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Annual Review of Immunology Volume 5 1987

Before and After Elvin A. Kabat. Vol. 6: 1–25

Transgenic Mice and Oncogenesis Suzanne Cory and Jerry M. Adams. Vol. 6: 25–48

Kinin Formation: Mechanisms and Role in Inflammatory Disorders D Proud, and A P Kaplan. Vol. 6: 49–83

Immunobiology of CR2, the B Lymphocyte Receptor for Epstein-Barr Virus and the C3d Complement Fragment N R Cooper, M D Moore, and G R Nemerow. Vol. 6: 85–113

Veto Cells Pamela J. Fink, Richard P. Shimonkevitz, and Michael J. Bevan.Vol. 6: 115–137

Antigenic Variation in Lentiviral Diseases Janice E. Clements, Susan L. Gdovin, Ronald C. Montelaro, and Opendra Narayan.Vol. 6: 139–159

Structure, Organization, and Regulation of the Complement Genes R D Campbell, S K Alex Law, K B M Reid, and R B Sim. Vol. 6: 161–195

Normal, Autoimmune, and Malignant CD5+ B Cells: The LY-1 B Lineage? K Hayakawa, and R R Hardy. Vol. 6: 197–218

Opioid Peptides and Opioid Receptors in Cells of the Immune System N E S Sibinga, and A Goldstein. Vol. 6: 219–249

Structure and Function of Human and Murine Receptors for IgG J C Unkeless, E Scigliano, and V H Freedman. Vol. 6: 251–281

Melanoma Antigens: Immunological and Biological Characterization and Clinical Significance M Herlyn, and H Koprowski. Vol. 6: 283–308

The Developmental Biology of T Lymphocytes H V Boehmer. Vol. 6: 309–326

V Genes Encoding Autoantibodies: Molecular and Phenotypic Characteristics C A Bona. Vol. 6: 327–358

Role of the Major Histocompatibility Complex Class I Antigens in Tumor Growth and Metastasis K Tanaka, T Yoshioka, C Bieberich, and G Jay. Vol. 6: 359–380

The Immunoglobulin Superfamily—Domains for Cell Surface Recognition A F Williams, and A N Barclay. Vol. 6: 381–405

Lymphotoxin N L Paul, and N H Ruddle. Vol. 6: 407–438

Regulation of Cytokine Gene Expression T Taniguchi. Vol. 6: 439–464

Unique Tumor-Specific Antigens H Schreiber, P L Ward, D A Rowley, and H J Stauss. Vol. 6: 465–483

Molecular Regulation of B Lymphocyte Response T Kishimoto, and T Hirano. Vol. 6: 485–512

IgE-Binding Factors and Regulation of the IgE Antibody Response K Ishizaka. Vol. 6: 513–534

Nonprecipitating Asymmetric Antibodies R A Margni, and R A Binaghi. Vol. 6: 535–554

Three-Dimensional Structure of Antibodies P M Alzari, M B Lascombe, and R J Poljak. Vol. 6: 555–580

Prospects for Gene Therapy for Immunodeficiency Diseases P W Kantoff, S M Freeman, and W F Anderson. Vol. 6: 581–594

C1 Inhibitor and Hereditary Angioneurotic Edema A E Davis, III. Vol. 6: 595–628

The T Cell Receptor/CD3 Complex: A Dynamic Protein Ensemble H Clevers, B Alarcon, T Wileman, and C Terhorst. Vol. 6: 629–662

To the Malaria Circumsporozoite Protein: An Immunological Approach to Vaccine Development M F Good, J A Berzofsky, and L H Miller. Vol. 6: 663–688

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Annual Reviews Ann.Rev. Immunol.1988.6: 1-24

IBEFORE AND AFTER

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Elvin A. Kabat Departmentsof Microbiology, Genetics and Developmentand the Cancer Center/Institute for Cancer Research, ColumbiaUniversity College of Physicians and Surgeons, NewYork, NewYork 10032, and the National Institute of Allergyand InfectiousDiseases,NationalInstitutes of Health, Bethesda, Maryland20892 Introduction Theearlier portion of myautobiography(1) dealt with the period from the time I started to work with MichaelHeidelberger as a laboratory helpe~.on January 2, 1933 until mygrants from the USPublic Health Service weresummarilycancelled in 1952.This chapter continues mystory from that point. First though, I wouldlike to add a few notes about my origins andearliest days. Beginnings Mymother and father had married in 1913 and I was born on September 1, 1914, Both of myparents had cometo the United States as small children. Myfather, Harris Kabatchnick,wasborn in 1872and emigrated to the UnitedStates fromLithuaniaas a small boy. His first recollection after landing in the United States by boat from Hamburg was that the flags wereat half mastbecausePresidentGarfield hadjust died. His family settled on the lower East Side. Mymother, DoreenOtesky, cameto the UnitedStates from Kievin 1893at the age of seven. Myfather completed elementaryschool, went to work, and with his two brothers, Samueland Joseph, started manufacturing women’sdresses. In 1908 they changed their nameto Kabat and the firm wascalled KabatBros. I knowlittle of mymaternal grandfather. Hewaskilled in an accident before I wasborn. Mygrandmotherand the children lived with his brother Morris. It seemsthat the generationthat emigratesto a foreign country, andespeciallythe first generationbornin the UnitedStates, tends to ignore ~ The USGovernmenthas the right to retain a nonexclusive, royalty-free license in and to any copyright covering this paper.

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2 KABAT its history and background until a complete story becomes difficult or impossible to get. Mymother completed high school and played the piano very well. Her mother lived with us until her death, whenI was 11 or 12 years old. We had a five-room apartment on the fourth floor at 24 West 11 lth Street. Both my mother’s and father’s families were very close. Manylived in Harlem within four or five blocks of our home; others lived in the Bronx or in Brooklyn. Aunts, uncles, and their children visited one another frequently. Mycousin Saul Meylackson, a physician who had been a captain in the Medical Corps, US Army, during World War I, became my role model. Myinterest in medicine grew largely from him, and when he visited, as he and his wife Pearl did frequently, I constantly plied him with questions about his patients and about medical research. I used to visit his office frequently on Saturdays, especially during myhigh school and college years, and looked at the smears he made of urethral exudates to diagnose gonorrhea. Pearl’s brother was Dr. Murray Peshkin, a wellknownallergist at Mount Sinai hospital, whomI also questioned incessantly about medicine. One grewup in the 1910s and 1920s keenly aware of the role of infectious disease. I lost a brother whodied of pneumoniaat a few weeks of age in 1918; a cousin died of polio in the 1918 epidemic; myfather was very sick in the influenza pandemic of 1917; a friend in our apartment house died of diphtheria, and manyfamilies lost a child or youngrelative. Epidemics of whoopingcough, chicken pox, scarlet fever, measles, and diphtheria were frequent. Whenthe Schick test and immunization with diphtheria toxin-antitoxin were first introduced in NewYork City Schools in 1924, I was Schick negative, an early indication of mypotentiality as an antibody former. Myparents were very devoted to me and to mysister Harriet, born May 8, 1920. I had everything I wanted for the first 12 years of mylife. My mother tended to be somewhatover-protective. At the age of 10 or 11 I went to a school on 117th Street, and had to cross Lenoxand St. Nicholas Avenues on the way. She wanted to accompany me, but I absolutely refused. She then followed me at some discrete distance. WhenI turned around and saw her, I laid down in the middle of the road and motioned to her to go back before I would stand up. WhenI was seven or eight I went through a religious phase and asked mymother to get me a Hebrewteacher; I studied with him until I became Bar Mitzvah at 13. Unfortunately, I was taught to read and memorizebut never to knowthe structure of the language. This knowledgewould have been of great value in myextensive contacts with the WeizmannInstitute and Israel.

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Myinterests in chemistry began early. Oneof myolder friends who lived on the samefloor wasgiven a chemistryset and let mehelp him. I soongot myownset and was alwaysexperimenting.Manyof the chemicals in the sets kids used in those days are nowhighly restricted even for laboratories. Myfather wouldstop off at Macy’son Saturday afternoon to buy mesomescience books.I wasgreatly influenced by Paul de Kruif’s MicrobeHunters. WhenI wasthirteen life becamevery difficult for us. Thefamily firm had prospered during the second and muchof the third decade of the century, manufacturingdresses selling for $24.95 to $89.95 wholesale. Thencheapdresses beganto flood the market, and the firm went bankrupt in 1927. They tried to get started again with moneyborrowed from relatives, but this enterprise also failed. From1929to 1933myfather did odd jobs, our incomedeclined continuously, and we were dispossessed from one apartment after another because we could not pay the rent. Whenwe movedinto a small apartment, wehad to put goods in storage and these were lost whenwe could not pay the monthlycharges. They containedmostof the papersof myearly life. Onelandlord turned off our electricity so we were in the dark. Wehad no food but a small piece of butter whichwekept from getting rancid by wrappingit and letting cold water run over it. Myfather and I went to court and the judge ordered the electricity turned on. Oneapartmenthotel held on to our furniture, so wethen had to find a furnished apartment. Twocousins whowere dress manufacturers,Arthurand Dick Shill, gave myfather a small job, helping to wait on customersin their showroom. Hestarted in 1932at five dollars per week, but as things improvedduring the NewDeal he continued to work and ended up with a more respectable salary--perhaps $40 to $50 per week. Mysurvival and ability to continue myeducation were largely consequencesof the educational concepts of the early 1920s. LewisM. Terman at Stanford during WorldWarI had emphasizedthe use of intelligence testing to select gifted children for special attention. TheNewYorkPublic SchoolSystemwasallowing bright children to skip grades (half years) they felt that they could do the work. WhenI entered elementaryschool in September1920, I wasalready able to read and to do arithmetic. This was in part becausethe kids in our apartmenthouse played school, with someof the older girls as teachers. Byskipping four times in elementary school I saved two years. WhenI entered DeWittClinton HighSchool at the age of 12, Terman’sextensive study of 1000gifted children whowere to be followedfor a goodportion of their lives waswell underway (2). The Termanwavehad reached high school. Bygetting goodgrades and taking somesummerclasses, I completedhigh school in three years instead of

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four. Thus, in the fall of 1929, at the age of 15 I entered City College. I had applied for a Pulitzer Scholarship to Columbiabut was not selected. At City College I also did well. Since I received extra credits for A and B grades, applied toward the 128 required for the bachelors degree, and since I took one or two summercourses, I was again able to complete the four year course in three. By June of 1932, I was graduated from City College with a B.S. degree. I applied to two medical schools, Cornell and Columbia, whose catalogs said that they had scholarships. Indeed, Columbia had a scholarship specifically for a City College graduate, but it was not being awarded in those days. (It is of interest that I was subsequently on the medical faculty of both these institutions.) Regardless of the extensive subsequent changes in psychologists’ ideas about the limitations and validity of IQ tests, the Termanideas had a very crucial influence on what happened to me. From 1931 to the time I was graduated, the relatives who were helping to support us applied substantial pressure on meto quit college and get a job. I was picking up small amounts of moneyby working during college registration, and I worked as an usher in the old Loew’s NewYork movie theatre during one summer. However, the pressure kept growing for me to quit college. I finally told one of mymother’s brothers to "get the hell out and don’t come back." It was many years before we made up. Twogood friends, Joseph Silagy and Joel Hartley, graduated in 1931 and went to NewYork University Medical School. I used to visit them and sit in on their classes on Saturday mornings. I heard lectures by Homer Smith and R. Keith Carman. I later became very friendly with Cannan while he was at NewYork University as chairman of the Biochemistry Department and at the National Research Council. Wealso met during summers at Woods Hole. While at City College, I had the opportunity to work with Professor Leo Lehrmanin analytical chemistry. Later I started working with Michael Heidelberger, but I continued at City College on Tuesday and Thursday evenings, when Leo taught evening classes. Myearliest papers were published with him. I also becamevery friendly with the professor of organic chemistry, W. L. Prager, and with a biology instructor, Alexander S. Chaikelis. I took his very popular course in physiology during one summer. As a way of trying to help me financially, we constructed a large flow chart of synthetic reactions of aromatic compoundsand tried, unsuccessfully, to interest publishers in it. I did showit to MichaelHeidelberger while I was looking for a job; he liked it and pointed out sections that represented his earlier work. It probably influenced his decision to take me. Charts of this type later becamepopular teaching tools. I was a good Germanstudent and became very friendly with my German teacher, Mark Waldman. Leo

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Lehrman tried, also unsuccessfully, for us to get the rights to translate Fritz Feigl’s bookon spot tests into English. I visited the Feigls many yearslater in Brazil. As a freshmanat City College, I brashly decided to volunteer for the curriculum committee, a student group that madesuggestions to the faculty. Theywere generally juniors and seniors; they had never had a freshmanon the committee,but they took me. I enjoyedtheir meetingsand did makesomesuggestions. DavidRittenberg was also on the committee duringmyfirst year. The Story Continues I will nowtake up mystory wherethe first chapter concluded. Onlyin 1981did I learn, underthe Freedomof InformationAct (Figures 1, 2), that the CriminalInvestigationDivisionof the Department of Justice had placed meon a "list for the apprehensionand detention of prominent individuals considered dangerousto the security of the UnitedStates." The FBI documentimplies that by 1954they were at least looking for information that might lead to myremovalfrom this Security Index. I include this since most Americansare probablyunawareof the existence of such a list. Considering the admittedinjustice of the forcedresettlement of Japanese Americansduring WorldWarII, one wonders howuseful sucha list actually is. Needlessto say, I wasneverapprehended or detained. The last monthsof 1953 and 1954were spent trying to continue work without support from the Public Health Service. Thestudies on cerebrospinal fluid gammaglobulin had proven very useful as an aid in the diagnosis of multiple sclerosis, and HoustonMerritt arranged for the Presbyterian Hospital to makeit a routine clinical laboratory deterruination. Theyprovidedmewith funds for a technician and someparttime help in washingglassware, drawnfromthe fees chargedfor the tests. My laboratory continued to do this until the late 1970s whenautomated methodsweresubstituted. I received a small grant from United Cerebral Palsy, whichhelped. I activated myONRcontract, whichallowed us to continue work on the blood group and dextran problems. The NSFgrant in 1954madeit possible to keepmytechnicians, graduateand postdoctoral students. The University took responsibility for mysalary (1). It was impossible, however,to support the monkeycolony; the allergic encephalomyelitis work was discontinued, just whenwe were planning to isolate the encephalitogenicantigen. Fortunately, this problemwastaken up by manyother workers. Blood Group Substances Weresumedworkon the structure of the BloodGroupA, B, H, Le", b, Le I and i glycoproteins. Mybook Blood GroupSubstances(3) was written

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Figure 1

during several summers at WoodsHole and was published in 1956. Oligosaccharides were isolated, after mild acid hydrolysis, and later by alkaline borohydride degradation. The use of periodate oxidation followed by Smith degradation made it possible to propose (with Kenneth O. Lloyd)

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BEFORE

Figure 2

a composite structure for the carbohydrate moiety of the blood group substances. Later, DonCarlson developed improvedconditions that prevented peeling after alkaline elimination and madepossible isolation of oligosaccharides with reducedN-acetylgalactosaminitol, so that we could

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isolate intact reduced oligosaccharide chains. Walter Morganand Winifred Watkins were isolating oligosaccharides and determining blood group structures independently during the same period. Our results were in excellent accord, and we becamevery close friends. Our oligosaccharides isolated by 3H-alkaline borotritiide differed extensively; manyside chains resulted from incomplete biosynthesis, and this revealed extensive heterogeneity. The findings up to 1982 have been extensively described in a more personalized account (4). Later studies with Albert M. Wu, David Zopf, and Bo Nilsson, Flavio Gruezo & Jerry Liao on six grams of a humanovarian cyst blood group A substance subjected to Smith & Carlson degradations yielded many more oligosaccharide structures from the interior of the blood group substances and made possible extension of the composite structure. This also permitted placing on the compositestructure the oligosaccharides isolated earlier from various intact blood group substances. These were done by Kenneth Lloyd, Luciana Rovis, Byron Anderson, Marilynn Etzler, and by Sherman Beychok,who also studied their optical rotatory dispersion and circular dichroism. The development of the hybridoma technique by Kohler & Milstein led many groups to produce monoclonal anti-A, anti-B, and other blood group reagents. Dr. H. T. Chen and I mappedthe fine structures of the combining sites of monoclonal anti-A and anti-B produced commercially in Canada, England, and Sweden. All were equivalent as blood grouping reagents by the usual hemagglutination tests, but their fine structures differed substantially. We have made cDNAsand have cloned and sequenced them to correlate sequence differences with variation in site structure. Interest in the blood group Ii system began with the studies of Donald Marcus and Richard Rosenfield in the early 1960s. These showed that enzymesof Clostridium tertium destroyed I activity of red ceils, liberating galactose and N-acetyl-glucosamine. Subsequent studies, with Ten Feizi, classified I and i antigens into groups. Anti-IMa(group 1) specificity was shown to involve DGalfl(l ~ 4)DGlcNAcfl(1 ~ OCH2-. With Ray Lemieux’s laboratory we have mapped the fine structure of monoclonal anti-IMa sites more extensively. Weused a substantial number of synthetic oligosaccharides, each modifying a distinct portion of the antigenic determinant, to evaluate its contribution to binding. Mostingenious was Lemieux’s replacement of one of the two H in the -OCH2moiety by deuterium to give two optical isomers, one of which was highly active and the other inactive. After leaving our laboratory, Dr. Feizi continued studies on other I and i determinants, and Dr. Marcusclarified the nature of the determinants of the blood group P system.

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Interlude The Josiah MacyFoundation ran a series of conferences on Polysaccharides in Biology during 1955-1959.Weall exchangedviews, using the Frank Fremont-Smithprinciple, "Don’t speak while I am interrupting." Oneamusingincident in 1955comesto mind. K. G. Stern and I differed on the question of whetherthe microheterogeneity of proteins wasascribable to mistakesin synthesis. I took the position that this could not be unequivocallyestablished since small amountsof impurities mightgive the appearanceof microheterogeneity. Stemreplied, "I cannothelp but feel that nature cannotworkso precisely that it can reproducea moleculeof a million or twentymillion molecularweightexactly in its own imageas, for example,the imprint of a gramophone record. Bywayof example,if two typewriters of the samemakeand the sametype are used, and the sameletters are writtenwiththem,at first glance,it will be said that the imprintis identical, but it is a well-knownfact that criminological investigation can distinguish betweendifferent individual typewriters by microscopicinvestigation, becausethere exist alwaysminute irregularities; so I wouldsay this is a microheterogeneity,somethingwhichat first approximationlooks homogeneous but cannotstand up to the mostsearching criteria." Boyd:"Youmeanthat nature does not always produce homogeneity,or can never produce homogeneity?" Stem:"I wouldsay that at the level of the maeromolecule, it is too muchto expect of nature that such structures shouldbe absolutelyidentical. This is a carry-overfrom the experiencewith small molecules." Kabat:"I think I wasvery careful to say that the evidencedid not permitus to infer whether or not nature produced a homogeneousmacromolceule.However,for the record I amperfectly willing to state that Dr. Stemhas completelyconvincedmeof the microheterogeneity of typewriters."

GeorgSpringerwhowasediting the conference,indexedthis under: "Typewriters, microheterogeneity of, p 163." Lectins I had beeninterested in lectins since the studies on ricin duringWorldWar II carried out with MichaelHeidelberger and AdaBezer. WhileI was in Swedenin 1967, Sten Hammarstrrm,from Peter Perlmann’s laboratory, proposed to becomea postdoctoral student. He mentionedthat he had isolated a lectin fromHelix pomatiawhichagglutinated A red cells, so I told himto bring somealong. Westudied its combiningsite and determined its association constant by equilibriumdialysis. MarilynnEtzler studied the site specificity of Dolichusbiflorus. ArneLundbladcamefromUpsala, bringing someof his blood group oligosaccharides fromurine whichwere of great help in mappingA, B, Hspecific combiningsites of lectins and antibodies.

