Exp. Cell Res. 212, 344-350. Lundstrom, M., Orlando, R. A,, Saedi, M. S., Woodward, L., Kurihara, H., and Gist Farquhar, M. (1993). Immunocytochemical and biochemical characterization of the Heymann nephritis antigenic complex in rat L2 yolk sac cells. Am. J. Pathol. 143, 1423-1435. Maack, T., and Kinter, W. B. (1969). Transport of protein by flounder kidney tubules during long-term incubation. Am. J. Physiol. 216, 1034-1043. Maack, T., Mackensie, D. D. S., and Kinter, W. B. (1971). Intracellular pathways of renal reabsorption of lysozyme. Am. J. Physiol. 221, 1609-1616. Maack, T., Park, C. H., and Camargo, M. J. F. (1985). Renal filtration, transport and metabolism of proteins. In “The Kidney: Physiology and Pathophysiology” (D. W. Seldin and G. Giebisch, eds.), pp. 1773-1803. Raven Press, New York. Macdonald, R. G., Pfeffer, S. R., Coussens, L., Tepper, M. A., Brocklebank, C. M., Mole, J. E., Anderson, J. K., Chen, E., Czech, M. P., and Ullrich, A. (1988). A single receptor binds both insulin-like growth factor I1 and mannose-6-phosphate. Science 239,1134-1137. Matherly, L. H., Wong, S. C., Angeles, S. M., Taub, J. W., and Smith, G. K. (1994). Distribution of the reduced folate carrier (RFC) versus the high affinity membrane folate binding protein (mFBP) in human tumors and tissues. Proc. Am. Assoc. Cancer Res. 35, 307. [Abstract] Maunsbach, A. B. (1966a). Absorption of I’25-labeled homologous albumin by rat kidney proximal tubule cells. A study of microperfused single proximal tubules by electron microscopic autoradiography and histochemistry. J. Ultrastruct. Res. 15, 197-241. Maunsbach, A. B. (1966b). Observations on the segmentation of the proximal tubule in the rat kidney. J. Ultrastruct. Res. 16, 239-258. Maunsbach, A. B., and Christensen, E. 1. (1992). Functional ultrastructure of the proximal tubule. In “Handbook of Physiology: Renal Physiology” (E. E. Windhager, ed.), 2nd Ed., pp. 41-107. Am. Physiol. SOC.,Washington, DClOxford Univ. Press, New York. Mayor, S., Rothberg, K. G., and Maxfield, F. R. (1994). Sequestration of GPI-anchored proteins in caveolae triggered by cross-linking. Science 264, 1948-1951. McMartin, K. E. (1984). Increased urinary folate excretion and decreased plasma folate levels in the rat after acute ethanol treatment. Alcohol Clin. Exp. Res. 8, 172-178. McMartin, K. E., Shiao, C. Q., Collins, T. D., and Redetzki, H. M. (1985). Acute ethanol ingestion by humans and subacute treatment of rats increase urinary folate excretion. Alcohol 2,473-477. McMartin, K. E., Collins, T. D., and Bairnsfather, L. (1986a). Cumulative excess urinary excretion of folate in rats after repeated ethanol treatment. J. Nutr. 116, 1316-1325.