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WhenMiercio Pereira, with an M.D. degree, came to the laboratory from Brazil, he expandedthe lectin studies to purify and characterize the combiningsites of blood group specific lectins from Lotus, Ulex, soybean, and peanut. Hagen Bretting studied sponge lectins from Axinella; Jerzy Petryniak and Pereira studied Evonymuseuropeus; Shunji Sugii did l, Vistariafloribunda; and Paul Kaladas studied l~icia villosa with Kimuraand Erson from Hans Wigzell’s laboratory in Sweden. Hagen Bretting and Stephanie Phillips studied the mutageniceffects of,4xinella lectin. Charles Woodwith S. Ebisu, L. A. Murphy,and Irwin Goldstein investigated the lectins of Bandeireae simplicifolia A4, B4, and BSlI. Santosh Sikder with C. J. Steer and Gil Ashwellstudied the chicken hepatic lectin, and he did lima bean lectin with Goldstein and Dave Roberts. Wewere very fortunate to have collaborations with Nathan Sharon of the WeizmannInstitute and Irwin Goldstein at Ann Arbor and their colleagues on many of these studies. Manysimilarities exist between the fine structures of antibody combining sites to A, B, H glycoproteins and those of the blood group specific lectins as determined by immunochemicalmapping. Of special interest is the finding that lima bean lectin (5) and a hybridoma antiblood group (6) are very similar in reactivity with a monofucosylhexasaccharide from humanurine but behave entirely differently whena second fucose is substituted on the third sugar from the nonreducing end. The comparative mapping of the fine structures of lectin and antibody sites of similar specificities and correlating these differences with amino acid sequences and X-ray crystallographic structures should throw much light on the evolutionary emergence and functions of these two very important and diverse groups of molecules. Studies

on Antipolysaccharides

The immunochemical mapping of the combining sites and later recombinant DNAstudies on antibodies to ~(1 ~ 6)dextran became the predominant problem of the laboratory, with ancillary studies on anti-~(1 3)~(1 -~ 6)dextran (B1355S), antilevan, and anti-SIII. Rose Magestudied rabbit anti-or(1 ~ 6)dextran and could also showthat the rabbit anti-SIII site was complementaryto a hexasaccharide. Gerald Edelman studied the various antibodies I produced in myself--antidextran, antilevan, anti-A-as well as other humanantidextrans, after reduction and alkylation, using starch gel electrophoresis. These were studied in acrylamide, by Bill Yount and Marianne Dorner, and their subgroup compositions and genetic factors were determined by James Allen, Bill Yount, Marianne Dorner, and Henry Kunkel. Myantilevan turned out to be a monoclonal IgG; the others were pauciclonal. Aminoacid sequencing of antibodies was just

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beginning. Hopingto be able to get enough of myantibodies to sequence, Dick Rosenfield kindly took 15 one-liter plasmaphereses from me, each a week apart, so that eventually I had about 7.5 liters of my serum to fractionate. Stuart Schlossman, Justus Gelzer, and Marianne Dorner tried to fractionate my antidextran to obtain monoclonal populations. While some fractionation into populations with smaller and larger antidextran sites was achieved, monoclonal populations were not obtained, and the amounts produced by fractionation were too small for the sequencing methods of the 1960s. Michael Potter at the National Institutes of Health and Melvin Cohn and Martin Weigert at the Salk Institute had isolated mouse monoclonal anti-~(1 -~ 6)dextrans. The fine structures of the combiningsites of three IgAs were studied with John Cisar, Jerry Liao, Marianne Dorner, and Michael Potter. In 1970 John Cisar had taken a summercourse that I gave at OregonState University at Corvallis, and he had cometo the lab after completing his PhDdegree. Weshowed that the combining sites of W3129 and QUPC52differed. W3129 and W3434were complementary to five ~(1 -~ 6) linked glucoses, whereas QUPC52was complementary to six. Despite this, the binding constant of QUPC52was only 1/30 that of W3129.By competition assays by equilibrium dialysis, methyl ~-D-glucoside and isomaltose contributed 60%of the binding energy of the pentasaccharide with W3129,but only 5%of the binding energy of the hexasaccharide with QUPC52.This indicated that the specificity of W3129 was directed toward the terminal nonreducing end of the ~(1 -* 6) linked oligosaccharide, whereas QUPC52 was specific for the internal chain of ¯ (1 -~ 6) linked glucoses, loosely termed cavity-type but also called end binders and groove-type sites, respectively. This distinction could be readily demonstrated with a synthetic linear dextran synthesized by Ruckel & Schuerch. Since the linear dextran had but one nonreducing end, it inhibited precipitation of W3129by dextran as well as the pentasaccharide; toward QUPC52,however, it was multivalent and so formed a precipitate with hybridomaascitic fluids. This provides a rapid screening methodfor classifying the two kinds of monoclonalanti-~(1 -~ 6)dextrans. Human Monoclonal

Antibodies

The demonstration by Waldenstr6m that macroglobulins and gamma globulins could occur as monoclonal homogeneousproteins in the serum of individuals with various blood dyscrasias, and by Edelmanand Gally that Bence Jones proteins were the light chains of immunoglobulins, led manyinvestigators to search for antibody activities..A numberwere soon reported. Several laboratories acquired large collections of humanmonoclonal proteins, but unlike the mousegammopathies, relatively few of

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12 KABAT the humanmonoclonalshad identifiable antibody activities. Myinterest in screening such humanmonoclonalsarose from two directions. Elliott Ossermanhad a large collection of sera from individuals with multiple myelomaand Waldenstrrm macroglobulinemia. Wedecided to screen these serums with mycollection of polysaccharides, blood group substances, etc. EdwardFranklin at NewYorkUniversity also had a large collection of humansera that had been screened for various antibody activities, but without success. MarianKoshlandhad asked him for a humanIgM;in an attempt to purify it over a Sepharosecolumn,it did not comeoff. It seemedto her that somemight be eluted with a number of sugars, so she thoughtI wouldbe interested. In screeningby gel diffusion we found that the IgMreacted strongly with a water extract of agar preparedat 20°C.Since agar contained4,6-pyruvylatedgalactose, I asked MichaelHeidelberger, whowas studying rabbit antisera to Klebsiella polysaccharides, for a numberof samples. The purified Franklin monowEAreacted muchmore strongly with Klebsiella K21than it clonal IgM did with agar in quantitative precipitin assays; only about one twentieth as muchof these Klebsiella K21polysaccharideswasneeded. At the same time we had found another humanmonoclonalIgM~AYin the Osserman collection that also reacted with agar and with the sameKlebsiella polysaccharide. Unlike IgMwEAMAy which was studied as purified IgM, IgM was studied using whole serum. Wetested other Klebsiella K polysaccharides, and K30and K33reacted in both sera in a wayidentical to K21.Onthe basis of these studies, whichhadbeen acceptedby the Journal of Experimental Medicine (7), the AmericanCancer Society gave me research development grant for a postdoctoral fellow, Dr. Arapalli S. Rao, whocamefromCalcutta. Since he wasto learn the quantitative precipitin methodto continue the work, I thought it wouldbe logical for him to MAy.Wehad run out of the first repeat the studies on IgMwEAand IgM sample of K21and madeup a solution from another sample. He learned the techniquerapidly and wasable to confirmall of our data except that K21gave no precipitate with IgMWEA; he becameconvincedthat our curve with K21waswrong.By this time the proof of the JEMpaper was due to arrive, and I had to decide whether to take out the curve of K21with wEA.In worrying about this I remembered IgM that MichaelHeidelberger had first sent mesix samples, of whichonly K21,whichhad the same4,6pyruvylatedgalactoseas agar, reacted, thus giving us our first clue as to wEA.Onlylater did we get K30and K33whichhad the specificity of IgM 3,4-pyruvylatedgalactose and precipitated equally well per unit weight. I told Raothat there must be someother explanation for the difference betweenthe two K21samples, since if the reaction with 1(21 had not providedthe initial clue to the specificities of the twoIgM,I wouldnever

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have been able to apply for the grant and he would not have come to the laboratory. The explanation finally emerged. It turned out that the first K21 was the neutral sodium salt, but the second K21 preparation had been isolated as the free acid and was progressively lowering the pHof the unbuffered purified IgMwEAbut was not altering the pH of the serum of IgMs~gv. Rao carried out a study of the effect of pH on IgMTM and IgMMAyand found that IgMwEggave identical precipitin curves with K21, sfgv K30, and K33 at pH 4, but K21 did not react at higher pH. With IgM all three polysaccharides reacted identically from pH7.0 to 4.0, indicating differences in the structures of the two antibody combiningsites. Subsequent screening of the Ossermancollection revealed a number of other humanIgM monoclonals with various anticarbohydrate specificities including antibodies specific for the interior of the water soluble blood group A and B substances and for chondroitin sulfates. Most exciting was the discovery of IgMN°v (8), an antibody specific for poly-~(2 ~ 8)Nacetylneuraminic acid, the specific capsular polysaccharide of the group B meningococcusand of E. coli K1, against which vaccination has not been successful in humans. This antibody is as protective per #g antibody in rats infected with E. coli as is a standard horse antigroup B meningococcal serum. IgM~°v also reacts with poly A, poly I, and with denatured DNA probably ascribable to similar oligomeric patterns of the carboxyls in the group B polysaccharide and the phosphates in the polynucleotides and in DNA,permitting reactivity in the IgMN°v antibody combining site. Michael Heidelberger in 1983 put forward a similar explanation of the cross-reaction of type 8 and 19 pneumococcalpolysaccharides; he suggested it was due to the ability of negatively charged phosphoryl-fl-D-Nacetylmannosamineto enter the type 8 site specific for cellobiuronic acid with its negatively charged COO-and vice versa. IgMN°v has potential as adjunct serotherapy together with standard antibiotic therapy for group B meningococcalmeningitis, a possibility suggested by studies in the first three decades of this century of reductions in the death rate due to intrathecal horse antimeningococcal serum. GiampaoloMerlini, Steve Birken, and Marian Gawinowiczare determining the amino acid sequence of the Vn and VL regions of IgMr~°v so that the antibody can be synthesized by recombinant DNAtechniques. Stanley Hoffman and Gerald Edelman have shown that IgMN°v is antibody to N-CAM,and Karl Pfenninger has found it to show the histochemical distribution of N-CAM. The Variability

Plot

Careful amino acid composition analyses of antibodies of different specificities by Marian Koshland had revealed differences. In 1965 Hilschmann and Craig presented the first amino acid sequences of two humanBence

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14 KABAT Jones proteins and showedthat they differed extensively in the first 107 residues fromthe aminoterminusbut wereessentially identical throughout the C-terminal half of the molecule. Theseare nowtermed variable and constant regions. At an antibody workshop(9) at the Weizmann Institute in 1965Dreyer & Bennettsuggestedthat these two domainswere codedby separate genes; additional sequences were presented by Putnam, Milstein, Hood, Gray, Dreyer, and their colleagues. I becameinterested in these developments and in 1966-1967began a Sabbatical in Pierre Grabar’s laboratory at the Institut Pasteur, writing the first edition of "Structural Conceptsin Immunology and Immunochemistry."I thought it would be important to include the sequencedata. At that time two mousex and several human k BenceJones sequenceshad been published. Onaligning the data it was evident that in variable regions of humanand mousevery few aminoacid differencesdistinguish mousefromhuman,e.g. there werevery few speciesspecific residues (nowtermed"species-associatedresidues," as later suggested by Capra). The constant region, however,contained43 differences between humanand mouse; this was comparable to findings with hemoglobins, cytochromes,etc, from different species. A sequenceof a human 2 Bence Jones protein by Titani, Wikler, and Putnamappeared, which further reducedthe numberof species-associated residues and was cited in a "note added in proof." Francois Jacob and VernonIngram both thought the finding important, so I sent it to the Proceedingsof the NationalAcademy of Sciences. Shortly thereafter, in comparingthe V and C regions, I noted the occurrence of moreinvariant glycines in the Vregion, including those at residues 99 and 101 whichhavenowbeenfound in homologouspositions of V-regionsin essentially all immunoglobulin light and heavychains, in the a and fl chains of the T-cell receptor for antigen, andin T cell surface antigens T4andT8. I suggestedthat the role of these two variant glycines mightbe to provide flexibility and permit movementof the rest of the V-region to makemaximum contact with an antigenic determinant. Cesar Milstein, Niall & Edman, and the Hood group published sequencesof several V~chains whichindicated that there weresubgroups largely based on the first 23 aminoacids from the N-terminus. It then becameevident fromthe frequencyof identical repeats that this segment of the chain did not showthe variability neededfor it to be involved in generating antibody complementarityand diversity. This led to my suggesting that the first 23 aminoacids were involved only in threedimensionalfolding. Also, Milstein had noticed that beyondaminoacid 94 he could no longer define subgroupsand suggestedthat frequent crossing over mightbe occurring beyondresidue 94, a finding prophetic of V-

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BEFORE AND AFTER

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J joining, which was largely ignored. The 1967 Cold Spring Harbor and Nobel Symposia provided forums for these exciting developments on aminoacid sequences of antibodies. In 1969 I received a letter from a Dr. Tai Te Wu, then at Cornell University Medical College and Sloan-Kettering in NewYork, asking if he could spend some time in the laboratory to learn quantitative immunochemistry. Whenhe visited the laboratory I asked about his background. He said he was a mathematical biophysicist and had some computer experience. I proposed that we collaborate on the sequence variability. I had previously been entering and aligning the sequence data by hand using pencil and paper. Webegan to meet one day a week to evaluate and discuss results. By this time more complete sequences were beginning to pour into the literature from the laboratories of Capra, Edelman, Hilschmann, Hood, Metzger, Potter, Milstein, and their coworkers. In June 1969 at a symposiumin Prague, Edelman, Franek, and I discussed positions containing more substitutions than were seen in the subgroups. Franek had tabulated dissimilarity in the subgroups, whereas Wu and I had searched for maximumvariability, and our data were in good agreement for three hypervariable regions. In June 1970 (10) we introduced an equation measuringvariability at each position in a set of VLsequences aligned for maximumhomology as follows: Numberof different aminoacids at a given position Variability = Frequencyof the most common aminoacid at that position" At this time some 77 complete and partial sequences of VLregions of humanV~ and V~ and of mouse V~ chains were available. This led to the recognition of three linear stretches of hypervariability, whichwe predicted would fold to form the antibody combiningsite. Whena sufficient number of VHsequences were examined, they too had three hypervariable regions (11). X-ray crystallographic studies on Fab fragments, BenceJones dimers, and more recently on the lysozyme-antilysozyme site by the Poljak group at the Institut Pasteur have all confirmed this prediction almost on a residue-by-residue basis. These hypervariable regions are nowtermed complementarity-determining regions (CDR). Wuand I proposed (10) in an insertional mechanismby which nucleotides coding for the CDRswould be recombined into the frameworkresidues of the various subgroups. The variability plot was based on the assumptionthat, since the antibody forming system was universal among vertebrates, combining data from different subgroups, species, etc, was justifiable, and the combiningsites would be in one place rather than in different portions of the variable regions. WhenMartin Weigert and Melvin Cohn with Cesari and Yon-

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16 KABAT kevich described the mouseV~chains, they found that most of their sequenceswere identical throughoutthe Vxregion. Theypostulated that these werethe productof a single germ-linegene. Thosechains that showed variation from the germ-line sequence were localized to the CDRsand could have been formedby point mutations. This providedthe first evidence for somatic mutation in immunoglobulinV~regions. The assumption is still valid for the very large numberof VLand VHchains now available. Variability plots havesince beenusedto define better allelisms in the MHC class I and II systemsand also to locate sites of variability in AIDS viruses. Variability plots for the T cell receptor require further analysis. Variability plots for other proteins, such as cytochromes,did not show hypervariable regions. A statistical examinationof each residue in the CDRsmadeit possible to designate certain positions as structural and others as probablyin contact with the antigenic determinant.

Compilationof Sequencesof ImmunoglobulinChains In 1973Harry Rose retired as chairmanof the MicrobiologyDepartment, and Harold S. Ginsburg whomI knew very well from WoodsHole was appointed.Since he wasjust getting started, I delayedmySabbatical one year so that I could participate in the teaching. I waschosenas a Fogarty Scholar and spent 1974-1975 at the NationalInstitutes of Health, writing the second edition of Structural Concepts in Immunologyand Immunochemistry. As it is customaryfor Fogarty Scholars to meet with the director of NIH,I was given an appointmentwith Dr. Robert S. Stone, whoinquired as to myresearch interests. WhenI replied that I was interested in the structure of antibody combiningsites and had been tabulating variable region sequences, he suggestedthat NIHhad the PROPr~Tcomputersystem and that this might help me. DeWittStettin, Jr., then DeputyDirector for Science at NIH, introduced me to WilliamF. Raub, then with the Division of Research Resources. NowDeputyDirector of NIH,he took a great interest in myactivity. To evaluate PROPrmT, HowardBilofsky came downfrom Bolt Beranek and Newman to help me in preparingthe tables of sequencesto be used in Structural Concepts.It soonbecameclear that PROPI-IEX had greatly superior potentialities for tabulating and keepingtrack of variable region sequences. Whenmyterm as a Fogarty Scholar was up, I wasasked to serve as an Expert, first to the NationalCancerInstitute andlater to the NationalInstitute of Allergy and Infectious Disease, spendingtwo days a weekat NIHto organize and maintain a data bank of variable region sequences.I rented an apartment in Building 20 at NIH. T. T. Wucame from Northwestern and Howard Bilofsky from Cambridgeto spend two days a monthkeeping track of and

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checkingthe data that I collectedfromthe literature. Thefirst edition (12) appeared in 1976 and contained only aminoacid sequences of variable regions; the secondedition (13) wasexpandedto include constant regions; the third (14) also containednucleotide sequencesand derived aminoacid sequences and included other membersof the immunoglobulinsuperfamily. Thefourth edition (15) appearedin April 1987, included T cell receptors, and had grownto 804 pages; Wuand I, along with Harold M. Perry and KayGottesman,are nowworkingon a fifth edition. Keeping track of exponentiallyincreasingdata is extraordinarilydifficult. Thecompendiumhas been distributed free to workers in the field and has been consideredvery useful. WhileI amvery happythat so manycolleagues find it valuable, I would not have undertaken it had I not felt it was essential to myresearch interests. In addition to providing an enormousbodyof data supporting our prediction (10, 11) that the CDRs in the light and heavychains would fold up to form the antibody combiningsite, wewere able to demonstrate independentassortment of framework(FR) aminoacids (16). This led to formulate the minigenehypothesis, in whichnucleotides coding for the three CDRswere assorted by recombination or insertion into the frameworknucleotides. This hypothesis was put forwardjust as Tonegawa et al. (17) had shownthat, in 12-day-old mouseembryoDNA,the region nucleotides only codedthrough aminoacid 95. Since adult myeloma coded through amino acid 107, we postulated a mechanismof somatic joining of a minigenethat codedfor the remainingaminoacid to give the intact V gene. The J minigene coding for amino acids 96 to 107 was found shortly thereafter by Tonegawa in 12-day-old embryoDNA.In the expressed assembledgene from adult myelomaDNA,it had been joined to the V-region(18). D and J minigenescoding for segmentsof the genewerefoundshortly thereafter (19). In the T-cell receptor for antigen, J minigenesare also present in e, fl, and ~ chains and Dminigenesin the fl chains. Thesefindingshold for all species examined. Since genescodingfor the rest of the VLand VHoccurredin the germline as single stretches of nucleotides, considerablecontroversyexisted as to whetherthe minigenehypothesisapplied to the V-region. DavidBaltimore and RichardEgel proposedthat geneconversionscould explain our assortmentdata. The Rajewskylaboratory (20) described a double recombinant in which CDR1of one germline gene was recombined with CDR2and 3 from another. H. G. Zachau’s laboratory (21) showedby nucleotide sequencingand comparisonsof V-regionsthat our assortment data could be accounted for by gene conversions in the DNAcoding for the FR segments.Mostrelevant are the studies of Reynaudet al (22) in J. Weill’s laboratoryat the Institut Pasteuron chickenVachains. Thechicken

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18 KABAT

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has only a single functional germline V~gene and a large numberof pseudogenes;thus diversity andthe antibody repertoire must be generated by rescue of functional CDRsof pseudogenes by gene conversions-essentially our original minigenehypothesis(16). Work with WHO In 1963, the WorldHealth Organizationbecameinterested in research and training in immunology. Theyorganizedfive meetingsof scientific groups. I wasinvited to participate at one of these at Ibadanin December 1964. Niels Jerne was one of the prime movers.I met Professor Ian Mclntyre, then Deanof the Faculty of Veterinary Science at University College, Nairobi, and Professor and later Dean in the Veterinary School at Glasgow.Webecomeclose friends. Ahigh priority wasassigned to setting up research and training centers in immunology in developingcountries in various parts of the world. Thesewereto be coordinatedwith centers in Geneva and elsewhere. HowardGoodman,Zdenek Trnka, and I went to Nigeria, Uganda,and Gambia.Weselected Ibadanas the first center. Ada E. Bezer, whohad been a technician with me for manyyears, and Dr. Ivan Riha fromPraguestaffed the center, whichwasin the Department of ChemicalPathology.A course in Immunology wasgiven for six to eight monthsfollowedby opportunities for students to do research. Thecenters initially took students fromall of WestAfrica and a numberof students completed PhDs. In 1966 HowardGoodmanand I went around the world and selected Singaporeas another center. Centerswereultimately selected in Sao Paolo, MexicoCity, Nairobi, Beirut, Teheran,and NewDelhi, with support centers in Lausanneand Geneva.From1968to 1971, I spent two weekseachyear (the first three years) with Dr. Otto G. Bier, andthe last year with lvam Mota, directors at the Sao Paolo center located at the Escola Paolista and later movedto the Institute Butantan. I conducteda reviewof the coursework, gavethe final exam,and tried to get a regular research seminarstarted. It wasvery difficult to maintaina critical mass and sustained enthusiasm for continuing the seminars whenI was not there. Dr. VasekHoubaand AdaBezer staffed the Nairobi center for several years. Later, regional centers tended to drawstudents moreexclusively from the country in whichthey were located, and Thailandset up its own immunological center. Dr. GeorgioTorrigiani is currently in chargeof the program,but WHO has difficulties supportingthe centers (23). I continue as a memberof the WHO Expert Advisory Panel on Immunologyand have recently been involved with work on immunizationwith bacterial polysaeeharides. A further developmentwas the setting up of ILRAD (International Laboratoryfor Researchin AnimalDiseases) in Nairobi.

Annual Reviews BEFORE

AND AFTER

19

This began work in 1978 under the auspices of the WorldBank, the Rockefeller Foundation, and the Agencyfor International Development, to develop vaccines against east coast fever and trypanosomiasis. I was involvedin the early planning.

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Interlude OnDecember26, 1970, the front page of the NewYork Times contained an appealfrom 14 Soviet scientists to President Nixonto see that Angela Davisreceiveda fair trial. I thought:"Wehavea free judicial system,the trial will be open to the public; President Nixonoughtto invite themto comeand see for themselves."I reflected on howone mightget himto do this. It became clear that onecould not write to the Presidentdirectly; he wouldnever see the letter. I decidedthat one hadto addressthe suggestion to someone on his staff, whoat least could bring it to his attention if he thoughtit a goodidea. I sent the followingtelegram: Dec. 26, 1970 John Ehrlichman The White House Suggest President Nixon reply to fourteen Soviet scientists’ appeal about Angela Davis by inviting them or any Soviet lawyers they nominate to attend her trial. Stop. Also call attention to request of five NewYork City District Attorneys to attend trials of Jews accused by Soviet Union of plotting highjacking. Elvin A. Kabat Member, National Academyof Sciences

Theidea evidently was favorably received. Figure 3 is the reply from John Ehrlichman.On Sunday,January 3, 1971, the six o’clock evening newsannouncedthat President Nixonhad invited the 14 Soviet scientists to attend AngelaDavis’s trial. Figure 4 showsa portion of Monday’s New YorkTimesarticle andits editorial of January8. It still seemsremarkable that a suggestionfroma private individual could reach the highest levels and be acted uponso rapidly. Current Activities and Future Plans AlthoughI becameEmerituson December31, 1984, I have been permitted to keep mylaboratories and to maintain mygroup at about the samesize. I decideda few years backthat the laboratory had to go into cloning and sequencingif wewereever to be able to formulatea detailed conceptof the interactions in the CDRsof antibodycombiningsites to explain antibody diversity. AccordinglyI spent a short time in MarkDavis’ laboratory, then at NIH, learning to makeand done cDNA.This was followed by about 10 days at the Weizmann Institut learning nucleotide sequencingin DavidGivol’s laboratory. I workedclosely with the carbohydratechemists

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20 KABAT

1970

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Dear M3:. Kabob: Thanks very much for your telegram in which ~’ou put fo~h your thoughts Presidenttsreply to the appeal ~de So%e% se~tis~s ~ be~lf Of ~gela

of December Z7th regarding ~e by fourteen Da~s.

i am sendln~ your wire to Henry Kissingerfor his

Thanks again

for letting

me have your thoughts.

7{) i{ave~Avenue New York 10032

Fiyure 3

in the laboratory,SantoshSikderandPradipAkolkar,to establish the making,cloning, andsequencingof cDNAs on a routine basis. Theythen taughtthe otherpost-doctorals,graduatestudents,andtechnicians. Weset ourselvesthe goal of trying to probethe repertoireof antibody sites formedto a single antigenicdeterminant, the epitope of a(1 -, 6)linked glucoses fromdextranB512.Graduatestudents JacquelineSharon and BarbaraNewman had been engaged in mappingthe antibody combiningsites ofhybridoma antibodiespreparedby injecting ~(1 -~ 6)dextran

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BEFORE ANDAFTER 21

Fiyure4 into BALB/cand C57BL/6mice. Eric Lai had carried out similar studies of C57BL/6hybridom~is from mice immunizedwith stearylisomaltosyl oligosaccharides. H. T. Chenand D. Makovercharacterized hybridomas to the stearylisomaltosyl oligosaccharides in a nude mouseand in C58 mice. Thus, we had a sampling of hybridomas with groove-type sites complementaryto six and seven ~(1 ~ 6)-linked glucoses. These were about equally divided between IgM and IgA, but two IgG3 hybridomas

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22 r,~d~Ar were obtained. Both antigens used for immunizationyielded T-independent hybridomas. Flavio Gruezo prepared mRNA and Drs. Sikder, Akolkar, Bhattacharya, and Jerry Liao have been cloning and sequencing them. Sherie Morrisonis collaborating. She and her colleagues pioneered in preparing transfectomassubstituting C-regiongenes of other species onto mouseVngenes. WithAmeliaBlack, a graduate student, Sherie and I are trying to express the transfection products in E. coli and in mammaliancells. TsukasaMatsudais studyingthe repertoire by the use of the isomaltose oligosaccharides coupledto BSAor to KLHto obtain a set of hybridomasto T-dependentantigens with an ~(1 ~ 6) epitope sufficiently large to fill the antibodycombiningsite. Paula Bordenhas preparedantiidiotypic hybridomasand is studying the idiotypic determinants of the various anti-a(1 ~ 6)dextrans. Oneimportant finding is that the T-independenthybridomashave Via chains belonging to three major germline gene families of Brodeurand Riblet, J558, J606, and J36-60and that the light chains also belong to several germline gene families although their combiningsites are very similar. This wouldtend to indicate that the antibody-formingsystemis very well protected against germlinegeneloss. Since mostother epitopes prepared by coupling small moleculesto a protein create heterogeneous populationsof antibodies, the findingthat different germlinegenefamilies were used could not be interpreted unambiguously. Paula Bordenfound that IgG3antiidiotypes to an anti-a(1 ~ 6)dextran hybridomaprecipitated with antidextran. The reaction differs from the usual precipitin reactions in that it is highly dissociated, and its role in idiotype antiidiotype reactions requires further study. H.-T. Chenhas mappedthe combiningsites of various antiblood group A and B hybridomas and, with Arne Lundblad and R. M. Ratcliffe, has cloned and sequencedseveral. These blood group epitopes are more complicatedstructurally and should also provide an intimate picture of those antibody combiningsites. Wesorely need high resolution X-ray studies of one or more antibody combiningsites to carbohydrate epitopes to be able to correlate immunochemicalmappingdata with the interactions of the dextran or blood groupoligosaccharidesin their respective sites. Wealso intend to study anti-antiidiotypes in this systemin relation to the "internal image"antibodyto a carbohydrate.I intend to stay in the laboratoryfor as long as I feel I can do creative work. Mylife at Columbiahas alwaysbeen most satisfying. Overthe years I have been able to collaborate with manycolleagues at Columbiaand throughout the world. The University provided mewith the opportunity to do as I wishedscientifically and alwaysdefendedacademicfreedomin

Annual Reviews BEFORE AND AFTER

23

general and mine in particular. I am indebted to the Office of Naval Research for seventeen years of support; to the molecular biology section of the National Science Foundation for 36 years of continuous support; and since 1974 to the National Institutes of Health for providing the opportunity for me to pursue my interests there as well as at Columbia. I thank Mr. Darryl J. Guinyard for typing the manuscript.

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Literature Cited I. Kabat, E. A. 1983. Getting started 50 years ago--Experiences,prospectives, andproblemsof the first 21 years. Ann. Rev. Immunol.l: 1-32 2. Boring, E. G. 1956. LewisMadisonTerman.FromBiographical Memoirsof the NationalAcademy of Sciences1959, Vol. 33: 414-61. NewYork: ColumbiaUniv. Press 3. Kabat, E. A. 1956. Blood GroupSubstances--Their Chemistry and Immunochemistry. NewYork: Academic 4. Kabat, E. A. 1982. Contributions of quantitative immunochemistry to knowledge of blood groupA, B, H, Le, I and i antigens. Am.J. Clin. Pathol.78: 28192 5. Sikder, S. K., Kabat,E. A., Roberts,D. D., Goldstein, I. J. 1986. Immunochemicalstudies on the combiningsite of the blood groupA-specific lima bean leetin. Carbohyd.Res. 151:247~i0 6. Chen,H.-T., Kabat, E. A. 1985. Immunochemical studies on blood groups. Thecombining site specificities of mouse monoclonalhybrldomaanti-A and antiB. J. Biol. Chem.260:13208-17 7. Kabat, E. A., Liao, J., Bretting, H., Franklin,E. C., Geltner, D., Frangione, B., Koshland,M.E., Shyong,J., Osserman, E. F. 1980. Humanmonoclonal maerogiobulins withspecificity for Klebsiella K polysaccharides that contain 3,4-pyruvylated-D-galactose and 4,6pyruvylated-D-galactose.J. Exp. Med. 152:979-95 8. Kabat, E. A., Niekerson, K. G., Liao, J., Grossbard, L., Osserman,E. F., Glickman,E., Chess,L., Robbins,J. B., Schneerson, R., Yang, Y. 1986. A humanmonoclonalmacroglobulin with specificity for ~z(1 ~ 8)-linked poly-Nacetyl neuraminic acid, the capsular polysaccharideof groupB meningococci and Escherichiacoli K1, whichcrossreacts with polynucleotides and with denatured DNA.J. Exp. Med.164: 64254 9. Porter, R. R. 1986. Antibodystructure

and the antibody workshop1958-1965. Symposiumon The Role and Significance of International Cooperationin the BiomedicalSciences,eds. G. Salvatore, H. K. Schachman,in Perspectives in Biology and Medicine29: Part 2, S161-S165 10. Wu,T. T., Kabat,E. A. 1970. Ananalysis of the sequences of the variable regions of Bence Jones proteins and myelomalight chains and their implications for antibody complementarity. J. Exp. Med. 132:211-50 11. Kabat, E. A., Wu,T. T. 1972. Attempts to locate complementarity-determining residuesin the variablepositionsof light and heavy chains. Ann. N.Y. Acad. Sci. 190:382-93 12. Kabat, E. A., Wu,T. T., Bilofsky, H. 1976. Variable regions of immunoglobulin chains. MedicalComputerSystems. Cambridge,MA:Bolt Beranek & Newman 13. Kabat, E. A., Wu,T. T.~ Bilofsky, H. 1979. Sequences of Immunoglobulin Chains. NIHPublication 80-2008. National Institutes of Health, Bethesda, Md. 14. Kabat,E. A., Wu,T. T., Bilofsky, H., Reid-Miller, M., Perry, H. 1983. Sequences of Proteins of Immunological Interest. NationalInstitutes of Health, Bethesda, Md. 15. Kabat, E. A., Wu,T. T., Reid-Miller, M., Perry, H. M., Gottesman, K. S. 1987. Sequencesof Proteins of Immunological Interest. U.S. Departmentof Health and HumanServices, Public Health Service, National Institutes of Health, No. 165-462,pp. 1-804. Washington, DC: USGPO 16. Kabat, E. A., Wu,T. T., Bilofsky, H. 1978. Variable region genes for the immunoglobulinframeworkare assembled from small segments of DNA--A hypothesis. Proc. Natl. Acad.Sci. USA 75:2429-33 17. Tonegawa,S., Maxam,A. M., Tizard, R., Bernard, O., Gilbert, W. 1978.

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24 roa~,a" Sequenceof a mousegerm-linegene for version. Proc.Natl. Acad.Sci. USA80: a variable region of an immunoglobulin 4997-5001 light chain. Proc. Natl. Acad.Sci. USA 21. Jaenichen, H.-R., Pech, M., Linden75:1485-89 maier, W., Wildgruber,N., Zachau,H. 18. Bernard, O., Hozumi,N., Tonegawa,S. G. 1984. CompositehumanVkgenes and 1978. Sequences of mouse immunoa modelof their evolution.NucleicAcids globulin light chain genes before and Res. 12:5249-63 after somatic changes. Cell 15: 1133- 22. Reynaud, C.-A., Anquez,V., Grimal, 40 H., Weill, J.-C. 1987. Ahyperconversion 19. Early, P., Huang, H., Davis, M., mechanism generates the chicken light chain preimmunerepertoire. Cell 48: Calame, K., Hood, L. 1980. An immunoglobulinheavychain variable region 379-88 geneis generatedfromthree segmentsof 23. Kabat,E. A. 1986.A tradition of interDNA:V~, D, and J~. Cell 19:981-92 national cooperation in immunology. 20. Krawinkel, U., Zoebelein, G., BrugProc. Int. Symp. "The Role and Si#gemann,M., Radbruch, A., Rajewsky, nifieance of lnternationalCooperation in K. 1983. Recombinationbetween antithe Biomedical Sciences,"" ed. G.Salvabody heavy-chain V and variable V tore, H. K. Schachman.Perspect. Biol. region genes: Evidencefor gene conMed. 29:S159-S160

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Annual Reviews www.annualreviews.org/aronline Ann. Rev. Immunol. 1988. 6 : 25-48 Copyright © 1988 by Annual Reviews Inc. All rights reserved

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TRANSGENIC MICE AND ONCOGENESIS Suzanne Cory and Jerry M. Adams TheWalterand Eliza Hall Institute of MedicalResearch,Post Office Royal MelbourneHospital, Victoria, 3050, Australia INTRODUCTION Thecreation of transgenic mice carrying specific cancer-promotinggenes has openedan exciting newera in oncology.Thebiological effects of an individual oncogeneon diverse cell types can nowbe assessed directly within the living animal. Whiletransgenic animals bear the introduced oncogenein every tissue, expressionof that genemayeither be widespread or directed to a particular cell lineage, dependinguponthe regulatory sequenceschosen, Thetransgene shouldbehaveidentically in every animal of an establishedlineageand, indeed, perhapsin everycell of a giventype. Thus, a well-characterized transgenic line becomesa permanentresource. Perhapsthe most significant opportunity providedby these transgenic animalsis the possibility of exploring the pre-neoplastic state. Onecan attempt to assess whetheran oncogenehas perturbeddifferentiation within particular lineages. The perturbations mayhelp to delineate early maturation stages and to clarify howdifferentiation is controlled. Thus,new insights mayemergeregarding the normalbiological functions of protooncogenes. The rules for oncogenecooperativity can also be evaluated within diverse cell types. For example,onecan isolate the relevant cells from a pre-neoplastic animal bearing an oncogeneand attempt to transformthemfully in vitro with retroviruses carrying other oncogenes.Alternatively, the second oncogenecould be introduced simply by breeding mice of two independenttransgenic lines. Whilethe studyof transgeniconcogenes is still in its infancy, majornew insights have already been gained. This chapter briefly summarizeshow transgenic animalsare produced,and then considers our current state of 25 0732~582/88/04104)025502.00

Annual Reviews 26

CORY & ADAMS

knowledgeconcerningthe effects of the different classes of oncogenesin transgenic mice. PRODUCTION

OF TRANSGENIC

MICE

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Gene Transfer to the Germline by Microinjection Introduction of newgenes into the mousegermlineis currently performed most efficiently by microinjection of DNA.Onlya brief outline of the technologyis presented here, since detailed reviews haveappearedelsewhere(1-5). Pronucleiof recently fertilized eggs are injected with a few hundredcopies of the cloned gene of interest, and the embryosare then transferred into the oviducts of pseudo-pregnantfemales and allowedto develop to term. In skilled hands, some25%of the mice that are born carry one or morecopies of the foreign gene. If integration occursprior to the first cell division,all cells in a transgenicpup,includingthe germcells, will carry the "transgene."In practice, a significant proportion(~ 30%) the transgenic pups are mosaic, indicating that integration often occurs subsequentto the first round of replication. Since the same degree of mosaicismis usually exhibited in somatic and germcells, lines reliably transmitting the transgene can usually be established even from mosaic foundersby breedingtheir transgenic offspring. The mechanismof integration is not known.Presumably on random breakage of a mousechromosome,the injected DNA is incorporated by the normalcellular repair enzymes(for a discussion, see 4). Typically, some1-50 copies of the injected gene are inserted in a tandem,head-totail array at a single chromosomalsite. On subsequent breeding, the transgenic array is inherited as a simple Mendeliantrait, usually in a remarkablystable manner.Morerarely, insertion occursat two positions, and these loci segregate amongthe progeny.Since the transgeneis inserted into only one homologueof the relevant chromosome, the founder mouse is heterozygousat that allele. Homozygous lines can be established by breedingonly if insertion has not interrupted a genenecessaryfor development(6). Expression of an inserted gene depends primarily on the regulatory regions that it bears. A majordeterminantis "strength" of the promoter/ enhancercombinationused, but the level and tissue distribution of expression mayalso be influencedby the site of chromosomal insertion and perhaps by unknownelements within the transgene, such as attachment sites for the nuclear matrix. Theexpressionof certain transgenes(but not others) is strongly inhibited by linked prokaryoticsequences(2). Manyof the problems associated with transgenic mouseproduction wouldbe alleviated by the developmentof vectors that can be maintained

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as autonomously replicating episomes.In principle, all injected eggs developing to term wouldbe transgenic, transmission to offspring wouldbe quantitative, problemsof insertional mutagenesisnonexistent, and most important, variation in expressionimposedby variable integration sites wouldbe absent. Attemptsto use bovine papillomavirus as an episomal vector have not been successful so far, however. In one study, many primarymice contained the vector in episomalform but weremosaic, and BPVDNA was apparently diluted further on subsequent breeding (7). two other studies, only integrated BPVDNA wasdetected (8). A promising advancehas been the serendipitous creation of autonomouslyreplicating elements containing defined segments of mousechromosomalDNAby injection of fertilized mouseeggs with a polyoma-based vector (9) together with trace levels of another plasmid(F. Cuzin, personal communication). Significantly, the incorporated mousesequences are homologousto conserved yeast centromeric sequences. Whetherthe propensity of the episomesto recombinationcan be overcomeremainsto be established. Retroviral

Vectors for Gene Delivery to the Germline

Considerableinterest exists in usingrecombinantretroviruses to introduce additional genes (including oncogenes)into the mousegermline. Amajor advantageof retroviral delivery is that it is technically less demanding, oncea suitable virus-producingline is available. Moreover,only a single gene is inserted per site, instead of a concatamer,whichmayadversely affect neighboringchromatinand transcriptional units (10). Severe problemsremain to be overcomewith the retroviral approach, however.To date, successful infection protocols use eight-cell stage embryos(11, 12)i hence, all founderanimalsare mosaic.Moreimportantly, however,expressionof the integrated provirus is usually suppressed,and this suppression is maintainedduring subsequentdevelopment(13). The mechanism of suppressionis not clear but mayinvolve methylation (14), inability of the long terminal repeat (LTR)to promotetranscription (1517), and/or transacting negativeregulatoryfactors (18). Twoapproaches have been taken to overcome suppression. One involved modifyingthe retroviral LTRby replacing the enhancerwith one knownto function within embryonalcarcinoma(EC) cell lines (12, 19). Theother involved redesign of the retrovirus to allow expressionof the geneof interest froman internal, cellular promoter(20-22). Despitepreliminarysuccesses,it is not yet clear howgenerallyusefulsuchvectorswill be. A potentially exciting use of recombinantretroviruses is to infect embryo-derived (ES) pluripotential cell lines capable of forminggermline chimeras(23). Since the viruses can be engineeredto express a selectable

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markergeneas well as the geneof interest, infected EScells can be selected in vitro prior to either injection into blastocystsor aggregationwith eightcell embryos.Preliminaryexperimentsindicate that provirusesselected in .vitro for expressionin ECand EScells are stably expressedthroughout development and within~all tissues (22, 23). Amajor probleminherent this approach,however,is the tendencyof the ESlines to becomeaneuploid duringthe in vitro culture period and henceto lose their ability to form chimeras. Annu. Rev. Immunol. 1988.6:25-48. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Choice of Regulatory Sequences In attempting to target expression, a numberof different promoter/ enhancersequencescan be attached to the gene of interest (see 4 for detailed discussion). Theexpressionpattern of the engineeredhybridgene often conformsto that of the gene from whichthe regulatory region was derived. Novel patterns are sometimes observed, however, perhaps becauseof the unforeseen creation of a "recombinantenhancer," and/or the influence of flanking DNA. Thebest studied regulator for generalized expression is that of the metallothionein (MT)gene. The endogenous MTgene is expressed virtually all cells and is responsiveto several regulators, includingglucocorticoids and metals. Whilemost MTfusion genes have been expressed wellin liver, intestine, kidney,heart, pancreas,andtestes, unusualpatterns of expressionhavealso beenobserved(4). Threetissue-specific regulators havebeenparticularly successful: the immunoglobulin enhancersfor expressionin B lymphoidcells; the 5’ region of the rat elastase-I gene, for expressionin the exocrinepancreas;and the 5’ region of the rat insulin II gene, for expressionin the//cells of the endocrinepancreas. Theidentification of "strong" tissue-specific regulators for other cell lineages will greatly increase the versatility of the transgenic approach. ONCOGENES AND THE PROGRESSION MALIGNANCY

TO

Cancerhas long been perceived to be a multistage process. In molecular terms, this progressionis currently believedto involvethe stepwiseacquisition of activated oncogenes(24). Theexperimentalevidencesupporting this view derives froman analysis of the transforminggenesin DNA tumor viruses (25, 26) and fromin vitro transfection studies with primarycells, principally fibroblasts (27-29). Transformationwas foundto require the concertedaction of two genes. Significantly, effective collaboration was usually achievedonly if one of the pair of oncogenescamefrom the class

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expressed in the nucleus and the other from the class expressed in the cytoplasm(24). Thecytoplasmicclass includes proteins related to growth factor receptors (erbB, fms), as well as proteins (e.g. ras) thought to regulate levels of critical second messengerssignalling interaction of growthfactors with their receptors. Thefunction of the nuclear class (e.g. myc, N-myc,fos) is unknownbut seemslikely to involve regulation of transcription. In markedcontrast to primaryfibroblasts, established but nontumorigenic lines such as NIH3T3can be readily transformed by a cytoplasmic oncogenewithout the mediation of a nuclear oncogene.This has led to the concept that the nuclear oncogeneprovides an "immortalization" function, while the cytoplasmiconcogenecompletesthe transformationprocess. Theconceptis an oversimplification, however,because transfection with a nuclear oncogenedoes not necessarily permit primary cells to growindefinitelyin vitro. Studieswith transgenicmicepermitdetailed investigationof the validity of the complementing oncogenemodelfor different cell lineages. Will a particular oncogeneprecipitate transformation as soon as expression is turned on, or does the disease progress in multiple stages? Are some transgenic strains essentially tumor-freeuntil crossed with a strain harboringa different oncogene?Cancells frompretumorigenictransgenic mice be readily grownas immortallines in vitro, or are they only able to do so if supplied with another activated oncogene?Are certain mousestrains moreprone than others to developingtumors from the action of a particular oncogene,and if so, do they harbor a susceptible proto-oncogene?

VIRAL ONCOGENES Introduction of a viral oncogeneinto the mousegermline opens up the possibility of testing its potential for transforming cell typesnot accessible to infection by the virus. Moreover,oncogenesisolated from viruses of other speciescan readily be investigated.