RENAL PROXIMAL TUBULE ENDOCMOSIS MEMBRANE RECEPTORS

279

McMartin, K. E., Collins, T. D., Shaiao, C. Q., Vidrine, L., and Redetzki, H. M. (1986b). Study of dose-dependence and urinary folate excretion produced by ethanol in humans and rats. Alcohol Clin. Exp. Res. 10,419-424. McMartin, K. E., Morshed, K. M., Hazen Martin, D. J., and Sens, D. A. (1992). Folate transport and binding by cultured human proximal tubule cells. Am. J. Physiol. 263, F841-FS48. McMartin, K. E., Morshed, K. M., Hazen Martin, D. J., and Sens, D. A. (1993). 5-methyltetrahydrofolate urinary excretion: Modeling by cultured human kidney cells. Adv. Exp. Med. Biol. 338,745-748. Mellas, J., Gavin, J. R., 111, and Hammerman, M. R.(1986). Multiplication stimulating activityinduced alkalinization of canine renal proximal tubular cells. J. Biol. Chem. 261, 1443714442. Moestrup, S. K. (1994). The alpha 2-macroglobulin receptor and epithelial glycoprotein-330: Two giant receptors mediating endocytosis of multiple ligands. Biochim. Biophys. Acta 1197,197-213. Moestrup, S. K., and Giemann, J. (1991). Analysis of ligand recognition by the purified alpha 2-macroglobulin receptor (low density lipoprotein receptor-related protein). Evidence that high affinity of alpha 2-macroglobulin-proteinase complex is achieved by binding to adjacent receptors. J. Biol. Chem. 266,14011-14017. Moestrup, S. K., Kaltoft, K., Sottrup-Jensen, L., and Gliemann, J. (1990). The human alfa2macroglobulin receptor contains high affinity calcium binding sites important for receptor conformation and ligand recognition. J. Biol. Chem. 265, 12623-12628. Moestrup, S. K., Holtet, T. L., Etzerodt, M., Th~gersen,H. C., Nykjaer, A,, Andreasen, P. A., Rasmussen, H. H., Sottrup-Jensen, L., and Gliemann, J. (1993a). a2-Macroglobulinproteinase complexes, plasminogen activator inhibitor type-1-plasminogen activator complexes, and receptor-associated protein bind to a region of the a2-macroglobulin receptor containing a cluster of eight complement-type repeats. J. Biol. Chem. 268, 13691-13696. Moestrup, S. K., Nielsen, S., Andreasen, P., J~rgensen,K. E., Nykjaer, A., Reigaard, H., Gliemann, J., and Christensen, E. I. (1993b). Epithelial glycoprotein-330 mediates endocytosis of plasminogen activator-plasminogen activator inhibitor type-1 complexes. J. Biol. Chem. 268, 16564-16570. Moestrup, S. K., Christensen, E. I., Nielsen, S., J~rgensen,K. E., Bjern, S. E., RQigaard, H., and Gliemann, J. (1994). Binding and endocytosis of proteins mediated by epithelial gp330. Ann. NY Acad. Sci. 737,124-137. Moestrup, S. K., Cui, S., Vorum, H., Bregengard, C., Bjern, S. E., Norris, K., Gliemann, J., and Christensen, E. I. (1995). Evidence that epithelial glycoprotein 330/megalin mediates uptake of polybasic drugs. J. Clin.Invest. 96, 1404-1413. Moestrup, S. K., Birn, H., Fischer, P. B., Petersen, C. M., Verroust, P. J., Sim, R. B., Christensen, E. I., and N e x ~ E. , (1996). Megalin-mediated endocytosis of transcobalamin-vitamin-B12 complexes suggests a role of the receptor in vitamin-B12 homeostasis. Proc. Nutl. Acad. Sci. USA 93, 8612-8617. Morshed, K. M., and McMartin, K. E. (1996). In vitro characterization of renal reabsorption and secretion of folate using primary cultures of human kidney cells. J. Nurr. Biochem. 7,276-281. Muldoon, R. T., and McMartin, K. E. (1994). Ethanol acutely impairs the renal conservation of 5-methyltetrahydrofolate in the isolated perfused rat kidney. Alcohol Ctin. Exp. Res. 18,333-339. Muldoon, R. T., Eisenga, B. H., Morshed, K. M., and McMartin, K. E. (1992). 5-Methyltetrahydrofolate is reabsorbed and metabolized in isolated perfused rat kidney. J. Nutr. 122,24152423. Muldoon, R. T., Ross, D. M., and McMartin, K. E. (1996). Folate transport pathways regulate urinary excretion of 5-methyltetrahydrofolate in isolated perfused rat kidney. J. Nutr. 126,242-250.

280

ERIK lLS0 CHRISTENSEN ET AL.

Nielsen, J. T., Nielsen, S., and Christensen, E. I. (1985). Transtubular transport of proteins in rabbit proximal tubules. J. Ultrustruct. Res. 92,133-145. Nielsen, J. T., Nielsen, S., and Christensen, E. I. (1986). Handling of lysozome in isolated, perfused proximal tubules. A m . J. Physiol. (Renal Fluid Electrolyte Physiol. 20) 251,F822F830. Nielsen, S., Nielsen, J. T., and Christensen, E. I. (1987). Luminal and basolateral uptake of insulin in isolated, perfused proximal tubules. A m . J. Physiol. (Renal Fluid Electrolyte Physiol. 22) 253, F857-F867. Novikoff, A. B. (1960). The rat kidney: Cytochemical and electron microscopic studies. In “Biology of Pyelonephritis” (E. L. Quinn and E. H. Kass, eds.), pp. 113-144. Little, Brown, Boston. Novikoff, A. B. (1963). Lysosomes in the physiology and pathology of cells: Contributions of staining methods. In “Ciba Foundation Symposium: Lysosomes” (A. V. S . De Reuck and M. P. Cameron, eds.), pp. 36-77. Little, Brown, Boston. Orlando, R. A., and Farquhar, M. G. (1993). Identification of a cell line that expresses a cell surface and a soluble form of the gp330/receptor-associated protein (RAP) Heymann nephritis antigenic complex. Proc. Natl. Acad. Sci. USA 90,4082-4086. Orlando, R. A,, Kerjaschki, D., Kurihara, H., Biemesderfer, D., and Farquhar, M. G. (1992). gp330 associates with a 44-kDa protein in the rat kidney to form the Heymann nephritis antigenic complex. Proc. Natl. Acad. Sci. USA 89, 6698-6702. Ottosen, P. D. (1978). Reversible peritubular binding of a cationic protein (lysozyme) to flounder kidney tubules. Cell Tissue Res. 194, 207-218. Ottosen, P. D., and Maunsbach, A. B. (1973). Transport of peroxidase in flounder kidney tubules studies by electron microscope histochemistry. Kidney Int. 3, 315-326. Ottosen, P. D., Bode, F., Madsen, K. M., and Maunsbach, A. B. (1979). Renal handling of lysozyme in the rat. Kidney Int. 15, 246-254. Ottosen, P. D., Madsen, K. M., Bode, F., Baumann, K., and Maunsbach, A. B. (1985). Inhibition of protein reabsorption in the renal proximal tubule by basic amino acids. Renal Physiol. 8, 90-99. Padykula, H. A., Deren, J. J., and Wilson, T. H. (1966). Development of structure and function in the mammalian yolk sac. I. Developmental morphology and vitamin 812 uptake of the rat yolk sac. Dev. Biol. 13, 311-348. Park, C. H., and Maack, T. (1984). Albumin absorption and catabolism by isolated perfused proximal convoluted tubules of the rabbit. J. Clin. Invest. 73, 767-777. Pietromonaco, S., Kerjaschki, D., Binder, S., Ullrich, R., and Farquhar, M. G. (1990). Molecular cloning of a cDNA encoding a major pathogenic domain of the Heymann nephritis antigen gp330. Proc. Null. Acad. Sci. USA 87, 1811-1815. Prasad, P. D., Mahesh, V. B., Leibach, F. H., and Ganapathy, V. (1994). Functional coupling between a bafilomycin Al-sensitive proton pump and a probenecid-sensitive folate transporter in human placental choriocarcinoma cells. Biochim. Biophys. Actu 1222, 309-314. Ragoussis, J., Senger, G., Trowsdale, J., and Campbell, I. G. (1992). Genomic organization of the human folate receptor genes on chromosome llq13. Genomics 14,423-430. Ramanujam, K. S., Seetharam, S., Ramasamy, M., and Seetharam, B. (1990). Renal brush border membrane bound intrinsic factor. Biochim. Biophys. Acra 1030, 157-164. Ramanujam, K. S., Seetharam, S., and Seetharam, B. (1992). Leupeptin and ammonium chloride inhibit intrinsic factor mediated transcytosis of [57Co]cobalamin across polarized renal epithelial cells. Biochem. Biophys. Res. Commun. 182, 439-446. Ramanujam, K. S., Seetharam, S.,Dahms, N. M., and Seetharam, B. (1994). Effect of processing inhibitors on cobalamin (vitamin B12) transcytosis in polarized opossum kidney cells. Arch. Biochem. Biophys. 315, 8-15. Ratnam, M., Marquardt, H., Duhring, J. L., and Freisheim, J. H. (1989). Homologous membrane folate binding proteins in human placenta: Cloning and sequence of a cDNA. Biochemistry 28, 8249-8254.

RENAL PROXIMAL TUBULE ENDOCMOSIS MEMBRANE RECEPTORS

281

Raychowdhury, R., Niles, J. L., McCluskey, R. T., and Smith, J. A. (1989). Autoimmune target in Heymann nephritis is a glycoprotein with homology to the LDL receptor. Science 244, 1163-1165. Rijnboutt, S., Jansen, G., Posthuma, G., Hynes, J. B., Schornagel, J. H., and Strous, G. J. (1996). Endocytosis of GPI-linked membrane folate receptor-alpha. J. Cell Biol. 132,35-47. Ritter, T. E., Fajardo, O., Matsue, H., Anderson, R. G., and Lacey, S. W. (1995). Folate receptors targeted to clathrin-coated pits cannot regulate vitamin uptake. Pror. Natl. Acad. Sri. USA 92,3824-3828. Rodman, J. S., Seidman, L., and Farquhar, M. G. (1986). The membrane composition of coated pits, microvilll, endosomes and lysosomes is distinctive in the rat kidney proximal tubule cell. J . Cell Biol. 102, 77-87. Ross, J. F., Chaudhuri, P. K., and Ratnam, M. (1994). Differential regulation of folate receptor isoforms in normal and malignant tissues in vivo and in established cell lines. Physiologic and clinical implications. Cancer 73, 2432-2443. Rothberg, K. G., Ying, Y. S., Kolhouse, J. F., Kamen, B . A., and Anderson, R. G. (1990). The glycophospholipid-linked folate receptor internalizes folate without entering the clathrincoated pit endocytic pathway. J. Cell Biol. 110, 637-649. Russell, D. W., Brown, M. S., and Goldstein, J. L. (1989). Different combinations of cysteinerich repeats mediate binding of low density lipoprotein receptor to two different proteins. J. Biol. Chem. 264,21682-21688. Sadasivan, E., and Rothenberg, S. P. (1989). The complete amino acid sequence of a human folate binding protein from KB cells determined from the cDNA [published erratum appears in J. B i d . Chem. (1990). 265 (3), 18211. J. Biol. Chem. 264, 5806-5811. Sadasivan, E., Cedeno, M. M., and Rothenberg, S. P. (1994). Characterization of the gene encoding a folate-binding protein expressed in human placenta. Identification of promoter activity in a G-rich SP1 site linked with the tandemly repeated GGAAG motif for the ets encoded GA-binding protein. J. Bid. Chem. 269,4725-4735. Sahali, D., Mulliez, N., Chatelet, F., Dupuis, R., Ronco, P., and Verroust, P. (1988). Characterization of a 280-kD protein restricted to the coated pits of the renal brush border and the epithelial cells of the yolk sac. J. Exp. Med. 167,213-218. Sahali, D., Mulliez, N., Chatelet, F., Laurent Winter, C., Citadelle, D., Roux, C., Ronco, P., and Verroust, P. (1992). Coexpression in humans by kidney and fetal envelopes of a 280 kDa-coated pit-restricted protein. Similarity with the murine target of teratogenic antibodies. Am. J. Pathol. 140,33-44. Sahali, D., Mulliez, N., Chatelet, F., Laurent Winter, C., Citadelle, D., Sabourin, J. C., Roux, C., Ronco, P., and Verroust, P. (1993). Comparative immunochemistry and ontogeny of two closely related coated pit proteins. The 280-kd target of teratogenic antibodies and the 330-kd target of nephritogenic antibodies. Am. J. Pathol. 142, 1654-1667. Said, H. M., Nguyen, T. T., Dyer, D. D., Cowan, K. H., and Rubin, S. A. (1996). Intestinal folate transport: Identification of a cDNA involved in folate transport and the functional expression and disrribuiion of its mRNA. Biochim. Biophys. Acta 1281, 164-172. Saito, A,, Pietromonaco, S., Loo, A. K., and Farquhar, M. G. (1994). Complete cloning and sequencing of rat gp330/"megalin," a distinctive member of the low density lipoprotein receptor gene family. Proc. Natl. Acad. Sci. USA 91, 9725-9729. Scott, K. C., and Gregory, J. F. (1996). The fate of [3H]folic acid in folate-adequate rats. J. Nutr. Biochem. 7, 261-269. Seetharam, B., Levine, J. S., Ramasamy, M., and Alpers, D. H. (1988). Purification, properties, and immunochemical localization of a receptor for intrinsic factor-Cobalamin complex in the rat kidney. J . Biol. Chem. 263,4443-4449. Seetharam, B., Christensen, E. I., Moestrup, S. K., Hammond, T. G., and Verroust, P. J. (1997). The target protein of teratogenic antibodies gp280 is identical to the intrinsic factorCobalamin receptor. J . Clin. Invest. 99, 2317-2322.