SV40 T Antigen Thefirst geneshownto exhibit oncogenicpotential in transgenic micewas the T antigen of the monkeypapovavirus SV40(30). The SV40virus transforms rodent and humancells in culture and induces tumors in hamsters but only rarely in mice. Newbornhamsters usually develop fibrosarcomasor, after intracerebral inoculation, choroid plexus papillomas, but adult animals can develop a wide range of tumors (30). The completetransformingpotential of SV40has been shownto be due to the large T antigen encodedby the early region. Since T antigen is expressed

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at the plasmamembraneas well as within the nucleus, it mayplay two different roles in the transformationprocess. Transgenicmice that carry the SV40early region linked to its natural regulator invariably developchoroid plexus tumorsat 3-5 monthsof age (30, 31). Thetumorsarise fromepithelial cells lining the ventricles of the brain. Tumordevelopmentdependson the presence of the SV40enhancer and the large T antigen codingsequencew~thinthe transgene, but not the small t antigen (31). Since most foundermicedevelopedthe samedisease, the chromosomallocation of the SV40antigen gene did not in general influencethe onset of disease (see below). T antigen wasreadily detected in tumorsbut not in unaffected tissues or in susceptible tissue prior to overt pathology.Withinthe tumors, the transgenic DNAwas usually amplified or rearranged. Nontumorigenic changes in SV40T antigen mice included thymic hypertrophyand kidney pathology;these tissues exhibited lowerlevels ofTantigen expressionthan did the choroid plexus tumors. It is not clear whetheramplified expression is sufficient to provoke tumorsor merelya prerequisite. Noris it knownwhychoroid plexustissue is permissiveto T antigen activation. TheSV40enhanceris thought to be inactivated during early development,and its reactivation in vivo maybe a rare event, dependentuponcell type. Reactivation mayreadily occur in vitro, however,becausemost apparently normaltissues from the SV40T antigen micereadily producedimmortalizedcell lines whenput into culture (30). Oneline of mice bearing an SV40T antigen gene remainedessentially free of tumors(32, 33). Significantly the level of expressionof T antigen in these micewas muchlowerthan in the other lines, both in vivo and in vitro (33), presumablydue to the chromosomal location of the transgene. Thecrucial check to tumor formation in this line may,however,be the immune response (32). Withthe low-incidencestrain, in whichonly 1 57 micedevelopeda choroidplexustumorwithin the first year of life, most mice mounta strong cytolytic T cell response after immunizationwith SV40virus. The developmentof cellular immunitypresumablyoccurs in response to chronic stimulation by the low levels of endogenousSV40T antigen expressedat the surface of cells. In markedcontrast, micewithin a strain with the normalhigh susceptibility to tumors failed to mount either a humoralor a cytotoxic response whenimmunized with virus, even though their response to vaccinia virus was normal. Suchstrains thus appearto be tolerant of T antigen, presumablybecauseantigen is expressed early in development.The high incidence of choroid plexus tumors thus appears to dependon the inability of mosttransgenic SV40T antigen mice to carry out immune surveillance.

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SV40 T Antigen Driven by Tissue-Specific Regulators TheSV40T antigen appears to be a potent tumorigenicagent for manycell types. Attachmentof a different regulatory sequenceavoids the negative regulation imposedby the S¥40 enhancer on transmission through the germlineand enablesexpressionto be directed to specific cell types. ~T/a" ANTIGEN Underthe control of the metallothionein enhancer, 13 of 16 primary transgenic mice exhibited a demyelinatingperipheral neuropathy associated with excessive numbersof Schwanncells (34). Myelination in the central nervoussystem wasapparently normal.Manyof the transgenic mice developedhepatocellular carcinomas(11 of 16) and islet cell adenomas (8 of 16), but only one exhibiteda choroidplexuspapilloma. Thosemice having a mild form of neuropathy and living long enoughto producelitters provedto mosaic.Their transgenic offspring all exhibited the severe peripheral neuropathy, all had hepatocellular carcinomasand 3 of 6 hadislet cell adenomas. ~t-CRYSTALLIN/T ANTIGEN Underthe

control of the murine ~-crystallin promoter/enhancer, transgenic SV40T antigen induced highly invasive lens tumors(35), eventhoughthis tissue has never beenobservedto become malignant under "natural" circumstances. T antigen expression and dysplasia wereobservedas early as midgestation, but it generally took 2 to 3 monthsfor invasive growthto occur outside the lens capsule. Whether the onset of malignancyrequired additional genetic changeis not clear. rNSULrN/T ANTIGEN Underthe control of the insulin regulatory region, S¥40T antigen transgene expression occurred exclusively in the fl pancreatic cells and culminatedin fl-cell tumors (36). On a normaldiet, transgenic mice frequently died suddenlyfrom hypoglycemia precipitated by hyperplasia of pancreatic fl cells. Thosespared by a high sugar diet succumbedlater (10-20 weeks)to pancreatic tumors. Presumablya second genetic alteration had occurred in such tumors, becauseno morethan 4 or 5 of the ~ 100 hyperplastic islets in a pancreas becameneoplastic. Lines developing "autoantibodies" against T antigen developed tumors considerablylater than did a line with no "autoantibodies." Again, this suggestsa role for immune surveillance (37). ELASTASE/T ANTIGEN Underthe

control of the elastase promoter region, SV40T antigen producedtumors of the exocrine pancreas (38). A marked hyperplasia wasevident in the pancreas within 17 days of gestation, but newbornmice retained pancreatic function and the DNAcontent of the cells wasdiploid. Onlyone monthlater, however,most cells had become tetraploid. The pancreas was soon riddled with nodules containing

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aneuploid cells, and the chromosome numbervaried betweennodules. The cells within these apparently independentclones were tumorigenicwhen transplanted. Theseresults suggesta role for SV40T antigen in the induction of karyotypicinstability via tetraploidization. Tumoronset maybe provoked by the subsequent loss of specific chr.omosome(s),perhaps becausecritical anti-oncogeneproducts are then either reducedin concentration or absent. T Antigen of Human Papovaviruses JCV and BKV The ubiquitous humanpapovaviruses JCV and BKVare structurally related to SV40.Themost divergent region of the three genomesis that encompassingthe region of replication and the enhancersequences, but homologybetween the T antigen genes is considerable (70-80%). The pathologyinducedby these viruses has beenreviewedby Smallet al (39). BKVreplicates preferentially in the kidney, producinga subclinical infection. Wheninjected into newbornhamsters, this virus causes brain tumors,insulinomas,and osteosarcomas. It is significant that in transgenic mice, the early region of BKVinducedrenal and liver tumors after 8-10 months(39). In humans,JCVhas beenlinked to the fatal demyelinatingdisease found in certain immunodepressed patients, someof whomalso develop glial tumors.Whilethe virus has not beenprovento cause these tumors, it does induce tumors in tissues of neural origin wheninjected into newborn hamsters. Significantly, transgenic mice mosaicfor the JCVearly region were found to develop metastasizing adrenal neuroblastomas(39). Those that were not mosaicdid not live long enoughto developtumors, because they developeda severe shaking disorder causedby dysmyelinationin the central nervoussystem(40). Thus, for both viruses, the pathologyinducedin transgenic mice by T antigen closely paralleled that thought to occur in immunosuppressed humansas a result of viral infection. Transgenicmice maythus provide excellent modelsfor humanvirus-induced disease. Bovine Papillomavirus Bovinepapillomavirus(BPV)(8) causes benignfibropapillomasin cattle. Ahigh proportionof infected animalsgo on to developtumorsif they graze on brackenfern, whichmayprovide a synergistic factor for oncogenesis. Certain other species, including C3HeB mice, developfibroblastic tumors wheninfected with the virus. Thegene(s) responsible for the transforming potential of the virus have not been fully characterized but are known from in vitro studies to reside within a fragmentcomprising69%of the genome.

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Aline of transgenic micehas beenestablished from an animalinjected with a plasmidcarrying a partial tandemduplication of the completeBPV1 genome(8). Themice display a dramaticpropensity to developmultiple skin tumors, starting at about 8 monthsof age. Thetumors, whicharise frequently in areas prone to abrasion and wounding,are characterized by disorganizeddermallayers. In addition, the skin is abnormallythickened over wide areas. Bycontrast, 15 lines of transgenic mice harboring the BPV-69%-transforming region failed to develop tumors whenthe mice weremonitoredfor 10 months(Palmiter& Brinster, cited in 8), so the 31% region presumablycontributes an essential function for transformationin vivo. The long latency prior to transformation is interpreted to mean that additional genetic change(s)are required to precipitate oncogenesis. Extrachromosomal replication is probably a crucial factor because the tumors harbor BPVDNA in episomalform, as well as an amplified number of integrated copies. Theintegrated transgenewasunchanged in unaffected tissue. Studies like these with humanpapillomavirusDNA mightprovide valuable animalmodelsfor cervical cancer.

CYTOPLASMIC

CELLULAR

ONCOGENES

Ras The family of ras proto-oncogenesencodes membrane-bound cytoplasmic proteins believedto transducethe proliferative signals deliveredto the cell membrane by growthfactors and mitogens(for a recent review, see 41). Mutationof a c-ras gene at certain critical positions converts it into a potent transforminggene for NIH3T3 fibroblasts. Similarly mutatedgenes have been identified within manyhumanand animal tumors. Recent transgertic mousestudies have dramatically underlined the transforming potential of mutatedras genes. ~LASrASE/c-Ha-RAS Transgenicmice were producedcarrying either a normal or a mutated c-Ha-ras gene driven by the elastase promoter (42). Expressionof the transgenesprovedto be specific to acinar cells, but the level wasonly a fewfold higher than that of the endogenous Ha-rasgenes. Withthe unmutatedHa-rastransgene, no tumorsdeveloped.Nevertheless, anaplastic changeswereobservedin the pancreas of adult animals after about 11 months. Withthe mutatedgeneencodingvaline at position 12 instead of glycine, the results weredramatic. Theonly transgenic pupsto survive (5 of 19) were probably mosaic--the other 14 were dead or moribundat birth. The surviving mice all eventually developedpancreatic tumors. Whenanother

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20 transgenic micewereexaminedas fetuses, 18 had an abnormalpancreas. Dysplasiawasapparentas early as 14 days of gestation, the time expected for onset of elagtase-drivenexpression.Byday20, large-scale disruption of pancreatic architecture had ensued:Islets wereno longer recognizable, and insulin-containing cells werescattered throughoutthe cellular mass. Differentiation of the acinar cells appearedto be blocked.Whilethe pathology resembledthat of a carcinoma,the acinar cells maynot have been malignant:There wasno evidenceof aneuploidy,invasiveness, or metastasis, and the cells did not initiate tumors in syngeneic or nude mice. Thus, the disease maymore properly be regarded as a lethal polyclonal proliferation than a malignancy. If the mosaicelastase/ras micecontainedevena fewtransgenic cells in the pancreas, howdid they survive for so long without developing a pancreatic carcinoma? One obvious possibility is that the level of expression mayhave been lower. Anotherpossibility is that ras has a muchmoreprofoundeffect in embryonicthan adult cells. A third, very intriguing, suggestion (42) is that the transformedphenotypewas suppressedin the presenceof an excessof normalcells, as postulated by Land et al (29) for fibroblast transformation. WAP/c-Ha-RAS TOinvestigate the consequencesof expressionof a ras oncogene in mammary epithelial cells in response to lactogenic hormones, transgenic lines were constructed bearing a mutatedHa-rasgene driven by the promoterregion of the gene encodingmurinewheyacidic protein (Wap)(43). Twolines exhibited no expression, but two others derived from female founders showedtissue-specific expression. A low level of Wap-rasRNAwas detected in the mammary glands (and brain) of lactating females, and no expression could be detected after lactation terminated.Maleswithin these lineages did not express the transgene. After 11 months, one lactating founder female developedmammary tumors that expressed elevated levels of Wap-rasRNA. In one unusual line, expression was obtained in the salivary gland of males, presumablydue to the unusual location of the transgene (the chromosome).Five animals developed adenocarcinomasof that gland, but only after 9 months; this suggests the need for additional genetic change. Again, Wap-RasRNAlevels were higher in the tumors than in nonmalignantsalivary tissue from the sameanimals. The transgene was neither amplifiednor rearrangedin the tumors,and its transcription rate was unchanged.Increased expression maythus have beendue to an alteration in mRNA stability. MMTV/v-Ha-RAS Thirteen strains of mice wereproducedcarrying the v-Ha-

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ras oncogene under the control of the mousemammarytumor virus (MMTV) promoter (78). As expected, the transgene was predominantly expressedin the mammary tissue and in the salivary glands, with variable expression in spleen, thymus, lung, seminal vesicle and the Harderian gland. Expression in the Harderian gland provokedmassive though nonmalignant hyperplasia, resulting in severe secondary bilateral exophthalmia. Most (10) strains developed adenocarcinomasof the mammary and salivary glands and, occasionally, lymphomas. The stochastic nature of tumor onset and the apparent monoclonality of the tumors strongly suggestedthat additional genetic changeshad occurred prior to the onset of malignancy. In summary,a transgenic mutant ras gene can provokeabnormallevels of proliferation in several different cell types. Theseverity of disease apparently dependson the level of expressionand cell type. Theonset of true neoplasia usually (perhapsalways)requires additional change. Transgenic Hernopoietic

Growth Factor Gene

Theproliferation, differentiation, and functionalactivation of the myeloid hemopoieticcell lineagesis controlled by a groupof glycoproteinsreferred to as colonystimulating factors (CSFs). The granulocyte/macrophage CSF (GM-CSF) stimulates proliferation and differentiation of granulocytes, macrophages,and eosinophils (44). Autocrinestimulation of factor-dependent but nontumorigenicimmortalizedcell lines renders themmalignant (45-47).Thus,it is of the greatest interest to ask whethertransgenicmice programmedfor constitutive CSFproduction are predisposed to tumor development. Twotransgenic mouselines were generated that contained the murine GM-CSF gene expressed from a retroviral LTR(48). The transgenic mice had elevated levels of GM-CSF in the serum, urine, peritoneal cavity, and eye. Striking pathogeniceffects ensued. Theeyes of newborntransgenic pups were opaque, due to an abnormalaccumulationof activated macrophages;the associated retinal damageled to blindness. Theperitoneal and pleural cavities accumulatedactivated macrophages in large numbers.The mice died at 2-4 months, and a high percentage exhibited progressive muscle wasting. Histological examination revealed macrophage-containing lesions in their striated muscletissue. Expressionof the transgene wasdetected in the peritoneal cell populationand in striated muscleand eye tissue harboring macrophagelesions. No tumors have yet been detected, but the severity of disease maynot allow sufficient time for the accumulationof additional genetic change(s).

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NUCLEAR

& ADAMS

CELLULAR

ONCOGENES

Fos The c-los proto-oncogene encodes a nuclear polypeptide implicated in the control of cell proliferation. One of the earliest responses of manycell types to growth factor or mitogenstimulation is a transient burst of c-fos synthesis. The gene mayhave other roles, however, since high constitutive levels have been found in extra-embryonic tissues and in macrophages(for a review, see 49). The gene was first identified as a viral oncogene. Two independent isolates of murine osteosarcoma viruses carry sequences homologous to c-fos: FBRmurine sarcoma virus, in which the transforming protein differs from c-fos mainly by a frameshift mutation at the C-terminus, and the more aggressive FBJ-murine sarcoma virus, which encodes a greatly altered version of the protein. Both viruses induce chondro-osseoussarcomas when injected into newborn mice. A mutation within the evolutionarily conserved central portion is necessary for immortalization, while COOHterminal changesare critical to their transforming properties (50). Transgenic mice have been produced bearing a murine c-fos gene controlled by the humanmetallothionein promoter/enhancer (51). Twoconstructs were used. Onehad the natural 3’ untranslated region of the c-fos gene, which contains a mRNA-destabilizing sequence. This region was replaced in the second construct by the 3’ LTRfrom the FBJ virus. In eight transgenic strains involving the natural 3’ end, very little expression could be detected, except after treatment with cycloheximide, which is thought to inhibit degradation of c-fos mRNA. In contrast, two of the five transgenic lines carrying the 3’ LTR-modifiedgene exhibited high levels of expression in pancreas, kidney, brain, heart, muscle, and lower levels in lung and salivary gland. Liver expression had been expected but was absent. While none of the animals developed tumors within the first 8 months, a marked abnormality in bone formation became apparent soon after birth. Swollen tibiae were evident in some2-3 weekold mice. Bonelesions were subsequently found in other mice by X-ray analysis. The swellings did not increase after 4 weeks of age. Histological analysis revealed a disturbance in the normal process involved in bone remodelling, with much more bone formation than bone resorption. The level of c-fos expression probably determines the severity of the disease, because a c-fos gene linked b promoter induced multiple swellings in all leg bones, not just to the H-2K the tibiae. These results point to a significant role for the normalfos gene in regulating bone morphogenesis. In the MT/c-fos mice, none of the other tissues expressing the transgene

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exhibited detectable abnormalities. However,H-2/c-fos miceexhibit perturbations of lymphopoiesis (E. Wagner,personal communication), quantitative effects maybe involved.ThethymicT cells exhibiteda marked depressionin the proportionof cells bearingboth L3T4and Lyt-2 markers. Dueto a defect in T cell maturation, these mice are immunologically incompetent. Thevery early onset of the bonelesions inducedby the c-fos transgenes, togetherwith their intriguing similarity to the frank osteosarcomas induced by v-fos bearingretroviruses, suggeststhat the lesions can be regardedas a hyperplastic state from which tumors could arise should additional genetic changetake place. Indeed,tumorshavearisen in someof the older c-fos mice. The ready induction of tumors by the retroviruses probably reflects the structural differences betweenv-fos and c-fos and/or a higher expressionlevel. Myc The c-mye proto-oncogeneis a nuclear phosphoproteinwith DNA binding activity. Its function remainsunclear but almostcertainly involves regulation of cell proliferation (52). Arapid increasein my¢expressionoccurs whenresting (Go)cells of several lineages are stimulatedto enter the cell cycle by mitogensor growthfactors. Recentevidencesuggests that c-myc protein plays a role in DNA synthesis (53). The fundamentalmechanism releasing the oncogenicpotential of c-myeis believed to be deregulation of its expression (54). Nochangein the sequenceof the polypeptide required. Totest the hypothesisthat deregulatedc-mycexpressionpredisposesto neoplasia, several strains of transgenic mice have been developedthat carry c-myelinked to different regulators (55, 56). Micebearinga normal mycgenetogether with several kilobases of 5’ and 3’ flanking DNA failed to developtumors(56). In contrast, those in whichmycexpressionhad been subjugated to a strong heterologouscontrol region exhibited a dramatic predisposition to tumors (55-57). Diverse tumor types were provoked, dependingon whichtissues expressedthe deregulatedgene. Beforereviewing these malignanciesin detail, however,it is worthnoting that a c-rnyc oncogenemaynot provoketransformation of all cell types. Thus, tumors did not developin lung, pancreas,and salivary gland expressingrelatively high levels of a deregulatedmy¢gene(42, 57). MMa’V/r,~YC Several strains of mice were developedthat bear a humancmyegene linked to the LTRof the mousemammary tumor virus (MMTV), whichprovides a hormonallyinducible promoter (55). The tissue distribution of expression varied betweenthe lines, presumablydue to the

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influence of the chromosomal insertion site. In mostlines, expressionwas restricted to the breast, salivary gland, and occasionallythe testis. Two multiparous founder animals developed malignant adenocarcinomasof the breast. The transgene was not amplified in the tumors but was expressed at higher levels than the endogenousc-mycgenes. In the strain studied in detail, all femaletransgenic offspring developedbreast tumors during their second or third pregnancy. Thus, mycexpression stimulated lactogenically appears to predisposethe mice to the onset of mammary adenocarcinomas. Not all the mammary glands in an individual becamemalignant;this strongly suggests that additional genetic changeis required(55). One exceptional MTV-myc lineage proved to express the transgene in diverse tissues, includingtestis, pancreas,lung, spleen, liver, and(in low amounts)kidney. This strain allowedan assessmentof the range of tissues susceptible to transformation involving c-mye (57). Some50%of the animals developedtumors, with a meanlatency of 14 months. Thevariety wasstriking: testicular tumors, B and T lymphomas, and mastcell tumors, as well as breast adenocarcinomas.Mice carrying a murine c-myc gene coupledto the SV40promoter/enhanceralso developeda range of tumors: a lymphosarcoma,a renal carcinoma, and a fibrosarcoma (56). Clearly deregulatedexpressionpredisposesdiverse cell types to becomemalignant. IG ENHANCER/MYCThe strong

association between c-myc deregulation and lymphoidneoplasia (54) provided a compellingcase for using transgenic mice to test the consequencesof constitutive mycexpressionwithin the lymphoidlineage. Twotransgenes were utilized, one involving the immunoglobulinheavy chain enhancer (E/~) and the other the kappa enhancer (Ex) (56). E#-myc geneused had been isola ted from an unusual plasmacytomain which 2.3 kb of the E# enhancer region had inserted just upstreamfrom the c-myc promoters (58). The Ex-mycgene wasa synthetic construct in whichmycexpression wascontrolled by the x light chain enhancerligated to the SV40promoter.TheE#-myctransgene proved remarkably potent: At least 13 of 15 primary mice developed lymphomas within a few monthsof birth. Thus, the heavy chain enhancer apparently dominatesexpression in manychromosomal contexts. The Exmyctransgene also induced lymphomas, albeit with lower frequency(6 of 17 primarymice) and longer latency. The lymphoidspecificity wasabsolute; no other tumor types were found either in the original study or subsequently. The high predisposition to tumor developmentin E#-mycmice has proven highly heritable. The mice typically succumbto a disseminated lymphoma affecting most lymphnodes, the spleen, and often the thymus;