282

ERIK lLS0 CHRISTENSEN ET AL.

Selhub, J. (1994). Folate binding proteins. Mechanisms for placental and intestinal uptake. Adv. Exp. Med. Biol. 352, 141-149. Selhub, J., and Franklin, W. A. (1984). The folate-binding protein of rat kidney. Purification, properties, and cellular distribution. J. Biol. Chem. 259, 6601-6606. Selhub, J., and Rosenberg, I. H. (1978). Demonstration of high-affinity folate binding activity associated with the brush border membranes of rat kidney. Proc. Natl. Acad. Sci USA 75,3090-3093. Selhub, J., Emmanouel, D., Stavropoulos, T., and Arnold, R. (1987a). Renal folate absorption and the kidney folate binding protein. I. Urinary clearance studies. Am. J. Physiol. 252, F750F756. Selhub, J., Nakamura, S., and Carone, F. A. (1987b). Renal folate absorption and the kidney folate binding protein. 11. Microinfusion studies. Am. J. Physiol. 252, F7577F760. Shen, F., Wu, M., Ross, J. F., Miller, D., and Ratnam, M. (1995). Folate receptor type gamma is primarily a secretory protein due to lack of an efficient signal for glycosylphosphatidylinositol modification: Protein characterization and cell type specificity. Biochemistry 34,5660-5665. Smart, E. J., Ying, Y. S., and Anderson, R. G. (1995). Hormonal regulation of caveolae internalization. J. Cell Biol. 131, 929-938. Smart, E. J., Mineo, C., and Anderson, R. G. (1996). Clustered folate receptors deliver 5methyltetrahydrofolate to cytoplasm of MA104 cells. J. Cell Biol. 134, 1169-1177. Spinella, M. J., Brigle, K. E., Sierra, E. E., and Goldman, I. D. (1995). Distinguishing between folate receptor-alpha-mediated transport and reduced folate carrier-mediated transport in L1210 leukemia cells. J. Biol. Chem. 270,7842-7849. Stefansson, S., Chappell, D. A,, Argraves, K. M., Strickland, D. K., and Argraves, W. S.(1995a). Glycoprotein 330/low density lipoprotein receptor-related protein-2 mediates endocytosis of low density lipoproteins via interaction with apolipoprotein B100. J. Biol. Chem. 270,1941719421. Stefansson, S., Kounnas, M. Z., Henkin, J., Mallampalli, R. K., Chappell, D. A,, Strickland, D. K., and Argraves. W. S. (1995b). gp330 on type I1 pneumocytes mediates endocytosis leading to degradation of pro-urokinase, plasminogen activator inhibitor-1 and urokinaseplasminogen activator inhibitor-1 complex. J. Cell Sci. 108, 2361-2368. Straus, W. (1959). Rapid cytochemical identification of phagosomes in various tissues of the rat and their differentiation from mitochondria by the peroxidase method. J. Biophys. Biochem. Cytol. 5, 193-213. Straus, W. (1962). Cytochemical investigation of phagosomes and related structures in cryostat sections of the kidney and liver of rats after intravenous administration of horseradish peroxidase. Exp. Cell Res. 27, 80-94. Straus, W. (1964). Cytochemical observations on the relationship between lysosomes and phagosomes in kidney and liver by combined staining for acid phosphatase and intravenously injected horseradish peroxidase. J. Cell Biol. 20, 497-507. Strickland, D. K., Ashcom, J. C., Williams, S., Battey, F., Behre, E., Mctigue, K., Battey, J. F., and Argraves. W. S. (1991). Primary structure of alpha 2-macroglobulin receptor-associated protein. Human homologue of a Heymann nephritis antigen. J. Biol. Chem. 266, 1336413369. Sumpio, B. E., and Maack T. (1982). Kinetics, competition and selectivity of tubular absorption of proteins. Am. J. Physiol. 243, F379-F392. Sun, X. L., Murphy, B. R., Li, Q. J., Gullapalli, S., Mackins, J., Jayaram, H. N., Srivastava, A,, and Antony, A. C. (1995). Transduction of folate receptor cDNA into cervical carcinoma cells using recombinant adeno-associated virions delays cells proliferation in vitro and in vivo. J. Clin. Invest. 96, 1535-1547. Takahashi, S., Kawarabayasi, Y., Nakai, T., Sakai, J., and Yamamoto, T. (1992). Rabbit very low density lipoprotein receptor: A low density lipoprotein receptor-like protein with distinct ligand specificity. Proc. Natl. Acad. Sci. USA 89, 9252-9256.

RENAL PROXIMAL TUBULE ENDOCYTOSIS MEMBRANE RECEPTORS

283

Verma, R. S., Gullapalli, S., and Antony, A. C. (1992). Evidence that the hydrophobicity of isolated, in situ, and de novo-synthesized native human placental folate receptors is a function of glycosyl-phosphatidylinositolanchoring to membranes. J. Biol. Chem. 267,41194127. Verroust, P. J., Christensen, E. I., Moestrup, S. K., Hammond, T. G., and Seetharam, B. (1997). The intrinsic factor cobalamin receptor expressed by yolk sac and proximal tubule epithelial cells is the target of teratogenic antibodies. In “Vitamin B12 and B12-Proteins.” Proceedings of the 4th European Symposium on Vitamin B12 and B-12 Proteins, Innsbruck, September 2-6, 1996. Section 7: B12 Medical Aspects, Ch. 32, pp. 491-504. Wiley-Verlag Chemie, Weinheim. Wahlstedt, V., and Grasbeck, R. (1985). Cobalamin-binding proteins in human urine: Identification and quantitation. J. Lab. Clin. Med. 106,439-446. Wang, X., Shen, F., Freisheim, J. H., Gentry, L. E., and Ratnam, M. (1992). Differential stereospecificities and affinities of folate receptor isoforms for folate compounds and antifolates. Biochem. Pharmacol. 44,1898-1901. Warshawsky, I., Bu, G., and Schwartz, A. L. (1993). Identification of domains on the 39-kDa protein that inhibit the binding of ligands to the low density lipoprotein receptor-related protein. J. Biol. Chem. 268,22046-22054. Westerhof, G. R., Jansen, G., van Emmerik, N., Kathmann, I., Rijksen, G., Jackman, A. L., and Schornagel, J. H. (1991). Membrane transport of natural folates and antifolate compounds in murine L1210 leukemia cells: Role of carrier- and receptor-mediated transport systems. Cancer Rex 51,5507-5513. Westerhof, G. R., Rijnboutt, S., Schornagel, J. H., Pinedo, H. M., Peters, G. J., and Jansen, G. (1995). Functional activity of the reduced folate carrier in KB, MA104, and IGROV-I cells expressing folate-binding protein. Cancer Res. 55, 3795-3802. Williams, W. M., and Huang, K. C. (1982). Renal tubular transport of folic acid and methotrexate in the monkey. Am. J. Physiol. 242, F484-F490. Willnow, T. E., Goldstein, J. L., Orth, K., Brown, M. S., and Herz, J. (1992). Low density lipoprotein receptor-related protein and gp330 bind similar ligands, including plasminogen activator-inhibitor complexes and lactoferrin, an inhibitor of chylomicron remnant clearance. J. Biol. Chem. 267,26172-26180. Willnow, T. E., Armstrong, S. A., Hammer, R. E., and Herz, J. (1995). Functional expression of low density lipoprotein receptor-related protein is controlled by receptor-associated protein in vivo. Proc. Natl. Acad. Sci. USA 92,4537-4541. Willnow, T. E., Hilpert, J., Armstrong, S . A., Rohlmann, A., Hammer, R. E., Burns, D. K., and Herz, J. (1996a). Defective forebrain development in mice lacking gp330/megalin. Proc. Natl. Acad. Sci. USA 93, 8460-8464. Willnow, T. E., Rohlmann, A., Horton, J., Otani, H., Braun, J. R., Hammer, R. E., and Herz, J. (1996b). RAP, a specialized chaperone, prevents ligand-induced ER retention and degradation of LDL receptor-related endocytic receptors. EMBO J. 15, 2632-2639. Yamamoto, T., Davis, C. G., Brown, M. S., Schneider, W. J., Casey, M. L., Goldstein, J. L., and Russell, D. W. (1984). The human LDL receptor: A cysteine-rich protein with multiple Alu sequences in its mRNA. Cell 39,27-38. Yochem, J., and Greenwald, I. (1993). A gene for a low density lipoprotein receptor-related protein in the nematode Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 90, 45724576. Yokota, S., and Kato, K. (1988). The heterogeneity of rat kidney lysosomes revealed by immunoelectron microscopic staining for cathepsins B and H. Histochemistry 89, 499504. Yokota, S., Tsuji, H., and Kato, K. (1986). Immunocytochemical localization of cathepsin H in rat kidney. Histochemistry 85, 223-230.

284

ERIK lLS0 CHRISTENSEN ET AL.

Yokota, S., Nishimura, Y., and Kato, K. (1988). Localization of cathepsin L in rat kidney revealed by immunoenzyme and immunogold techniques. Histochemistry 90, 277283. Zheng, G., Bachinsky, D. R., Stamenkovic, I., Strickland, D. K., Brown, D., Andres, G., and McCluskey, R. T. (1994). Organ distribution in rats of two members of the low-density lipoprotein receptor gene family, gp330 and LRPIcY~MR, and the receptor-associated protein (RAP). J. Histochem. Cytochem. 42, 531-542.