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the disease is accompaniedby leukemia. All tumors so far analyzed (> 50) have proven to be of B lymphoid origin, even those primarily involving the thymus. All exhibit B lineage surface markers and immunoglobulin gene rearrangements; none carry Thy-1; and only one or two have undergone rearrangement of the a or fl T cell receptor loci. The absence of T cell tumors was initially surprising, since a complete immunoglobulin# transgene is expressed in T as well as B cells (59). But no expression of the Et~-mycgene has been detected in T cells (see below). A minority of the E#-rnyc tumors are comprised of mature B cells expressing sIg, while the rest can be assigned to different pre-B stages, exhibiting either D-J or V-D-J heavy chain rearrangement. None so far studied represents the very early maturation stage that lacks any heavy chain rearrangement. Most are relatively easy to establish as permanent lines in culture, and manyof the pre-B cell lines maturein vitro to sIg + B cell lines (A. Harris, W. Langdon, S. Cory, unpublished). The rearrangement patterns in certain mice suggested that maturation also sometimes occurs in vivo. Thus, myc-induced tumorigenesis need not prevent some subsequent differentiation. Compared to B lineage tumors generated by retroviruses bearing a variety of other oncogenes,including v-abl, a significant difference in the phenotype of El~-myc tumors is the absence of the 6C3 surface marker (W. Langdon, unpublished). This antigen (60) is also expressed at levels on stromal cells that support pre-Bcell proliferation and is speculated to be a growth factor or receptor (61). Tumors produced by myc retrovirus also lack this marker (62). Thus, it seems likely that myc-induced tumorigenesis involves a pathway different from that imposed by other oncogenes. Another distinctive feature of most E#-myctumors is the lack of the Lyl marker (W. Langdon, unpublished). This antigen is present on the surface of most conventional B lymphoid tumors and also on nontumorigenic B cell lines derived from normal mice (63). Most conventional lines therefore apparently derive from the minor, seeminglydistinct, Ly 1 + subset of B cells (64), whereas El~-mye-mediatedtransformation may garner a morerepresentative sample of all B lineage cells. To date, no plasmacytomas have arisen spontaneously in E#-mye mice, despite the fact that conventional plasmacytomas all carry chromosome translocations produced by recombination of the c-myc locus with an Ig heavy chain locus (54). This paradoxical observation maysimply reflect the different genetic background; the E#-myc gene is on a C57BL/6× SJL/J background, whereas plasmacytomas usually arise in BALB/eor NZB mice. Nevertheless, it maybe possible to induce plasmacytomasin E#-rnyc mice by providing the appropriate milieu. Experiments are currently in

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progress (A. Harris, unpublished results) to determine whether pristane injection, which induces granulomas, will promote plasmacytoma formation in El~-myc mice as it does in BALB/cmice. E/z-myc induced tumorigenesis involves additional genetic change Despite the almost invariant onset of tumors in E#-mycmice, several observations argue that deregulated myc expression is almost certainly not the only critical factor. First, the lymphoidtissues of young E#-mycmice lacking any overt sign of disease express the transgene at the samelevel as do E#myc tumor cells (65). Nevertheless, in contrast to the tumor cells, they do not provoke malignancy on transplantation (66). Second, while the transgene disturbs B cell development in all E#-myc mice, even before birth (66), lymphomasarise sporadically--typically between one and six months of age (56, 67). Third, the pattern of immunoglobulin gene rearrangement indicates that nearly all the lymphomasare monoclonal (56). Finally, and most definitively, DNAfrom several of the El~-myc tumors transforms NIH3T3fibroblasts (S. Cory, W. Alexander, J. Adams, unpublished results). Since the EIz-myc gene does not transform NIH3T3 cells, these tumors must harbor an additional activated oncogene, presumably one that can collaborate with c-myc. The nature of the transforming gene(s) is presently under study. While all E#-myc mice eventually develop tumors, a minority succumb faster than most, and another group lag conspicuously. It seems possible that the variation in rate of onset reflects the variable genetic background of the mice. Because transgenic mice are produced more efficiently from hybrid eggs (2), each founder was an F2 derived from the C57BL/6and SJL/J strains, and the major colony has been maintained as an F2 pool. Preliminary backcross experiments (A. Harris, unpublished results) have revealed a major influence of strain: E#-myc mice of a largely C57BL/6 backgroundall still develop tumors, but the rate of onset is significantly lower. It appears relevant that old SJL mice can develop a lymphoid malignancy,perhaps as a result of a single recessive trait (68). The pre-neoplastic state in E#-mycmice To investigate howconstitutive c-myc expression establishes a high predisposition to malignancy, we analyzed B cell lymphoid differentiation in prelymphomagenic EIz-myc mice (66). The most conspicuous change was a remarkable polyclonal expansion of pre-B cells, somewhatat the expense of mature B cells. By 18 days of gestation, the proportion of pre-B cells in the fetal liver was already twice that in normal littermates. The number of pre-B cells increased dramatically after birth, reaching a plateau of about 65%of the bone marrowcells at 3 to 4 weeks of age. Pre-B cells were also found in the spleen; this is not the case in normal mice. The cells mayhave been

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generatedin situ as a result of increased splenic hemopoiesis.It is significant, however, that pre-B cells were not detected in the lymph nodes or thymus, even though these are the most commonsites for tumors. Thus, acquisition of the ability to invade the lymph nodes and thymus may be a critical step in progression towards malignancy. No significant changes were detected within the thymus of prelymphomagenicEl~-myc mice: The proportions of the four major T cell populations were unaltered. Because of reduced numbers of sIg + B cells, the proportion of T cells in the lymph node was greater than normal. The size of these cells was normal, however. Both the pre-B and B cells in the El~-mycmice were notably larger than most equivalent cells in normal mice. Late pre-B cells are thought to enter a quiescent nonproliferative phase as they mature to B cells. In El~-myc mice, however,no small pre-B or B cells were detectable. In addition, more than twice as manyof the pre-B and B cells were actively cycling in E#myc than in conventional mice. Since the analysis did not distinguish betweennoncycling cells and those traversing G1, it is conceivable that as a result of constitutive c-myc expression, none of the cells ceased cycling prior to their death. A large proportion of the cycling cells must in fact die to account for the steady state level found in adult El~-mye mice. The factors regulating the plateau level are not known,but one obvious possibility is that the supply of the requisite growthfactor(s) is limited. Overall, the total numberof pre-B cells in El~-myc mice was elevated over 4-fold, while the numberof mature sIg + B cells was depressed about 30% (66). Moreover, fewer splenic B cells had matured to surface IgD expression. These observations raised intriguing questions regarding the immunologicalcompetenceof B cells continually subjected to constitutive myc expression. Et~-mycB cell function was therefore investigated both in vitro and in vivo (68). While in vitro stimulation with either mitogens antigens led to proliferation and antibody production, the frequency of responsive B cells in E~-myccultures was only 30%that of B cells from control cultures. In vivo responses were sometimesdelayed but eventually reached normal levels, and switching from IgM to IgG production occurred. Thus, deregulated myc expression appears to retard but not prevent B cell differentiation; consequently, immunologicalfunction is not grossly impaired. To account both for the increased numberof cells early in the B lineage in Et~-mycmice and for the maintenance of at least a high proportion of the cells in cycle, it was proposed that c-myc normally plays an important role in control of differentiation as well as mitogenesis (66). The level c-myc expression was envisaged to set the balance between self-renewal and maturation within a cell lineage, the maturation requiring decreased

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levels. Constitutive expressionwouldthus favor self-renewal. Additional evidencesupportingthis modelis that deregulated mycexpressionwithin mouseerythroleukemiacells blocks the terminal differentiation induced by dimethylsulfoxide (69, 70). The model maynot hold for all lineages, however, because differentiated keratinocytes maintain high myc expression(71). All changesnoted in the E#-mycmice reflect alterations within the B lymphoidcompartment,consistent with evidence that the transgene is apparentlynot expressedin other lineages (65). It is significant that the level of transgene expression was found to be comparableto that in activated normallymphocytes.Thus, constitutive expression at physiological levels of a normalcellular genecan elicit all the profoundbiological effects cited above. This conclusion dramatically underlines the importance of understanding the molecular mechanismthat normally regulates c-myc expression. Astriking feature of all E#-mycB lymphoidcells, whetheror not they have becometumorigenic,is that the endogenousmycalleles are transcriptionally silent (56, 65), as is the untranslocatedmycallele in plasmacytomasand Burkitt lymphomas (72, 73). These results strongly support a negative feedbackmodelfor control of mycexpression(74, 75). A myc polypeptide concentration above a threshold level (which may vary betweenlineages or differentiation stages) is hypothesizedto repress further myctranscription, either directly or indirectly. TheE#-myctransgene, like a translocated mycallele, presumablyis recalcitrant to this repression. In viewof the immortalizingrole ascribed to the c-rnyc oncogene,it was surprising to find that pre-neoplasticE#-mycB lineage cells wereno easier to growin culture than their normalcounterparts. Whencultured under conventional conditions, with no source of growth factors other than serum, E#-myepre-B marrowcells in fact died more rapidly than their normal counterparts (W. Langdon, unpublished results). Moreover, splenic B cells from E#-mycmice did not display unlimited proliferative capacity, even in the presence of mitogens and rich sources of growth factors (68). Thus, the description of my¢as an immortalizinggene may be an oversimplification. At present, E#-mycpre-B cells can only be maintainedin vitro within Whitlock-Wittecultures (W. Langdon,unpublished results), in which unidentified growth factors are provided by an adherent stromal layer grownfrom normal bone marrowcells. In such cultures, the transgenic cells were again found to be larger than normalpre-B cells in parallel cultures. At least twiceas manywerein cycle, but theygrewto cell densities only slightly higher than those of their normalcounterparts, presumably

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because growth factor levels were limiting. Whenisolated from bone marrow,manyEla-myc pre-B cells display Ia (66), a marker normally confined to more mature cells within the B lineage. After one weekin culture, however,Ia expressionwaslost, perhapsbecausethe cultures lack factor(s) essential for Ia expression.Thus, all evidenceto date suggests that B lymphoidcells constitutively expressing c-myc maintain their requirement for growth factors, although perhaps at somewhatreduced levels (see also 76). Theseresults contrast markedlywith the report that infection with a v-myccontaining retrovirus abrogates the factor dependenceof certain hemopoietic cell lines (77). It is of great interest to see whetherthe entire E~-myctumorigenic processcan be reconstructedin vitro. Thecells in several long-termcultures of transgenic marrowcells markedlychangedtheir growthcharacteristics after several monthsof growth(W.Langdon,unpublished), reaching 10fold higher saturation densities but still requiting the stromallayer. One such culture finally becamefactor independentand tumorigenic.Thus, the events required in vivo for onset of tumors maywell include mutations subvertingfactor requirements. In summary,analysis of prelymphomagenic E#-mycmice has provided someinsight into whydysregulated mycexpression creates tumor-prone mice. Whenimposed on the B lymphoid lineage, constitutive myc expressioncauses a 4-fold elevation in cell numbers,and morethan twice as manycells are replicating. Since the total numberof divisions has been increased by an order of magnitude,the risk of genetic accident is also significantly increased. Similar conclusions presumablyhold for other lineages expressing a deregulated myc gene. The immunoglobulin rearrangement mechanismcould be an added risk factor for lymphoid cells if, as in E#-mycmice, the expandedpopulationis primarily composed of cells actively undergoingDNA rearrangement.These factors maynot, however,fully account for the very high predisposition to tumoronset, and it maywell provethat mycacts moredirectly to increase the frequency of mutation. ONCOGENE

COOPERATIVITY

Theconclusion frommost of the transgenic oncogenestudies discussed is that expressionof one oncogeneis insufficient for malignancyto develop. (Ras oncogeneexpressionin pancreatic acinar cells maybe an exception-see 42.) Thetransgenic miceoffer uniqueopportunitiesto investigate the consequenceof exposingthe relevant cells to the action of additional oncogenes.Oneobvious wayis via retroviral infection. Neonatal mice carrying one oncogeneas a transgene can be infected with a virus bearing

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the secondoncogeneand monitoredfor tumoronset. Alternatively, cells isolated fromthe transgenic mice can be infected in vitro and their subsequent growthproperties monitoredin vitro and in vivo. Studies initiated along these lines with pre-B cells from E#-mycmice are proving most informative (W. Alexander, J. Adams,W. Langdon,S. Cory, unpublished results). Whenneonatal EIz-mycmice wereinfected with a helper virusfree virus carrying a mutated Ha-ras gene, lymphomainduction was greatly accelerated. Analternative strategy to investigate cooperativityis to cross lines of transgenic mice that express different oncogeneswithin the sametissue. Myc/rascooperativity has been demonstratedin breast epithelial cells by breeding a strain of MMTV-ras transgenic mice with an MMTV-myc line (78). The latency for onset of mammary carcinomas was considerably reducedbut their time of onset wasstill variable and the tumorsdid not involve the entire breast. Thusadditional somatic events appearedto be required for the onset of malignancyeven in the presence of both a deregulated mycgene and an activated ras gene. CONCLUSIONS

AND FUTURE

PROSPECTS

It is alreadyclear that transgenicmicewill substantially deepenour understanding of oncogeneaction. The efficacy of several genes as tumorpromotingagents in the living animalhas been demonstratedconclusively. In general, activity of morethan one cancer-promotinggene seemsto be required for the emergenceof a tumorigenic clone. The myc and ras oncogeneshave been shownto collaborate in the transformation of two cell types. Wecan look forwardto seeing the results of a great variety of such experiments, and these should establish the "rules" for oncogene cooperativityin diversecell lineages. Theclear demonstrationof an altered but preneoplastic phase in the transgenic animalsis particularly important. Duringthis stage, the biological effect of the primaryoncogenecan be analyzed.Moreover,attempts can be madeto alter the courseof the disease and perhapsevento prevent the onset of malignancy.Just as important,the effect of a rangeof chemical agents can be investigated for their tumor-promoting activity. Thecharacterization of additional tissue-specific promoter/enhancer sequenceswill extend the versatility of experimentstargeting oncogene expressionto specific cell lineages. Conversely,it will be of considerable interest to comparethe biological effects of different oncogeneswithin the same cell type. Already a comparisonhas been madeof the effects of mycand ras within mammary epithelial cells (55, 78). Moreover,several different oncogeneshave been coupledto the immunoglobulin heavychain

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enhancer (S. Cory, A. Harris, J. Adams,unpublished results), including N-myc, N-ras, v-myb, v-abl, and bcr-v-abl, a construct mimicking the hybrid oncogenethought to play a central role in chronic myeloid leukemia. Most of these genes have provoked lymphoid neoplasia in transgenic mice. The tumors observed to date include plasmacytomas and T lymphomas, as well as pre-B and B lymphomas. Transgenic mice harboring different growth factor genes will be an area for fruitful investigation, irrespective of whether their deregulated expression provokes tumors. In fact, certain oncogenes maywell prove to be growth factors expressed at very specific times of development: Int-2 has already been implicated as an embryonicgrowth factor (79). Finally, transgenic mice mayprove crucial to analysis of the recessive oncogenes, genes encoding products that normally prevent the emergence of tumors. High-level expression of an antisense copy of such genes in tumorigenic mice might mimic their loss and thereby induce malignancy. Since a strong candidate for the recessive retinoblastoma (Rb) susceptibility gene has been cloned recently (80), anti-Rb transgenic mice may well be the first of such analyses attempted.

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8. Lacey, M., Alpert, S., Hanahan, D. 1986. Bovine papillomavirus genome elicits skin tumors in transgenic mice. Nature 322:609-12 9. Rassoulzadegan, M., L6opold, P., Vailly, J., Cuzin, F. 1986. Germline transmission of autonomous genetic elements in transgenic mousestrains. Cell46:513-19 10. Wagner,E. F., Covarrubias, L., Stewart, T. A., Mintz, B. 1983. Prenatal lethalities in mice homozygous for human growth hormone gene sequences integrated in the germ line. Cell 35:647-55 11. Jaenisch, R. 1976. Germline integration and Mendelian transmission of the exogenous Moloney leukemia virus. Proc. Natl. Acad. Sci. USA73:1260-64 12. Van der Putten, H., Botteri, F., Miller, A. D., Rosenfeld, M., Fan, H., Evans, R., Verma, I. 1985. Efficient insertion of genes into the mouse germ line via retroviral vectors. Proc. Natl. Acad. Sci. USA 82:6148-52 13. Jaenisch, R., J/ihner, D. 1984. Methylation, expression and chromosomal position of genes in mammals. Biochem. Biophys. Acta 782:1-9 14. Stewart, C. L., Stuhlmann, H., J/ihner, D., Jaenisch, R. 1982. De novo methylation, expressionand infectivity of retro-

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27. Land, H., Parada, L., Weinberg,R. A. 1983. Tumorigenicconversion of primaryembryofibroblasts requires at least two co-operating oncogenes. Nature 304:596~00 28. Ruley, E. 1983. Adenovirusearly region 1Aenables viral and cellular transforminggenesto transformprimarycells in culture. Nature304:602~5 29. Land,H., Chen,A. C., Morgenstern,J. P., Parada,L. F., Weinberg,R. A. 1986. Behaviourof mycand ras oncogenesin transformation of rat embryofibroblasts. Mol.Cell. Biol. 6:1917-25 30. Brinster, R. L., Chert, H. Y., Messing, A., Van Dyke, T., Levine, A. J., Palmiter, R. D. 1984. Transgenicmice harboring SV40T-antigengenes develop characteristic brain tumors. Cell 37: 367-79 31. Palmiter, R. D., Chen,H. Y., Messing, A., Brinster, R. L. 1985. SV40enhancer and large-T antigen are instrumentalin developmentof choroid plexus tumors in transgenic mice. Nature 316:45740 32. Faas, S., Pan, S., Pinkert, C., Brinster, R., Knowles, B. 1987. Simian virus 40 SV40-transgenic mice that develop tumoursare specifically tolerant to SV40 T antigen. J. Exp. Med.165:417-27 33. VanDyke,T., Finlay, C., Levine, A. J. 1985. A comparisonof several lines of transgenic mice containing the SV40 early genes. Cold Spring HarborSymp. Quant.Biol. 50:671-78 34. Messing,A., Chen, H. Y., Palmiter, R. D., Brinster, R. L. 1985. Peripheral neuropathies, hepatocellular carcinomasandislet cell adenomas in transgenic mice. Nature 316:461~3 35. Mahon, K. A., Chepelinsky, A. B., Khillian, J. S., Overbeek,P. A., Piatigorsky, J., Westphal, H. 1987. Oncogenesis of the lens in transgenic mice. Science 235:1622-28 36. Hanahan,D. 1985. Heritable formation of pancreatic r-cell tumoursin transgenic miceexpressing recombinantinsulin/simian virus 40 oncogenes. Nature 315:115-22 37. Adams,T. E., Alpert, S., Hanahan,D. 1987. Non-toleranceand autoantibodies to a transgenicself antigenexpressedin pancreaticfl cells. Nature325:223-28 38. Ornitz, D., Hammer,R., Messing,A., Palmiter,R., Brinster, R. 1987.Science. In press 39. Small, J. A., Khoury, G., Jay, G., Howley,P. M., Scangos, G. A. 1986. Early regions of JC virus and BK virus inducedistinct andtissue-specific tumorsin transgenic mice. Proc. Natl. Acad. Sci. USA83:8288-92

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TRANSGENICMICE AND ONCOGENESIS 40. Small, J. A., Scangos,G. A., Cork,L., Jay, G., Khoury, G. 1986. The early region of humanpapovavirus JC inducesdysmyelination in transgenicmice. Cell 46:13-18 41. Barbacid, M. 1986. Humanoncogenes. In ImportantAdvancesin Oncology,ed. V. DeVita, S. Hellman,S. Rosenberg, pp. 3-22. Philadelphia:Lippincott 42. Quaife,C. J., Pinkert, C. A., Ornitz, D. M.,Palmiter,R. D., Brinster, R. L. 1987. Pancreatic neoplasia induced by ras expressionin acinar cells of transgenic mice. Cell 48:1023-34 43. Andres, A. C., Sehonenberger,C. A., Groner, B., Henninghausen, L., LeMeur,M., Gerlinger, P. 1987. Ha-ras oncogeneexpression directed by a milk protein gene promoter: Tissue specificity, hormonalregulation, and tumor inductionin transgenicmice.Proc.Natl. Acad. Sci. USA84:1299-1303 44. Metcalf, D. 1985. The granulocytemacrophage colony stimulating factors. Science 229:16-22 45. Lang, R. A., Metcalf, D., Gough,N. H., Dunn, A. R., Gonda, T. J. 1985. Expressionof a hemopoieticgrowthfactor eDNA in a factor dependentcell line results in autonomous growth and tumorigenicity. Cell 43:531-42 46. Rosenthal,A., Lindquist, P. B., Bringman,T. S., Goeddel,D. V., Derynck,R. 1986.Expressionin rat fibroblasts of a humantransforming growth factor c~ cDNA results in transformation.Cell46: 301-9 47. Hapel, A. J., VandeWonde,G., Campbell, H. D., Young,I. G., Robbins, T. 1986. Generationof an autocrine leukemia using a retroviral expression vector carrying the interleukin-3 gene. Lymphokine Res. 5(4): 249~50 48. Lang, R. A., Metcalf, D., Cutherbertson, R. A., Lyons,I., Kelso, A., Kannourakis,G., Williamson,J., Klintworth, G., Gonda,T. J., Dunn,A. R. 1987. Transgenic mice expressing a haemopoieticgrowth factor gene (GMCSF)develop an accumulationof activated macrophages, blindness and a fatal syndromeof tissue damage.Submitted 49. M/iller, R. 1986.Cellular and viral fos genes: Structure, regulation of expression and biological properties of their encoded products. Biochem. Biophys. Acta 823:207-25 50. Jenuwein,T., M/iller, R. 1987. Structure-function analysis offos protein: A single aminoacid changeactivates the immortalising potential of v-fos. Cell 48: 647-57