A

in monkey, 92-93 in mouse, 92 histamine tag, 121 human CLC protein distribution AND-I basophil, 169-171 AND-I1 basophil, 171-173 CDB phenotype, 173 in control basophils, 164-165 FMLP-induced redistribution, 183-187 granule numbers, 163-164 phenotypes and kinetics, 191-192 PMD-I basophil, 166-168 PMD-I1 basophil, 168-169 profiles, 187-191 recovering basophil I, 174-175 recovering basophil 11, 175-183 temporal changes, 157-163 vesicle numbers, 163-164 degranulation, 155-157 granules, histamine distribution, 192-193 FMLP-stimulated release, 193-194 recovery from FMLP-stimulated secretion, 194-197 transport vesicles, CLC protein-loaded FMLP stimulation, 127-142, 144-148 histamine, 151-153 TPA stimulation, 142-144, 148-151 initial identification, 90 piecemeal degradation, 103-104 recovery from secretion, 198 secreted cellular products, 96-97 secretion, ultrastructural kinetic models, 111-114

Amphibians, limb regeneration preceding events, 1-4 wound healing, 2-3 Arylsulfatase, effect on VoZvox ECM, 70-71 Axolotls, regenerative capabilities, 2

B Basophils anaphylactic degranulation, 100-103 cell contents, 95-96 cell markers, 94-95 CLC protein as tag, 119-121 cytoplasm in multiple species, 122-125 degranulation, morphometric analysis anti-IgE-stimulated basophils, 200-203 basophil similarities, 210-211 control basophils, 199-200 degradation stimulation, 198-1 99 exposure differences, 207-209 GVA-supported vesicle transport, 212 histamine-releasing secretogogues, 209-210 MCP-1-stimulated basophils, 204-207 PMD secretogogues, 209-210 rHRF-stimulated basophils, 204 in disease, 97-98 early degradation model, 104-107 early experiments, 125 early tools for studies, 110-111 electron microscopy, 91-92 in human. 93-94

205

286

INDEX

Basophils (continued) secretogogues, 96-97, 114-118 as secretory cells, 98-100 secretory granules, uptake and storage abilities, 107-109 tools for increasing supplies, 111 ultrastructural subcellular tracers, 121-122 vesicular transport, 125-127 Biogenesis, Volvox ECM algal cell adhesion molecule, 63-64 asexual life cycle, 66 glycoprotein ISG, 64-66 microscopy studies, 62-63 sexual development, 66-70 stress-induced modifications, 70-72 Biosynthesis, gp280-IFR, 261-264 Blastema cell proliferation, 3-4 characterization, 2 growth control, 19-24 molecules in regeneration cell-cell adhesion molecules, 18 collagen type I, 17 collagen type XII, 17 fibronectin, 17 keratin, 16 tenascin, 18 transcription factors, 16-17 Bone morphogenetic proteins, in limb regeneration, 24-25 Boundary zone, in Votvox ECM, 56-58 Buffers, incubated basophils, 164-165

C Cell-cell adhesion molecules, in blastema, 18 Cell lines, secretory, storage of exogenous proteins, 109-110 Cell markers, in human basophils, 94-95 Cellular zone, in Volvox ECM, 56-58 Charcot-Leyden crystal protein as basophil tag, 119-121 distribution in basophils AND-I basophil, 169-171 AND-I1 basophil, 171-173 CDB phenotype, 173 in control basophils, 164-165 FMLP-induced redistribution, 183-187

granule numbers, 163-164 phenotypes and kinetics, 191-192 PMD-I basophil, 166-168 PMD-I1 basophil, 168-169 profiles, 187-191 recovering basophil I, 174-175 recovering basophil 11, 175-183 temporal changes, 157-163 vesicle numbers, 163-164 loaded human basophil transport vesicles FMLP stimulation, 127-142, 144-148 histamine, 151-153 TPA stimulation, 142-144, 148-151 vesicular transport in basophil, 154-155 Chemokines, stimulation of basophil degranulation, 198-199 Chondroitin sulfate, in preblastema, 13-14 CLC protein, see Charcot-Leyden crystal protein Collagen, in preblastema, 12 Collagen type I, in blastema, 17 Collagen type XII, in blastema, 17 ' Crosslinking, in Volvox ECM, 72-74 Cytokines, stimulation of basophil degranulation, 198-199 Cytoplasm, basophils, in multiple species, 122-125

D Deep zone, in Vofvox ECM, 56,61-62 Degranulation basophils anaphylactic degranulation, 100-103 CLC protein distribution AND-I basophil, 169-171 AND-I1 basophil, 171-173 CDB phenotype, 173 in controI basophils, 164-165 FMLP-induced redistribution, 183-187 granule numbers, 163-164 phenotypes and kinetics, 191-192 PMD-I basophil, 166-168 PMD-I1 basophil, 168-169 profiles, 187-191 recovering basophil I, 174-175 recovering basophil 11, 175-183 temporal changes, 157-163 vesicle numbers, 163-164 '

287

INDEX

early degranulation model, 104-107 piecemeal degranulation, 103-104 basophils, human, 155-157 FMLP-induced degranulation, 111-112 TPA-induced degranulation, 112-114 basophils, morphometric analysis anti-IgE-stimulated basophils, 200-203 basophil similarities, 210-211 control basophils, 199-200 degranulation stimulation, 198-199 exposure differences, 207-209 GVA-supported vesicle transport, 212 histamine-releasing secretogogues, 209-210 MCP-1-stimulated basophils, 204-207 PMD secretogogues, 209-210 rHRF-stimulated basophils, 204 Development, sexual, Volvox, 66-70 Diaminobenzidine reaction, effect on basophil peroxidase activity, 122 Disease, basophils in, 97-98