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51. Riither, U., Garber, C., Komitowski, D., Muller, R., Wagner,E. F. 1987. Deregulatedc-fos expressioninterferes with normalbone developmentin transgenie mice. Nature325:412-16 52. Kelly, K., Siebenlist, U. 1985. Therole of c-mycin the proliferation of normal andneoplasticcells. J. Clin. Immunol. 5: 65-77 53. Studzinski,G. P., Brelvi,Z. S., Feldman, S. C., Watt, R. A. 1986. Participation of c-myc protein in DNAsynthesis of humancells. Science 234:467-70 54. Cory, S. 1986. Activation of cellular oncogenesin haemopoietic cells by chromosome translocation. Adv. CancerRes. 47:189-234 55. Stewart,T. A., Pattengale,P. K., Leder, P. 1984. Spontaneousmammary adenocarcinomas in transgenic mice that carry and express MTV/mycfusion genes. Cell 38:627-37 56. Adams,J. R., Harris, A. W., Pinkert, C. A., Corcoran,L. M., Alexander,W.A., Cory, S., Palmiter, R. D., Brinster, R. L. 1985. The c-myc oncogenedriven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice. Nature 318:533-38 57. Leder,A., Pattengale,K., Kuo,A., Stewart, T. A., Leder, P. 1986.Consequence of widespreadderegulationof the c-myc gene in transgenic mice: Multiple neoplasms and normal development. Cell 45:485-95 58. Corcoran,L. M., Cory, S., Adams,J. A. 1985. Transposition of the immunoglobulin heavychain enhancerto the myc oncogene in a murine plasmacytoma. Cell 40:71-79 59. Grosschedl,R., Weaver,D., Baltimore, D., Costantini, F. 1984. Introductionof a # immunoglobulin gene into the mouse germline: Specific expressionin lymphoid cells andsynthesisof functionalantibody. Cell 38:647-58 60. Tidmarsh,G., Dailey, M., Whitlock,C., Pillemer, E., Weissman,I. L. 1985. Transformed lymphocytes from Abelson-derivedmice express levels of a B lineage transformationassociated antigen elevated from that foundon normal lymphocytes. J. Exp. Med. 162: 142130 61. Whitlock,C. A., Tidmarsh,G. F., MUller-Sieburg, C., Weissman,I. L. 1987. Bonemarrowstromalcell lines with lymphopoieticactivity expresshighlevels of a pre-B neoplasia-associatedmolecule. Cell 48:1009-21 62. Morse,H. C., Tidmarsh,G. F., Holmes, K. L,, Fredrickson, T., Hartley, J., Pierce, J., Langdon,W.Y., Dailey, M.,

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Weissman, I. L. 1987. Expressionof the line. Nature322:748-50 6C3 antigen on murine hematopoietic 71. Dotto, G. P., Gilman, M. Z., Maruneoplasms. J. Exp. Med.165:920-25 yama,M., Weinberg,R. A. 1986. c-myc and c-fos expressionin differentiating 63. Braun, J., Forouzanpour,F., King, L., Teheranizadeh,T., Bray, M., Kliewer, mouseprimary kerotinocytes. EMBO J. ÷ S. 1986. B-Lyl cells: ImmortalLy-1 5:2853-57 B lymphocytecell lines spontaneously 72. Bernard, O., Cory, S., Gerondakis,S., arising in maturesplenic cultures. ImmuWebb,E., Adams,J. M. 1983. Sequence nol. Rev. 93:5-21 of the murine and humancellular myc 64. Hayakawa,K., Hardy, R., Herzenberg, oncogenesand two modesof mye transcription resulting from chromosome L. A., Herzenberg, L. A. 1985. Progenitorsfor Ly-1Bcells are distinct from translocation in B lymphoidtumors. progenitors for other B cells. J. Exp. EMBOJ. 2:2375-83 Med. 161:1554~58 73. Nishikura,K., Ar-Rushidi,A., Erikson, 65. Alexander,W., Schrader, J. W., Adams, J., Watt, R., Rovera, G., Croce, C. M. J. M. 1987. Expression of the c-myc 1983.Differential expressionof the normal and of the translocated humanconcogene under control of an immunoglobulin enhancer in E#-myctransmyc oncogenesin B cells. Proe. Natl. genic mice. Mol. Cell. Biol. 7: 1436Acad. Sci. USA80:4822-26 74. Leder,P., Battey,J., Lenoir, G., Moulding, L., Murphey,W., Potter, H., Stew66. Langdon,W. Y., Harris, A. W., Cory, S., Adams,J. A. 1986. Thec-myc oncoart, T., Taub, R. 1983. Translocation gene perturbs B lymphocyte develamong antibody genes in human opmentin E#-myctransgenic mice. Cell cancer. Science 222:765-71 47:11-18 75. Rabbits, T., Forster, A., Hamlyn,P., 67. Harris, A. W.,Pinkert, C. A., Crawford, Baer, X. 1984. Effect of somaticmutaM. C., Brinster, R. L., Adams,J. M. tion within translocated c-mycgenes in 1987. The E#-myctransgenic mouse:A Burkitt’s lymphoma.Nature 309: 592modelsystem for high incidence spon97 taneous lymphoblastic lymphomaand 76. Cory, S., Bernard,O., Bowtell,D., Schleukemiaof early pre-Bcell origin. Subrader, J. W.1987. Murinec-myc retromitted viruses alter the growthrequirementsof 68. Vaux,D. L., Adams,J. M., Alexander, myeloidcell lines. Oncogene1:61-76 W.S., Pike, B. L. 1987. Immunological 77. Rapp,U., Cleveland,J., Brightman,K., competence of B cells subjected to Scott, A., Ihle, J. 1985. Abrogationof IL-3 and IL-2 dependence by recomconstitutive c-rnyc oncogeneexpression in E#-myctransgenic mice. J. Immunol. binant murineretroviruses expressingvIn press myc oncogenes. Nature 317:434-38 68a. Bubbers,J. E. 1984. Identification and 78. Sinn, E., Muller, W., Pattengale, P., linkage analysis of a gene, Rcs-I supTepler, I., Wallace,R., Leder, P. 1987. pressing spontaneous SJL/J lymphoma Coexpression of MMTV Iv-Ha vas and expression.J. Natl. CancerInst. 72: 441MMTV I c-myc genes in transgenic mice: 46 synergistic action of oncogenesin vivo. 69. Coppola, J. A., Cole, M. D. 1986. Cell 49:465-75 Constitutive c-myconcogeneexpression 79. Dickson,C., Peters, G. 1987. Potential oncogeneproduct related to growthfacblocks mouse erythroleukemia cell differentiation but not commitment. tors. Nature326:833 Nature 320:760-63 80. Lee, W.-H., Bookstein, R., Hong, F., Young,L.-J., Shew,J.-Y., Lee, E. Y.-H. 70. Dmitrovsky,E., Kuehl, W.M., Hollis, G.F., Kirsch,I. R., Bender,T. P., Segal, P. 1987. Humanretinoblastoma susS. 1986. Expression of a transfected ceptibility gene: Cloning,identification humanc-myconcogeneinhibits differand sequence. Science 235:1394-99 entiation of a mouseerythroleukemia

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Ann. Rev. lmmunol. 1988. 6 : 4943 Copyright © 1988 by Annual Reviews Inc. All rights reserved

KININ FORMATION: MECHANISMS AND ROLE IN INFLAMMATORY DISORDERS David Proud Division of Clinical Immunology,Johns Hopkins University School of Medicine at The Good Samaritan Hospital, 5601 Loch Raven Boulevard, Baltimore, Maryland 21239

Allen P. Kaplan Division of Allergy, Rheumatologyand Clinical Immunology, State University of NewYork, Stony Brook, NewYork 11794-8161

INTRODUCTION Bradykinin and lysylbradykinin are potent vasoactive peptides liberated from ~2 globulins, called kininogens, by the actions of various proteases, known collectively as kininogenases. The pharmacologic properties of kinins--including their abilities to increase vascular permeability, to cause vasodilatation and pain, to contract most smooth muscle preparations, and to stimulate arachidonic acid metabolism--have led several investigators to suggest that these peptides may be important inflammatory mediators in humans(1-3). Only in recent years, however, with improved assay technologies and an increased awareness of the mechanismsregulating kinin levels, has meaningfuldirect evidence to support a role for the kinin system in humaninflammatory disorders begun to accumulate. In the present chapter we review our current knowledgeregarding the three different pathwaysthat maylead to kinin formation in inflammatory events: (a) Generation of bradykinin as a result of the activation of the Hagemanfactor-dependent pathways and production of plasma kallikrein, (b) the generation of lysylbradykinin by tissue kallikreins, and (c) the potential role of cellular proteases in kinin formation. Wealso critically 49 0732-0582/88/0410-0049502.00

Annual Reviews 50

PROUD & KAPLAN

evaluate the evidence of activation of one or more pathways and the involvcment of kinins during inflammatorydisordcrs in humans.

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MECHANISMS

OF KININ

FORMATION

Hageman Factor Dependent Pathway (4) The formation of bradykinin in humanplasmais dependentuponthe interactionof certain negativelychargedsurfaces withthree plasmaproteins; namely,FactorXII (Hageman factor) (5), prekallikrein(6-8), high molecularweight (HMW) kininogen(9-11) (Figure 1). These proteinsare also the initiating factors requiredfor the intrinsic blood coagulationandfibrinolytic pathways.In plasma,prekallikreinandHMW kininogencirculate as a complex with1 : 1 molarstoichiometry (12), while Hageman factor is not complexed.These proteins bind to initiating surfaces, the Hageman factor becomes activated, prekallikreinis converted to kallikrein, andHMW kininogenis digested to release the vasoactive peptidebradykinin(Figure1). A secondmajorplasmasubstrateof activated Hageman factor, factor XI, also circulates boundto HMW kininogen(13); it is convertedto factorXIa(14), whichcontinuesthe intrinsic coagulationcascade. Hageman factor (HF)(coagulationfactor XII) is a single-chainglobulin of molecularweight80,000(15). It is activateduponcontactwithcertain

surfoce

FACTOR Xl HMWKininogen ~ [

"PRO"-KALLIKREIN Intr~cellulorEnzymes ~ Plosmin PIosmoKollik~’~in

~

TISSUEKALLIKREIN(SECRETED)

FACTOR ~IA

LMWKININOGEN1:~] PREKALLIKREIN - ~ KALLIKRF’IN

i COAGULATION

~<~N~NO~N ~ BRAD¥~:!N, IN IH~w

I

LYSYLBRADYKININ (KALLIDIN)

Corboxypeptidose N (C/aN)

l,~m/nopeptidose

DES -ARC9- BRAOYKININ~ )RRAOYKININ)

l

l ngiotensin Converting Enzyme Other Plosmo Proteoses

INACTIVEPEPTIDES,AMINOACIDS Figure 1

Mechanisms of bradykinin formation

by plasma and tissue

pathways.

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KININS AND INFLAMMATION

51

negativelychargedsurfaces, for example,glass, silicates, Lipid Aof endotoxin, dextran sulfate, sulfatides, and heparin. TheHFbecomescleaved (16, 17) and expresses enzymaticactivity against low molecularweight synthetic substrates as well as against a numberof plasmaproteins. As seen in Figure1, the major protease capable of activating Hageman factor i~ kallikrein (18). Otherweakeractivators are plasminor factor XIa, each of whichis formedas a consequenceof Hagemanfactor activation. If one examinespurified preparations of Hageman factor and prekallikrein, minimalenzymaticactivity is seen if precautions are taken to eliminate contaminating activating enzyme. Whenmixed and exposed to macromolecular anionic materials--"surface"--rapid activation of both proteins results. However,surface-dependentactivation of Hageman factor can occur in the absence of prekallikrein, and the activated Hageman factor can convertfactor XI to factor XIa so that coagulationcan proceed. Congenitalprekallikrein deficiency(Fletcher Trait) behavesin this fashion (19, 20). Bycontrast HF-deficientplasmadoes not activate uponcontact with a surface. Thus, Hageman factor is essential for contact activation, while prekallikrein contributes a prominentaccelerating action which, however,is not absolutely required. Hageman factor can autoactivate (21-23), and this mayexplain the gradual evolution of coagulantactivity in prekallikrein deficient plasma. The kinetics of appearanceof activity in purified Hageman factor when exposedto a surface ha~e been analyzed by an iterative computermodel (24), an explicit secondorder kinetic model(25), and by demonstration exponentialinitial rates (22), all consistent with an autoactivationmechanism. Activated Hageman factor (HFa)has been shownto cleave native Hageman factor to generate more HFain the cases of both rabbit (23) and humanproteins (22, 26). The dilemmaposed by an autoactivation mechanism is whereor howthe initial activated moleculesare created so that native HFmaybe digested. Someproposals consideredthe possibility that surface binding of HFcould create an active site by a conformational changeprior to any cleavage (27, 28). Other workers suggested that substrate-induced conformational change occurs in HFwhenHFplus substrate is boundto the surface (29, 30). However, proof of the creation of active sites prior to proteolysisis very difficult to obtain. Therates of activation seen in mixturesof HFand prekallikrein accelerate rapidly due to the positive feedback.Tankersley& Finlayson(24) estimatedthe kinetic constants for the componentreactions and calculated that, at plasma concentrations of HFand prekallikrein, the introduction of a surface in whichonemoleculeof active enzymeis present per milliliter (5 x 10-13 total protein) will yield 50%activation in 13 sec. Since the best preparations of HFhave about 0.02%active enzyme,it is not possible to

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52

PROUD & KAPLAN

achieve or measuresuch infinitesimal activation. Wehave used a rapidly acting chloromethylketone to destroy all traces of activated HFin preparations of HFand reducedenzymaticactivity to less than 1/4200of that of HFa(31). Eventhen, HFcould autoactivate and convert prekallikrein to kallikrein, albeit with a very low initial rate. Wethen proposedthat enzymaticactivity containedin native HF(if any) is too low to measure and that contact activation mayinvolve participation of extremelylow levels of active enzymethat are continuouslypresent. Thepossibility that native HFhas enzymaticactivity analogousto that seen in trypsinogen(32) (comparedto trypsin) is not excludedbut is subject to the aforementioned constraints. Thesurface appears to accelerate contact activation in two ways. It causesa conformationalchangein native HFsuch that its rate of cleavage is increased(33). This suggeststhat boundHFis a better substrate regardless of whetherHFa(autoactivation) or kallikrein effects its cleavage. addition, the catalytic effect of the surface maybe to create a local milieu in whichreversible binding of protein componentsresults in an increase in local concentration,therebyacceleratingtheir rate of interaction. Once someactivated HFconverts prekallikrein to kallikrein, the kallikrein rapidly activates the bulk of HF.Since the latter is far morerapid, this pathwayis kinetically dominant;hence the thickened arrow in Figure 1. Recentevidenceindicates that kallikrein also has to bind to the surface to activate HFwith maximalefficiency (34, 35). The idea of a reciprocal reaction betweenthe zymogenand activated forms of Hageman factor and prekallikrein was complicatedby the realization that yet another component is required for optimal contact activation (36, 37). The plasmaof patients not deficient in either Hageman factor or prekallikrein had a markedlyprolongedkaolin-inducedpartial thromboplastintime and generated no bradykinin. This led to the identification of HMW kininogen as a non-enzymaticcofactor that augments the activation of both prekallikrein and factor XI by activated Hageman factor and increases the rate of Hageman factor activation by kallikrein (38, 39). One critical observation regarding the mechanismby which HMW kininogen functions as a cofactor is the demonstrationthat both prekallikrein (12) and factor XI (13) circulate in plasma bound to kininogenand that these complexesare boundto initiating surfaces via the HMW kininogen moiety. In plasma, HMW kininogen augments the attachmentof prekallikrein and factor XI to surfaces such as kaolin (40, 41). Whenthe HMW kininogenis cleaved, this effect is augmented(42). Thus, HMW kininogen maybe an activatable cofactor (42) although the magnitudeof this effect is uncertain. Adhesiveglycoproteinssuch as

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KtNINS

AND INFLAMMATION

53

fibrinogen may compete with HMW kininogen for binding to initiating surfaces, e.g. antigenic fibrinogen in clot promoting surfaces becomes undetectable after l0 rain when HMW kininogen is present, an effect not observed in HMW kininogen deficient plasma. Perhaps cleaved HMW kininogen is a more effective competitor. Yet in buffer systems (41), prekallikrein and factor XI bind to kaolin equally well in the presence or absence of HMW kininogen, but activation of each by HFa is still accelerated upon addition of HMW kininogen. Thus, aside from quantitative binding effects seen in plasma, HMW kininogen must also place the substrates of activated Hagemanfactor upon the surface in a conformation that facilitates their cleavage. Moredifficult to explain is the ability of HMW kininogen to augment the cleavage and activation of Hageman factor by kallikrein since it has no demonstrableeffect uponthe enzymatic activity of kallikrein (using synthetic substrates) and has no knowninteraction with Hagemanfactor. The dissociation constant for the prekallikrein (kallikrein)-HMW kininogen complex is 15 nM(43), and plasma concentration, about 10-20%of prekallikrein circulates free (44). Thus, kallikrein formed by activation of surface-bound prekallikreinHMW kininogen-complexescan dissociate from the surface. It is then able to digest Hagemanfactor molecules on the same or other particles (41, 45). In this fashion, contact activation is disseminated along the surface. Thus, the effective ratio of kallikrein/Hagemanfactor is increased in the presence of HMW kininogen, and this may account for the augmentation seen. The magnitude of this contribution of HMW kininogen varies depending upon the nature of the surface used and whether the reaction is studied using plasma or purified components. Hageman factor (Figure 2) is initially cleaved within a disulfide bridge so that a single chain of 80,000 is converted to a two-chain enzymein which a heavy chain of 50,000 is disulfide linked to a light chain of 28,000 (46). Noloss in size results, and the enzymeis called HFa(or ~ HFa). The binding site for the surface is on the heavy chain (47), and the light chain contains the active enzymatic site. Further cleavage by kallikrein digests HFaat two sites (2 and 3) in sequence (48) to form HFf (or fl HFa) in which a heavy chain 28,000 is disulfide-linked to a light chain of either 2500 or 500 (64). mixture of these two forms accounts for the doublet typically seen in alkaline disc gels (12) or unreduced SDSgels (16). Enzymesother than kallikrein are able to cleave and activate Hageman factor, including plasmin (17, 33, 49) and factor XIa (18), but efficiencies of these reactions are muchlower. The cleavage of Hageman factor by HFaduring autoactivation is potentially of greater significance CLEAVAGE OF PROTEINS DURING CONTACT ACTIVATION

Annual Reviews 54

PROUD & KAPLAN SURFACE BINDINGSITE HzN ...........................