E ECM, see Extracellular matrix Electron microscopy, basophils in human, 93-94 in monkey, 92-93 in mouse, 92 studies, 91-92 Embryogenesis, Volvox algal cell adhesion molecule, 63-64 ECM glycoprotein ISG, 64-66 Endocrine cells, secretory granules, basophils as, 98-100 Endocytic apparatus, renal proximal tubule, 239-243 Endocytic receptor, megalin function as, 252-257 Exocrine cells, secretory granules, basophils as, 98-100 Extracellular matrix higher plant, 74-75 nomenclature, 51-54 v01v0x biochemical characterization, 56-57 boundary and flagellar zone, 57-58 cellular zone, 58-61 deep zone, 61-62

biogenesis and remodeling, 62-63 algal cell adhesion molecule, 63-64 asexual life cycle, 66 crosslinking, 72-74 ECM lysis, 74 glycoprotein ISG, 64-66 sexual development, 66-70 stress-induced modifications, 70-72 ultrastructure, 54-55 boundary zone, 56 cellular zone, 56 deep zone, 56 flagellar zone, 55-56

F Ferritin, cationized, as tracer method for basophils, 122 Fibroblast growth factor in limb regeneration, 24-25 mitogenic effect, 22 Fibroblast growth factor receptor, in limb regeneration, 24-25 Fibronectin, in blastema, 17 Flagellar zone, in Volvox ECM, 55-58 Folate-binding protein, identification, 268-269 Folate receptor expression, 268-269 function, 269-272 role, 268 structure, 268-269

G Genes c-myc, 21 control of morphogenesis in limb regeneration bone morphogenetic proteins, 24-25 fibroblast growth factor receptors, 24-25 fibroblast growth factors, 24-25 homeobox-containing genes, 24-25 homeoboxes, 31-36 retinoid receptors, 24-31 retinoids, 24-31 segment polarity genes, 24-25, 36-38 p53, 21

288

INDEX

Glycoprotein 280-intrinsic factor receptor expression, 261 function, 264-265 intracellular traffic biosynthesis, 261-264 internalization and recycling, 261 localization, 259-260 structure, 260 Glycoproteins gp280-IFR, see Glycoprotein 280-intrinsic factor receptor ISG, in Volvox embryogenesis, 64-66 SSG 185 in Volvox asexual life cycle, 66 in Volvox ECM cellular zone, 58-60 gp280-IFR, see Glycoprotein 280-intrinsic factor receptor Granules basophil, histamine distribution, 192-193 FMLP-stimulated release, 193-194 recovery from FMLP-stimulated secretion, 194-197 number in FMLP-stimulated basophils, 163- 164 Growth, control in blastema, 19-24

H Healing, wounds, in amphibians, 2-3 Histamine-releasing factor, stimulated basophils, 204, 207-211 Histamines as basophil tag, 121 distribution in basophil granules, 192-193 FMLP-stimulated release, 193-194 recovery from FMLP-stimulated secretion, 194-197 secretogogues releasing, PMD basophil exposure, 209-210 vesicular transport, 151-153 in basophil, comparison with CLC protein, 154-155 Homeoboxes, in limb regeneration, 24-25, 31-36 HRF, see Histamine-releasing factor Hyaluronate, in preblastema, 13-14

I Immunoglobulin E, anti-IgE, exposed basophils. 200-203,207-211

Insulin-like growth factor 11-mannose-6-phosphate receptor, localization, 265-268 Intracellular traffic, gp280-IFR biosynthesis, 261-264 internalization and recycling, 261

K Keratin in blastema, 16 in wound epidermis, 10-11 Kinetic analysis basophils, 191-192 CLC protein, 183-184

L Life cycle, asexual, Volvox, glycoprotein SSG 185,66 Limb regeneration morphogenesis, controlling genes homeoboxes, 31-36 retinoid receptor, 26-31 retinoids, 26-31 role, 24-25 segment polarity genes, 36-38 preceding events, 1-4 Lysis, in Volvox ECM, 74

M Mast cells, secretory granules, basophils as, 98-100 Matrix metalloproteinases, in preblastema, 12-13 MCP-1, see Monocyte chemotactic protein-1 Megalin definition, 243-246 expression, 247-252 function, 252-257 RAP ligand, 258-259 role in renal proximal tubule disease, 259 structure, 246-247 Mesenchyme, blastemal, regeneration blastema, 15-19 preblasterna, 12-15

289

INDEX

Microscopy electron microscopy, 91-94 in study of ECM, 62-63 Models kinetic, ultrastructural, for basophil secretion, 111-114 remodeling, see Remodeling Molecules, in regeneration blastema, 15-19 preblastema, 12-15 wound epidermis, 4-12 Monocyte chemotactic protein-1 exposed basophils, 207-21 1 stimulated basophils, 204-207 Morphogenesis, in limb regeneration, controlling genes homeoboxes, 31-36 retinoid receptor, 26-31 retinoids, 26-31 role, 24-2.5 segment polarity genes, 36-38 Morphometric analysis, basophil degranulation anti-IgE-stimulated basophils, 200-203 basophil similarities, 210-211 control basophils, 199-200 degranulation stimulation, 198-199 exposure differences, 207-209 GVA-supported vesicle transport, 212 histamine-releasing secretogogues, 209-210 MCP-1-stimulated basophils, 204-207 PMD secretogogues, 209-210 rHRF-stimulated basophils, 204