~

SITE 4 (?) mmHFo---~40,OOOMW i.-~I ~ activefragment ~ I L

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

-I I

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SITE, (~53.__.__~)

,.,N..~Ar V~OI g-

~l / SITE.~2 (334__.~)

SITE~_.(343)

V ~ Thr Mt r r - e-Se-Se-Leu-Ser-Lys-Arg-Leu-Arg-Gly-Cys-Ser-Leu-Pro-Gly-Asn

Gly"GIn

HOOCSer-VoI-Thr

Cys

(~

Fi#urc 2 Cleavagesites of Hagcman factor during activation. Initially, cleavagesitc converts the single chain HF to a two-chain disulfide linked enzyme (HFa). Theheavychain contains the surface binding site andthe light chain has the active site. TheScr, Asp, His forming the "charge relay" of scrine proteasesa~e circled. Cleavageat sites 2 and then converts HFato HFr. The light chain of HF~(1) is 18 amino acids (site 1 to site 2) and

light chainof HFr(2) is 8 amino acids(site 3 to site than these reactions. Autoactivation produces, in addition to HFa and HFr, a third form in which the serine protease domainis linked to a 12,000 mol wt fragment of heavy chain to give a molecule ofmol wt 40,000 before reduction (17, 26; Figure 2), but kallikrein rapidly converts it to HFr. The complete amino acid sequences of the heavy chain of HFa (50) and of both chains of HFr (51) is nowknown.The latter study found the light chain of HFf to have nine amino acids presumably corresponding to the short segment remaining after cleavage at site 3. The heavy chain was found to possess a variety of structural domains with homologyto other proteins. Twoshort domains resemble fibronectin, two regions are homologous with epidermal growth factor, and a kringle structure is seen, as reported in plasminogen, tissue plasminogen activator, urokinase, and prothrombin. The light chain of HFa(or heavy chain of HFr) containing the active site has a high degree of homology with tissue plasminogen activator and, in decreasing order, with urokinase, plasmin, trypsin, factor X, and factor IX. Strong homologywith proteins involved in fibrinolysis is apparent. More recently, the complete amino acid sequence has been deduced using factor XII cDNAisolated from a cDNAlibrary prepared from human liver mRNA(52). HFr lacks the binding site for the surface and, once formed, is released

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KININS AND INFLAMMATION

55

into the fluid phase. It is not a coagulationfactor (it possessesonly 2-5% of the coagulantactivity of HFa)(16, 17), becausefactor XI activation strictly surface dependent(53). However, HFfis an effective prekallikrein activator (16, 17,. 53) and can continue to lead to bradykinin formation until Cl-inactivator (CI-INH)binds andinactivates it (54, 55). Prekallikrein is a single chain a globulin with two forms at mol wts 85,000and88,000(56), both of whichare present in all plasmastested. manyas seven formswith isoelectric points 8.5 and 8.9 can be identified by isoelectric focusing(57). Prekallikrein is convertedto kallikrein cleavage within a disulfide bridge such that a heavychain of 52,000 is disulfide linked to light chains of either 33,000and 36,000(one for each molecularform) (56, 58). Theheavychain interacts with the surface binds to HMW kininogen (34), while the light chain has the enzymatic active site (56). Purified light chainretains enzymatic activity on synthetic substrates (34) or in fluid-phase kinin generationbut is ineffective as surface-dependentcoagulationfactor. Factor XI is unusual amongcoagulation factors in that it consists of two identical disulfide-linked chains (59, 60). Uponactivation each chain is cleavedto yield disulfide-linkedheavyandlight chains; thus, the active enzymehas four chains. The heavychains bind to the surface (61), while each light chain has an active site (62) and, wheninactivated, can bind one moleinhibitor (60). Also, it has recently beenshownthat the molarratio of binding of HMW-kininogen to factor XI is 2 : 1 (63). The HMW kininogenis cleaved by plasmakallikrein at two sites (LysArg, and Arg-Ser)to liberate the nonapeptidebradykinin. Bothcleavages occur within a disulfide bridge (64~6) so that kinin-free HMW kininogen consists of a heavychain of molwt 42,000-65,000disulfide linked to a light chain variously reported as havinga molwt of 56,000-62,000.Further proteolysis of the light chainby kallikrein convertsit to a light chainof 45,000-47,000(64, 66, 67). Theheavy chain of HMW kininogen, which identical to the heavy chain of low molecular weight (LMW) kininogen (64, 68, 69), has beenshownto inhibit cysteine proteases (70-72)such cathepsin L, papain, or platelet calpain 2 (73). In fact, cDNA analysis a2 thiol proteinase inhibitor demonstratedthat it is identical to LMW kininogen(71). Thelight chain of H MW kininogenis the coagulantportion of the molecule(64, 74) andpossessesa binding site for the surface (74) as well as a bindingsite for the heavychain of prekallikrein (12, 43, 44, 74, 75) or factor XI (12, 74, 76) (Figure 3). Thesebinding constants beenestimated to be 3.4 × l07 M-1 and 4.2 x 108 M-1, respectively (74). CONTROL MECHANISMS Regulation of the Hagemanfactor pathways occurs by both intrinsic and extrinsic mechanisms.Cleavageof HFato

Annual Reviews 56

PROUD

& KAPLAN BRN}YKININ ~

CYSTEINE

PROTEASE

2

INHIBITOR H2N

I 5

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CLEAVAGE

t

SURFACE 3 SITE

LluMetLys Ar(]---ArgSer

BINDINGSITE FOR

BINDING PREKALLIKREIN OR FACTOR Xl

I S

RESULT

SITE I

¯ ¯ ¯

FORMS"NICKED" HMWKININOGEN AUGMENTATIONOF COAGULATIONCOFACTORFUNCTION REDUCTION~BRADYKININ AT C-TERMINUS OF H CHAIN

SITE 2

¯ ¯

RELEASEOF 8RADYKININ REDUCTION~62,000 H CHAIN ÷ 56,000 L CHAIN

SITE 3

¯

CONVERTS L CHAIN FROM56,000 TO 47,000 AND RELEASES PEPTIDEOF 8,000 RETAINS COAGULANT ACTIVITY THE LOCATION OF THIS SITE IS UNCERTAIN,BUTIS DEPICTED AT AMINO TERMINALEND OF L CHAIN

¯ ¯

Figure 3 Mechanism of digestion

of HMW-kininogcn to release

bradykinin.

functional domains of the molecule are shown and the consequences of cleavage is described.

The various at each site

HFf is one example of an intrinsic control in which coagulation via the surface dependent activation of factor XI is limited while bradykinin generation via fluid phase activation of prekallikrein can continue. Likewise, the digestion of kinin-free HMW kininogen light chain by factor XIa limits its coagulant activity (77). Extrinsic controls are provided by plasma inhibitors of each enzyme. The only major plasma inhibitor of HFa or HFr is CI-INH(54, 55, 78, 79). There are two main kallikrein inhibitors in plasma; namely a2 macroglobulin (80) and CI-INH (81, 82). kallikrein is added to plasma, approximatelyhalf is boundto each inhibitor at equilibrium (83, 84). However,when a surface such as kaolin is added to plasma, 70-80%of the kallikrein formed is bound to CI-INH(85); the mechanismresponsible for this change in inhibition ratio is unknown. Factor XIa is inhibited primarily by a~ antitrypsin (86-88) and CI-INH (78), while plasmin is inhibited by a2 antiplasmin (89, 91) and a2 macroglobulin (92-94). AntithrombinIII (ATIII) accounts for only a few percent of plasma inhibition of each of these factors, but its activity is augmented in the presence of heparin. Although this augmentation is less than that shownwhen the kinetics of thrombin inactivation are examined, there is clearly a prominent effect, the magnitudeof which is controversial. These reactions need to be reassessed using purified components,and the rate of inhibition of each enzymeshould be carefully determined in heparinized

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KININS

AND INFLAMMATION

57

vs citrated plasma.For such studies the dose of heparin and definition of the activity of the fraction usedwill be critical. Bradykininis an exceedinglypotent vasoactive peptide whichcan cause venular dilatation, increased vascular permeability, hypotension, constriction of uterine and gastrointestinal smoothmuscle, constriction of coronaryand pulmonaryvasculature, bronchoconstriction,and activation of phospholipaseA2to augmentarachidonic acid mobilization. Thus, its regulation is of prime importance, and plasma contains a variety of enzymesthat serve this function. Theaminoacid sequenceof bradykinin and the mechanisms by whichit is degradedare shownin Figure 4. First, the C-terminalarginine is removedby carboxypeptidase N(95, 96) to leave the residual octapeptide. Thedes-Arg9 bradykininthus formedis inactive in the skin or gastrointestinaltract but displaysactivity in severalin vitro vascular preparations (3). Separate receptors for these two moieties have 9 bradykinin)and B2(bradykinin) beenproposedand are called B1(des-Arg (97). Therate of Arg removalin serum, however,far exceedsthe rate plasma(98), whilethe level of carboxypeptidase N is unaltered. It appears that either a cofactor that augmentsthe activity of carboxypeptidase Nor a carboxypeptidaseB-like enzymeis generated during coagulation. DesArg9 bradykinin is then degraded, by angiotensin-convertingenzyme,to the pentapeptide Arg-Pro-Pro-Gly-Pheplus the tripeptide Ser-Pro-Phe (99). The C-terminal phenylalanine is then cleaved from each peptide, leaving Arg-Pro-Pro-Gly and Ser-Pro. The Ser-Pro is then digested to Ser and Pro while the C-terminal Gly is removedfrom the tetrapeptide. The final plasmadegradationproductsof bradykinin are, therefore, one mole each of Arg-Pro-Pro,Gly, Ser, Pro, and Arg, plus two molesof phenylalanine.

Arg Pro Pro Gly Phe Ser Pro Phe Arg

Brodykinin

CPN or CPB

~ Arg Pro Pro Gly Phe Set Pro Phe + ArcJ des - Arg 9- 1,4CE

Brodykinin

Arg Pro Pro Gly Phe + Set Pro Phe 7 enzymes (s Arg Pro Pro + Gly + Phe

? enzyme(s) Ser + Pro + Phe

Figure 4 Amino acid sequence of bradykinin and products obtained upon digestion human serum.

in

Annual Reviews PROUD & KAPLAN

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ASSESSMENT OF CONTACT ACTIVATION IN HUMAN DISEASE The

Hageman

factor-dependent pathways described herein are ~aot likely to contribute to thrombosis and hemostasis in a major way. Thus, although activation of Hagemanfactor leads to in vitro coagulation, in most thrombotic disorders activation of the extrinsic pathway occurs either alone or in addition to Hagemanfactor activation, and the effects of tissue factor predominate. Certainly no thrombosis is associated with hereditary angloedema, a disorder in which contact activation is prominent. Likewise, patients deficient in Hagemanfactor, prekallikrein, and HWM kininogen have no obvious bleeding diathesis, and factor XI deficient patients have a bleeding disorder that is highly variable and often mild. Observations such as these have led to considerations of physiologic mechanisms of factor XI activation that bypass the other contact activation components, but this remains unsettled. Most evidence suggests that the importance of the cascade relates to the pathogenesis of inflammatoryreactions, the local control of blood flow (in which bradykinin functions as a hormone), and perhaps control of blood pressure. To assess the Hagemanfactordependent pathways in humandisease, we would like ideally to measure each active enzyme, demonstrate cleaved HMW kininogen, and assay bradykinin. These determinations are difficult because the enzymesand the bradykinin are rapidly inactivated and often one has to determine the level of residual proenzyme. Thus, one can quantitate Hagemanfactor, prekallikrein, or HMW kininogen in plasma by coagulant assay, (100), immunologic determination of antigen levels (101-103), or by in vitro activation and evolution of enzymatic activity (104-107). Shifts in electrophoretic mobility indicative of complex formation between enzymes and their inhibitors can be assessed qualitatively. Thus far, monoclonal a~tibodies reactive solely with the active site of enzymeshave not been developed, although DeAgostini et al (108) have described a monoclonal antibody reacting with a neoantigen in the kallikrein-C1 INH complex. The aboveassays are useful if significant activation has occurred, leading to substrate depletion. But if only a small percentage conversion of each factor takes place, it is likely to be missed. Assays have recently been developed by Harpel for enzyme-inhibitor complexes based on the assay for plasmin-~2 antiplasmin complexes (109). These include double antibody ELISA methods for activated Hagemanfactor-C1 INH (110), kallikrein-C1 INH(111), and kallikrein--~2 macroglobulin complexes (85), which can detect as little as 1%activation of any of the components. Bradykinin can be determined by radioimmunoassay (112), and a radioimmunoassayfor des-Argg-bradykinin has been developed (113).

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Tissue Kallikreins Tissue kallikreins are single chain, acidic glycoproteins physicochemieally and immunologically distinct from plasma kallikrein. Thus, they have a reported tool wt range of 25,000 to 43,000 and isoelectric points close to pH4.0 (114, 1 ! 5). WhileHMW kininogenis clearly the preferred substrate for plasma kallikrein, tissue kallikreins readily release kinin from both HMW kininogen and LMW kininogen (116, 117). Immunoreactive tissue kallikrein has been detected in both rodent (118-120) and human(121), plasma, but most, if not all, of this material apparently represents biologically inactive kallikrein destined for clearance by the kidneys (119). Whilesomesystemic role for tissue kallikreins cannot be ruled out, therefore, it seems likely that these enzymesfunction principally in the local environmentof their tissues of origin. Originally tissue kallikreins were thought to derive only from the major exocrine organs such as the pancreas, salivary glands, and kidneys. More recently, however, kallikreins were discovered to be more widely distributed in tissues such as the gut (122) and the prostate gland (123). Althougha report that tissue kallikrein was also derived from the endocrine portion of the pancreas (124) has been vigorously disputed (125127), morerecent workdescribing the presence of kallikrein in the pituitary gland (128-~130)leaves little doubt that these proteases are not restricted to exocrine tissue. , Within a given species, the tissue kallikreins from all organs are immunologically identical (131-133). Furthermore, recent eDNAcloning and sequence analysis studies have shownthat humanpancreatic and renal kallikreins have identical aminoacid sequences, with the active enzymein each case consisting of 238 residues (134, 135). In addition, the same mRNA is expressed in the pancreas, kidney, and sublingual gland (134). Although in both mouseand rat, kallikrein activity is associated with the highly conserved members of a large multigene family located on chromosome 7 (136--138), studies suggest that there are no more than two or three closely related genes in humans(135). Kallikreins are synthesized in all tissues examinedto date in the form of the preproenzyme. Whenstudied by conventional biochemical techniques, however,kallikrein is detected in someorgans, such as the pancreas (139, 140) and kidney (141) primarily in the form of prokallikrein, while in submandibular gland only the active kallikrein is found (142). Further posttranslational differences also occur with regard to glycosylation of tissue kallikreins. Isoelectric focussingof purified kallikreins fromdifferent organs usually reveals microheterogeneityin terms of multiple isoelectric

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forms (115, 143, 144). Treatment with neuraminidase to removesialic acid abolishes this microheterogeneity (143). The distribution of kallikrein in a range of tissues of differing function has led to the suggestion that tissue kallikreins mayplay differing roles depending upon their tissue of origin (145). This is supported by evidence that synthesis and secretion of these enzymesare controlled by different factors in different tissues. It is well established, for example,that restriction of dietary sodiumcauses elevations in urinary kallikrein derived from the kidney (146, 147). This effect maybe mediated by mineralocorticoids, because patients with primary aldosteronism also have elevated urinary kallikrein levels (148), and administration of the synthetic mineralocorticoid fludrocortisone reproduces the effects of dietary sodium restriction (146, 147). Morerecently, parotid salivary kallikrein has been shownto respond similarly to mineralocorticoids and sodium restriction (149). Aldosteronereceptors are present in both the kidney and the parotid gland ’(150), and while it is possible under certain circumstances to dissociate aldosterone and kallikrein levels (151), mineralocorticoids may directly regulate renal kallikrein (152). In other tissues, kallikrein levels appear to be controlled by androgen concentrations. In the rat submandibulargland the tissue content of kallikrein is elevated by testosterone or thyroxine, but no such effects are observed in the kidney (153). The increased kallikrein content of the submandibular gland in response to androgenis due to increased synthesis (154). By contrast, kallikrein levels in the anterior pituitary are considerably higher in female rats than in males (155), and gene expression in this tissue, as determined by mRNA levels, is estrogen, not androgen, dependent (130). The secretion of kallikrein from different tissues is likewise controlled by a spectrumof stimuli. As mentioned above, mineralocorticoids appear to play a role in renal kallikrein secretion, apparently favoring the secretion of the active enzyme (156), while salivary kallikrein secretion is largely under sympathetic control (157, 158). By contrast, pancreatic kallikrein secretion appears to controlled by the vagus nerve and by cholecystokinin (159), but secretion from intestinal goblet cells is largely under cholinergic control (160). This confusing array of control mechanisms and the widespread distribution of kallikreins have led several investigators to suggest that these enzymes cannot serve primarily as kinin-forrning enzymes in all these tissues but must, in somecases, act principally on substrates other than kininogen. For example, it has been suggested that in the pituitary gland kallikrein mayfunction in prohormoneprocessing. The last few years have seen a flurry of effort to search for alternate substrates for tissue kallikreins. It has been suggested that tissue kallikreins can activate prorenin to renin (161,162), but this reaction appears to require noncatalytic quantities

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kallikrein. Likewise exogenoustissue kallikrein can process atriopeptinogen,the precursorof atrial natriuretic factor (163), but it has also beensuggestedthat kallikrein can inactivate atrial natriuretic peptides (164, 165). Anotherpotential substrate for kallikrein is apolipoprotein 100 whichcan be degradedby urinary kallikrein (166), but the physiological significanceof this reaction remainsunclear. Analternative hypothesis for the functionsof tissue kallikreins in different organsis that they mayserve to producekinins but that the primaryresponse to kinins may vary fromtissue to tissue. In the kidney, for example,kallikreins are synthesized in the connectingtubule (167, 168), and the primarysite kinin bindingis in the cortical andouter medullarycollecting tubule (169). Thus,kinins mayfunction in regulating sodiumexcretion and in regulating the hydro-osmoticresponse to vasopressin (170, 171). In the salivary glands kinins mayalso play a role in regulating the ionic compositionof secretions, or alternatively they mayfunction to control local blood flow (172). Bycontrast, in the intestine kinins mayregulate chloride transport (173, 174) or, perhaps,regulate nutrient absorption(175). Thetissue-specific roles of these enzymesclearly remainsa subject of considerable speculation. Regardinginflammatoryreactions, however,the widespreaddistributions of tissue kallikreins enhancestheir potential for involvementin kinin generation at multiple sites. Likewise, while kallikreins may,indeed, have roles outside of kinin-formation, there can be little doubtof the effectiveness of these proteases as kininogenases. Approximately70%of the total kininogen in humansis LMW kininogen. Unlike plasmakallikrein, therefore, whichfunctions principally on HMW kininogens,the ability of tissue kallikreins to react equally well with HMW and LMW kininogens (116, 117) provides these enzymeswith a larger pool of availablesubstrate. Theinteractionof tissue kallikreins with single-chain kininogens has always been fascinating from the biochemicalstandpoint because these enzymeshydrolyze two dissimilar bonds in the kininogen moleculeto liberate lysylbradykinin(Figure 3), or in a recently described variation, ala4-1ysylbradykinin(176). (This variant, however,apparently displays the samephysiological properties as does lysylbradykinin.) The ability of tissue kallikrein to hydrolyzetwodissimilar bondsandrelease lysylbradykininis still not fully understood.Onesuggestionis that tissue kallikreins mayhavetwoactive sites (177), but little firm evidencesupports this conceptas yet. Tissue kallikreins are knownto contain the normal charge-relay system found in serine proteases, and it has been suggested that the initial hydrolysismayoccur at the Arg-Serbondin kininogen,an idea in keepingwith the anticipated primarysubstrate specificity of tissue kallikreins (115). Althoughtissue kallikreins will hydrolyzemethionyl bonds in small peptides, the preceedingaminoacid sequencemayplay an

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importantrole. Thebulky leucine residue at position P2 in the kininogenderived peptide Ser-Leu-Met-Lys-bradykinin, for example,is thought to be responsiblefor directing the attack of kallikrein to the methionylbond to release lysylbradykinin (178). Toachievea high catalytic rate of hydrolysis of such a methionylbond, however,the intact kininogenmoleculeis necessary and presumably provides an essential stereochemical conformation. Afinal considerationin understandingthe generationof kinin by tissue kallikreins is the set of factors that controlthe activity of these enzymes. As mentionedabove, in manytissues kallikreins are present mainly in the formof prokallikrein. Whileit is knownthat prokallikrein can be activated by trypsin, plasmin, or even plasmakallikrein, the identity of such an endogenouskallikrein-activating enzymein tissues such as the kidney remains unknown.Onceactivated, however,tissue kallikreins are less susceptible to inhibition by plasmaprotease inhibitors than is plasma kallikrein. Indeed,in humans,only g ~-antiproteaseis a significant inhibitor of tissue kallikrein, and even then inhibition is weakand slow (179). Althoughbovine pancreatic trypsin inhibitor is an effective kallikrein inhibitor in ruminants,an equivalentinhibitor has yet to be isolated from humans.Monovalentcations havebeen reported to inhibit the esterolytic activity of tissue kallikrein (180, 181), but while onereport suggeststhat such ions also inhibit kininogenaseactivity (180), another suggests that they enhancethe kinin-generatingactivity of these enzymes(181). In summary,tissue kallikreins are extremelypotent kinin-generating enzymes,widelydistributed in humans.Their ability to interact with both HMW and LMW kininogens to generate lysylbradykinin and their relative resistance to inhibition enhancestheir potential for involvementin kinin formation during inflammatorydisorders. Cellular Proteases Acommon feature of several types of cells involvedin inflammatoryevents is their high content of proteases. It is clear that, should any of these cellular proteasesdisplay kininogenaseactivity, the potential wouldexist for a direct meansof kinin formation during inflammatorydisorders, independentof plasmaor tissue kallikreins. This concepthas attracted the attention of several investigators whohavestudied the ability of a number of cell types to liberate kininogenase activity. In the case of immediatehypersensitivityreactions, efforts havefocused on the basophil and the mastcell as sources of kinin-generatingenzymes. Preparations of humanleukocytes containing basophils have been shown,

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uponIgE-mediatedstimulation, to release arginine esterolytic activity (182). This activity, measuredby its ability to hydrolyzethe synthetic subs~rate N-~-p-tos3d-L-arginine methyl ester (TAME),showedrelease characteristics similar to those of histamine, suggestingthat the esterase is derived fromthe basophilgranule. Biochemical characterization of this activity showedthat TAME-esterase activity eluted on gel filtration with an apparent mol wt of 1.2 × 10 6 and was associated with both kiningenerating and Hageman factor-cleaving activities 083). The unusually high molecularweightof this entity together with its ability to display several functional activities raises the question of whethermorethan one enzymewasactually beingstudied. In the mastcell high salt concentrations are necessaryto disrupt the interactions of cellular proteases with the proteoglycancomplex(184, 185). Thus, since low salt concentrationswere used in the abovestudies, the apparentmolecularweightmaybe explained by a complexof several enzymesattached to a proteoglycan. In recent preliminarystudies using purified (5%to 84%)basophils, the release TAME-esterase activity in response to an IgE-mediatedstimulus has been confirmed,but not the presence of kinin-generating ability (D. Proud, unpublishedobservations). Antigen challenge of passively sensitized humanlung fragments has beenassociatedwith the generationof three enzymaticactivities: a kininogenase(186), a prekallikrein activator (187), and an enzymethat cleaves but does not activate Hageman factor (188). Generationof these activities in responseto antigen challengefocussedattention on the mast cell as a possible sourceof these enzymes.Prekallikrein activator activity has been detected fromstimulatedmastcells (189) althoughrelatively large numbers of mast cells are neededto generate significant amountsof activity. A Hageman factor-cleaving enzyme,apparently identical to neutrophil elastase, is also released by an IgE-mediatedmechanism from purified mast cells (190). Kininogenase activity is also releasedfromhighlypurified mast cells (191). Althoughit had beenreported that tryptase, the majorneutral protease from humanlung mast cells, does not possess kinin-generating activity (192, 193), recent characterizationhas demonstratedthat the mast cell kininogenaseis, indeed, tryptase. However,this enzymegenerates bradykininoptimallyat pH5.5 (194). Whiletryptase is a relatively weak kininogenase,it is present in mastcells in large amounts.It remainsto be determined whythis neutral protease generates kinin optimally at pH 5.5, but this acidic pHoptimumclearly raises questions regarding the physiologicalrole of tryptase as a kininogenase.Thepropertiesof tryptase suggest that it is not the enzymedescribed fromchoppedlung (186) and raise the possibility that this latter enzymeis not mastcell-derived. Since

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tissue kallikrein has been detected in bronchoalveolar lavage fluids from asthmatics (195), it is possible that the enzymemeasuredin choppedlung may represent tissue kallikrein released by an indirect mechanismor, perhaps, someother protease yet to be characterized. Calpains are calcium-activated cytosolic cysteine proteases present in manytissues and cells, including reticulocytes, keratinocytes, and platelets (196). Since platelets, for example, can be activated during certain inflammatory states such as asthma (197), a recent report that calpains can generate kinins (198) bears scrutiny. Kininogens are now known to thiol protease inhibitors (72, 199) and have been shownto inhibit platelet calpain (73). This potent inhibitory capacity presumably explains why kinin formation occurs only in a limited and high range of calpain to kininogen molar ratios (0.5 : 1 to 8 : 1). The report that lysylbradykinin liberated from kininog~ns by these enzymessuggests that calpains are the first enzymesother than tissue kallikreins to be able to hydrolyze the dissimilar bonds in kininogens to release lysylbradykinin. Only 20%of the kinin content of the kininogensused in this study was liberated by calpains, however, and it remains to be determined if this is due to subsequent inhibition by kininogens or to the presence of "nicked" two-chain kininogen in these preparations. Thus, more work is required to ascertain that calpains can liberate kinins from single-chain kininogen and to determine if the high molar ratios of calpain to kininogens used by Higashiyamaet al (88) are achieved during inflammatory events in vivo. The first report that neutrophils maycontain a neutral protease capable of generating kinin was in 1967 by Melmon& Cline (200) who incubated intact neutrophils with whole plasma or a crude kininogen preparation. These authors suggested that neutrophils contained both kinin-generating and kinin-destroying activities. While other workers were unable to reproduce these results with intact neutrophils, Movatet al were able to use fragmerited neutrophils with purified kininogens to demonstrate the presence of such a neutral protease from these cells (201). The enzymeappears to be distinct from elastase and cathepsin G (202, 203), but the nature the kinin generated by the purified protease remains uncertain. At this point the relative activities of the kinin-generating and kinin-destroying enzyme during inflammatory events also remain to be determined. In addition to this neutral kininogenase, neutrophils are amongthe cells containing an enzyme capable of generating leukokinins. This enzyme, which is almost certainly cathepsin D, interacts with a precursor, whichis distinct from HMW and LMW kininogens, to liberate pharmacologically active peptides of 21-25 aminoacids called leukokinins (204). This process occurs optimally at acidic pH and is rather slow. Thus, even though all the components of the leukokinin system are present, for example, in

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ascitic fluid, moreworkis neededto elucidate the role of leukokininsin humaninflammatoryprocesses.