Neuropeptides, in wound epidermis, 11 Newts, regenerative capabilities, 2

P Peroxidase, activity of basophil, 122 Phenotypic analysis basophils, 191-192 CLC protein, 184-187

Pherophorin, in Volvox ECM cellular zone, 60-61 Pherophorin 11, effect on Vohox development, 67-69 Pherophorin S, effect on Volvox development, 69-70 Plants, higher, ECM, 74-75 Polarity, segment, gene, in limb regeneration, 24-25, 36-38 Preblastema, molecules in regeneration chondroitin sulfate, 13-14 collagen, 12 hyaluronate, 13-14 matrix metalloproteinases, 12-13 Proteins bone morphogenetic proteins, 24-25 Charcot-Leyden crystal, see Charcot-Leyden crystal protein exogenous, storage by basophil secretory granules, 107-109 by secretory cell lineages, 109-110 folate-binding protein, 268-269 MCP-1, 204-211 RAP, 2.58-259 Proximal tubule, renal endocytic apparatus, ultrastructure, 239-243 megalin expression, 247-252 role of megalin in disease, 259

R RAP protein, as megalin ligand, 258-259 Recycling, gp280-IFR, 261 Regeneration associated molecules blastema, 15-19 preblastema, 12-15 wound epidermis, 4-12 limb amphibian, 2-3 morphogenesis. controlling genes, 24-2.5 homeoboxes, 31-36 retinoid receptor, 26-31 retinoids, 26-31 segment polarity genes, 36-38 Xenopus, gene upregulation, 20

290

INDEX

Remodeling, Volvox ECM algal cell adhesion molecule, 63-64 asexual life cycle, 66 crosslinking, 72-74 ECM lysis, 74 glycoprotein ISG, 64-66 microscopy studies, 62-63 sexual development, 66-70 stress-induced modifications, 70-72 Retinoid receptor, in limb regeneration, 24-31 Retinoids, in limb regeneration, 24-31

Secretion, basophils GVA-stimulated secretion, 212 recovery from, 194-198 ultrastructural kinetic models, 111-114 Secretory cells basophil granules, uptake and storage, 107-109 basophils as, 98-100 Segment polarity, gene, in limb regeneration, 24-25, 36-38 Storage, exogenous proteins by basophil secretory granules, 107-109 by secretory cell lineages, 109-110 Stress, effect on Volvox ECM arylsulfatase, 70-71 wounding response, 71-72

T Tenascin, in blastema, 18 Thymidine, in wound epidermis, 4-7 Tracers, subcellular, ultrastructural, for basophils, 121-122 Transcription factors, in blastema, 16-17 Transferrin, in blastema, 23-24 Transport vesicles basophils, 125-127 GVA-supported, basophils, 212 human basophil CLC protein and histamine, comparison, 154-155 FMLP stimulation, 127-142, 144-148 empty gold-labeled vesicles, 137

empty and gold-labeled vesicles, 135-136 empty vesicles, 133-134 full gold-labeled vesicles, 137-138 full and gold-labeled vesicles, 136-137 full vesicles, 134-135 gold-labeled empty vesicles, 138-139 gold-labeled full vesicles, 139-142 gold-labeled vesicles, 128-132 number of vesicles, 128 histamine, 151-153 "PA stimulation, 142-144, 148-151

U Ultrastructure renal proximal tubule endocytic apparatus, 239-243 Volvox ECM, 54-55 boundary zone, 56 cellular zone, 56 deep zone, 56 flagellar zone, 55-56 Urodeles, aquatic, regenerative capabilities, 2

v Vesicles, number in FMLP-stimulated basophils, 163-164 Vesicle transport basophils, 125-127 GVA-supported, basophils, 212 human basophil CLC protein and histamine, comparison, 154-155 FMLP stimulation, 127-142, 144-148 empty gold-labeled vesicles, 137 empty and gold-labeled vesicles, 135-136 empty vesicles, 133-134 full gold-labeled vesicles, 137-138 full and gold-labeled vesicles, 136-137 full vesicles, 134-135 gold-labeled empty vesicles, 138-139 gold-labeled full vesicles, 139-142

INDEX

gold-labeled vesicles, 128-132 number of vesicles, 128 histamine, 151-153 TPA stimulation, 142-144, 148-151 Volvox, extracellular matrix biochemical characterization, 56-57 boundary and flagellar zone, 57-58 cellular zone, 58-61 deep zone, 61-62 biogenesis and remodeling, 62-63 algal cell adhesion molecule, 63-64 asexual life cycle, 66 crosslinking, 72-74 ECM lysis, 74 glycoprotein ISG, 64-66 sexual development, 66-70 stress-induced modifications, 70-72 boundary zone, 56

291 cellular zone, 56 deep zone, 56 flagellar zone, 55-56 ultrastructure, 52-55

w Wounds epidermis, molecules regulated during regeneration, 4-12 healing, in amphibians, 2-3 response in Volvox, 71-72

X Xenopus, regeneration, gene upregulation, 21

This Page Intentionally Left Blank