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Rhinitis Clinical symptoms of thinitis can be inducedby a variety of allergic and nonallergic stimuli. In recent years data havebeenobtainedwhichsuggest that kinins maybe involvedin the pathogeneses of several types of rhinitis. A model of nasal challenge with allergen in which inflammatory mediatorscan be measuredin secretions obtainedby lavage (205) has been used to demonstrate that kinin formation occurs during the immediate response to allergen challenge (206). Kinin generation correlates with the onset of symptomsand with the production of other inflammatory mediatorsincluding histamine, prostaglandin DE(205, 206), and the sulfidopeptide leukotrienes (207). Unlikeplasma, in whichthe measurement of kinins is technically complex(208,209),it is possible to measurekinins simply and reproducibly in nasal lavages collected in the presence of EDTA.In part this maybe due to the large dilution factor created by lavage. Becauseof these advantages,the nasal systemhas also lent itself to biochemicalstudies of the mechanisms of kinin formationand metabolism during the immediateallergic response. The demonstrationthat the immunoreactive kinin generatedduring this reaction represented a mixture of bradykinin and lysylbradykinin (206) suggested that more than one enzymewasinvolvedin kinin formationor conversion.Duringthe allergic response, both HMW and LMW kininogens enter nasal secretions by transudation from plasma(210). Since prekallikrein circulates in plasma as a complexwith HMW kininogen (12), it was not surprising that antibody that recognizes plasmaprekallikrein and kallikrein detected increased immunoreactive plasma(pre)kallikrein in nasal secretions during the allergic reaction (211). Biochemicalanalysis revealed, however,that prekallikrein activation occursduringthis process, suggestingthat plasma kallikrein contributes to the generation of bradykinin before subsequent inactivation by plasmaprotease inhibitors. Thepresence of complexesof C1inactivator with both kallikrein andHageman factor in nasal secretions during the immediateallergic response has recently been confirmed(D. Proud & A. P. Kaplan, unpublishedobservations) using specific assays (110, 111). Thus,it seemscertain that conversionof prekallikrein to kallikrein occursas a result of contact activation of Hageman factor either upona surface altered by initial inflammatory events or by somenegatively charged mucusmacromolecules or mast cell heparin (212). Althoughmast cell activation clearly occurs during the immediateallergic reaction and

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tryptase can be detected in nasal secretions (211), the relative levels and activities of plasmakallikrein andtryptase wouldsuggest that the latter makes, at best, a minimalcontribution to bradykinin generation. Generation of lysylbradykinincannotbe explainedby the actions of either of these enzymes.This peptideis generatedby a tissue kallikrein that has been shown,for the first time, to be present in nasal secretions. Immunoreactive tissue kallikrein can be detected evenin resting nasal secretions, but concentrationsof this materialincreasein responseto antigenchallenge(213). Theidentity of this material as authentic tissue kallikrein wasconfirmed by physicochemical and immunological criteria as well as by its ability to generate lysylbradykinin. Althoughpresent in approximately10-fold lower concentrationsthan immunoreactive plasmakallikrein, tissue kallikrein is responsiblefor mostof the kininogenaseactivity in post-challengelavages (213), presumablydue to its resistance to inhibition relative to plasma kallikrein. This resistance to inhibition, together with a greater substrate availability, probablyexplains the fact that as muchas 45%of the kinin detected duringthis reaction represents lysylbradykinin(206, 214). Indeed, since postchallengenasal lavagesalso contain elevated levels of an aminopeptidase capable of converting lysylbradykininto bradykinin (215), the level of lysylbradykinin mayinitially be greater than 45%,emphasizing the importanceof tissue kallikrein in kinin-formationduringthe immediate allergic response. In addition to metabolismof lysylbradykinin by the aminopeptidase, both bradykinin and lysylbradykinin are metabolized by a carboxypeptidase N-like enzymewhich removes the C-terminal arginine from both peptides. Nasal lavages also contain low levels of angiotensin-converting enzyme.Althoughlittle, if any, bradykinin metabolismdue to angiotensin-converting enzymewas observed in lavages, this does not meanthat this enzymedoes not play a role in kinin metabolismin nasal secretions that have not been diluted by lavage. Thus, kinin metabolism in nasal secretions during the immediateallergic response showsseveral similarities to metabolismin plasmawhereinitial kinin hydrolysisis by carboxypeptidaseswith subsequentslower hydrolysis by angiotensin converting enzyme(96, 99). Interestingly, the actions of these peptidases nasal secretions could be expectedto lead to a temporalaccumulationof des (Arg9) bradykinin.This peptide is inactive in systemsregulated by the "classic" kinin receptor, called B2-typeby Regoliet al (97, 216)but is more active than bradykinin on the so-called B1receptor. Thus, dependingon 9) the type of kinin receptors in the nasal mucosa,formationof des (Arg bradykinincouldresult in terminationof biologicactivity (if B2receptors predominate)or the generation of a biologically active peptide (if receptors are present). This conceptis currently understudy.

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In addition to the immediate allergic response, bradykinin and lysylbradykinin have also been shown to be generated during the late phase reaction to nasal challenge (217). The mechanismscontrolling kinin formation and metabolism during the late reaction have not yet been studied, but several differences betwten the immediate and late responses suggest this would be worthwhile. The profile of inflammatory mediators in the late reaction differs slightly from the immediateresponse. Whilehistamine is present in both reactions, prostaglandin D2 (PGD2), the major cyclooxygenase product of humanmast cells (218), is present in the early reaction but is undetectable during the late response. Since basophils makevery little PGD2(218), this suggests that this cell type mayparticipate in the late reaction. Also, while levels of histamine and TAME-esteraseactivity are similar during the immediateand late reactions, kinin levels are significantly lower during the late response (217). This mayreflect altered rates or pathways of formation associated, perhaps, with basophil rather than mast cell activation or different rates or mechanismsof kinin metabolism, particularly since inflammatorycells accumulate in the nasal mucosa during the late response (219). Kinin generation also occurs during the inflammatory response to nasal challenge with cold, dry air. Again, kinin formation is associated with a symptomatic response and with the production of a spectrum of inflammatory mediators (220, 221) virtually identical to that observed during the immediate allergic response. Although symptomsof rhinitis in response to cold, dry air challenge clearly involve mastcell activation, this activation is independent of antigen and seems to occur via different mechanism. Thus, while the tricyclic antihistamine, azatadine, is effective in blocking IgE-mediatedmast cell mediator release in vitro and in vivo (222), it does not prevent mediator release or symptomsin response to cold, dry air challenge (223). In each of the three types of rhinitis described above, kinin formation is associated with mast cell or basophil activation, and it would seem reasonable to assume that the release of mast cell/basophil mediators initiates the inflammatory response allowing influx of kininogens and plasmakallikrein to nasal secretions. The large circulating pools of plasma kininogens would provide a source for prolonged kinin formation which could maintain chronic symptomatology. The relationship between mast cell/basophil activation and kinin formation does not always hold, however. It is knownthat exposure of mast cells or basophils to hyperosmolar environments in vitro will induce mediator release (224). Nasal provocation with hyperosmolar mannitol similarly induces increased concentrations of histamine and leukotrienes in nasal secretions, but this mast cell activation is associated with little, if any, significant degree of kinin

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formation (225). Thus, kinin generation does not appear to be an automatic consequenceof mast cell activation. It is also possible to have symptomatic episodes of rhinitis in which kinin formation occurs independent of mast cell activation. In recent studies of induced rhinovirus colds, kinin generation did not occur in individuals who were infected but not symptomatic. During symptomatic infections, however, concentrations of kinins in lavages correlated with symptomscores, and the strength of this correlation increased with the severity of illness, i.e. the moresevere the cold the greater the relationship between the measurements of kinins and symptoms. In contrast to the various types of reactions described above, the mast cell mediators histamine and prostaglandin D2 were not increased during rhinovirus colds (226). Indeed, to date, kinins are the only mediators detected that correlate with the symptomatic response to rhinovirus infection. Despite the lack of mast cell activation during these infections, kinin generation occurs via mechanismssimilar to those delineated for the immediateallergic response. Both bradykinin and lysylbradykinin are produced, although it would appear that lysylbradykinin represents a lower proportion of the kinin generated than during the allergic reaction. In keeping with this, tissue kallikrein levels do not increase to the degree observed for the allergic response (D. Proud, unpublished observations). While the presence of kinins in nasal secretions during these various types of rhinitis suggests that these peptides maycontribute to the symptomatologyof rhinovirus colds as well as allergic reactions, such a role for kinins is clearly dependent upon their ability to induce relevant symptoms whenapplied to the nasal mucosa. It has recently been demonstrated that nasal provocation with bradykinin does, indeed, induce symptoms of nasal obstruction and rhinorrhea. Of particular relevance to the possible involvement of kinins in the commoncold is the observation that nasal provocation with bradykinin also leads to the development of a sore throat, presumably as a result of mucocilliary transport causing some kinin to reach pain receptors in the throat. The ability of bradykinin to induce symptomsis not mediated by mast cell activation (227). Thus, the ability of kinins to induce relevant symptomsin the nasal cavity together with the evidence that kinin generation occurs during various types of rhinitis provides strong support for the hypothesis that kinins may be important mediators of several types of inflammatory disorders of the upper airways. The recent developmentof the first competitive antagonists of bradykinin (228.) mayprovide a basis to establish definitively the extent to which kinins contribute to the symptomatology of such reactions.

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Asthma The edema-promoting and smoothmuscle-contractingproperties of kinins must also lead to consideration of whether these peptides participate in the pathogenesis of inflammatoryconditions of the lower airways, particularly asthma.Bolusintravenousinjections of bradykininhavebeen shownto induce transient respiratory distress in asthmatics but not in normalsubjects (229). Similarly, several inhalational studies have corroborated the ability of bradykinin to provokebronchospasmin human asthmatics but not in normalhumans(230, 232); this is consistent with the airways hyperreactivity typical of asthmatics (233). Early reports elevated systemic plasmakinins during asthmatic attacks (234, 235) provide somesupport for the involvementof kinins in asthma but, unfortunately, in light of current knowledgeregarding the problemsof measuring plasmakinins (208, 209), the accuracyof these early measurements must be considered dubious. Recent studies of bronchoalveolar lavage fluids, however,showedthat kininogenaseactivity could be detected in 22 of 24 samples from 17 asthmatics whoeither responded to aerosolized allergen challenge or had symptoms of active asthma, while such activity wasabsent in samplesfrom 6 of 7 normalcontrols (195). Levels of free kinins in lavagefluids correlatedwiththe presenceof kininogenase activity. The principal componentof this kininogenase was shownto be tissue kallikrein; in a small numberof samples,someplasmakallikrein activity wasalso present. Theprincipal control of this tissue kallikrein appearsto be by ~ 1-antiprotease(236). Thus,mechanisms for the generationof kinins are present and active duringasthma.Preliminaryevidencealso indicates elevatedtissue kallikrein andkinin levels in lavagefluids fromsubjectswith other types of local pulmonaryinflammation(bronchitis and pneumonitis) (195); this suggestsperhaps,in analogywith the upperairways,that kinins maybe involvedin a range of inflammatory disorders of the lowerairways. Hereditary Angioedema Hereditary angioedemais causedby low levels of functional C 1-INHdue to low total protein levels or synthesis of an abnormalC I-INH. In this disorder simultaneousactivation occurs of the Hageman factor-dependent pathwaysand the classical complementpathways. Patients present with episodic swelling occurring virtually anywherein the body, attacks of several abdominalpain due to edemaof the bowel wall, and laryngeal edema.Althougha kinin derived from a cleavage product of the second complementcomponentwas thought to be responsible for the severe swelling(237), other studies suggestbradykininto be the critical peptide

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(238, 239). Onthe other hand, a kinin-like vasoactive substancemayyet be shownto be a complementderivative, a point suggested by recent reports (240). Duringattacks of angioedemathere is depletion of prekallikrein and HMW kininogen(241), formation of bradykinin (242), whenblisters are inducedin the skin of patients, they are found to have elevated plasmakallikrein levels (243). Thecomplement activation appears due to autoactivation 0f C1(specifically Clr) in the absenceof CI-INH (244, 245). However,Hageman-factorfragment (but not HFa) has shownto activate enzymaticallythe first componentof complement (246) whenit is directly incubated with purified C1 or added to plasma. C1 activation is due to cleavage of the Clr subcomponent by HFf(247). hereditary angioedema,HFactivation maycontribute to the complement consumptionseen during episodes of swelling. Other Disorders Although we routinely use dextran sulfates as model compoundsupon which Hageman-factor activation can occur, other sulfated mucopolysaccharides maysubserve similar functions in vivo (248, 249). The synovial fluid of patients with rheumatoidarthritis contains plasmakallikrein, whichhas been shownto activate neutrophil procollagenase to collagenase(250). Uric acid and pyrophosphatecrystals can act as surfaces in contact activation (251,252)and maycontribute to the inflammationof gout and pseudo-gout.Thus, a role for the contact activation system in inflammatoryarthritides is possible, and this mayinvolveother functions of the enzymesformed,in addition to any effects of kinins. Activation of Hageman factor is also inducedby the Lipid A component of endotoxin (253-255), and prominentdepletion of contact activation proteins occurs in endotoxic shock (256-259). The pooling of fluid into bodycavities, the intravascular volumedepletion, and the hypotension seen maybe caused by release of bradykinin. Likewise, other bacterial infections such as typhoid fever have been shownto be associated with prekallikrein depletion, formation of kallikrein-C1 INHcomplexes,and CI-INHdepletion as evidence of contact activation (260). In patients hospitalized becauseof trauma, the onset of sepsis has beenshownto be associated with kininogendepletion and determinationof serial kininogen levels has prognostic value (257). Anunfortunate circumstancehas dramatizedthe kinin-formingcapacity of HFf; trauma patients given plasma protein fractions (as plasmaexpanders) that were contaminatedwith HFf suffered profoundhypotension(261,262). In patients with disseminatedintravascular coagulation due to endo-

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thelial injury and/or endotoxemia(including gram-negativesepsis, grampositive sepsis, or viremia), decreasedplasmafactor XII, prekallikrein, and kallikrein inhibitory activity are seen (263). Thesechangeswerenot observedin disseminatedintravascular coagulation associated with leukemia, carcinoma, or abortion (264). These data suggest activation the Hagemanfactor~lependent pathways. A similar pattern of protein depletion has beenobservedin somepatients with polycythemia vera (265), type II hyperlipidemia(familial hypercholesterolemia)(266) and Rocky Mountainspotted fever (267). In somepatients with nephrotic syndrome, a similar diminutionof contact activation factors has beenreported (268) and this wasnot explicable simplyas a result of loss of proteins in the urine (269). Cirrhosis, as mightbe anticipated, is associated with diminished levels of plasmaprekallikrein, HMW-kininogen, and, to a lesser degree, Hageman factor, whichseem to be due to a diminished rate of protein synthesis(270).

SUMMARY Althoughconsiderable progress has been madein elucidating the molecular events occurring during kinin generation by both the plasmakininformingsystemandthe tissue kallikrein system,it is only in recent years that wehavecometo appreciate their potential role in inflammationin a wide variety of diseases. The importanceof the tissue kallikrein system dependsuponsecretion of the active form of the requisite enzymein the presence of a source of kininogen. Since tissue kallikreins are widely distributed in tissues, andsince lymphandinterstitial fluid containskininogen(271), a local milieu for potential kinin formationis alwayspresent. Theplasmasystemwill be activated secondaryto inflammationinitiated by someother process. There maybe endothelial or epithelial damage exposingconnectivetissue. Plasmaleakagecausedby release of someother permeabilityfactor (including kinin madeby tissue kallikrein) wouldthus lead to activation of the plasmacascadein manyformsof inflammation. As with all mediators,however,the contributionof kinins to an inflammatoryresponsecan only be definitively evaluatedif their actions can be selectively antagonized.Competitivereceptor antagonists have recently been synthesized (228) and will, we hope, soon be available for administration to humans.Shouldthese compounds proveeffective in vivo, they couldbe usedin conjunctionwith currentlyavailableassaysfor kallikreins, kininogens,kinins, and their various inactivated or degradedproducts, to provide newinsights into the role of these systemsin the pathogenesesof inflammatorydiseases.

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ACKNOWLEDGMENTS The authors thank Bonnie Evans for excellent Proud acknowledges support from grant National Institutes of Health.

secretarial assistance. Dr. number HL32272 from the

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Literature Cited 1. Spragg,J., Talamo,R. C., Austen,K. F. 1970. Immunochemistryof bradykinin and immunologic activation of the kinin system. In Handbookof Experimental Pharmacolo#y:Bradykinin, Kallidin and Kallikrein, ed. E. G. Erdos, 25:531-49.Berlin: SpringerVerlag 2. Garcia-Leme, J. 1978. Bradykinin system. In Handbookof Experimental Pharmacology. Inflammation,ed. J. R. Vane, S. H. Ferreira, 50: 464-522. Berlin: Spdnger-Verlag 3. Marceau,F., Lussier, A., Regoli, D., Giroud, J. P. 1983. Pharmacologyof kinins: Their relevanceto tissue injury and inflammation.Gen. Pharmacol.14: 209-29 4. Kaplan,A. P., Silverberg, M.1987.The coagulation-kinin pathway of human plasma. Blood 70:1-15 5. Ratnoff, O. D., Rosenblum, J. M.1958. Role of Hageman factor in the initiation of clotting by glass: Evidence that glass frees Hageman factor from inhibition. Am. J. Med.25:16(~68 6. Wuepper,K. D. 1973. Prekallikrein deficiency in man. J. Exp. Med.138: 1345-55 7. Weiss,A. S., Gallin, J. I., Kaplan,A. P. 1974. Fletcher factor deficiency. Abnormalitiesof coagulation, fibrinolysis, chemotacticactivity, and kinin generation attributable to absence of prekallikrein.J. Clin. Invest. 53:622-33 8. Saito, H., Ratnoff, O. D., Donaldson, V. H. 1974.Defectiveactivationof clotting, fibrinolytie, and permeability enhancing system in humanFletcher trait plasma.Circ. Res. 34:641-51 9. Wuepper,K. D., Miller, D. R., LaCombe,M.J. 1975.Flaujeac trait. Deficiency of human plasma kininogen.J. Clin. Invest. 56:1663-72 10. Colman, R. W., Bagdasarian, A., Talamo,R. C., Scott, C. F., Seavey, M., Guimaraes,J. A., Pierce, J. V., Kaplan, A. P. 1975. Williamstrait: Humankininogen deficiency with diminishedlevels of plasminogenactivator and prekallikrein associatedwith

abnormalities of the Hagemanfactor dependentpathways.J. Clin. Invest. 56: 1650-62 11. Donaldson, V. H., Glueck, H. I., Miller, M.A., Movat,H. Z., Habal,F. 1976. Kininogendeficiency in Fitzgerald trait. Role of high molecular weightkininogenin clotting and fibrinolysis. J. Lab. Clin. Med.87:327-37 12. Mandle, R. Jr., Coleman, R. W., Kaplan, A. P. 1976. Identification of prekallikrein and HMW-kininogen as a circulating complex in human plasma.Proc.Natl, Acad.Sci. USA73: 4179-83 13. Thompson,R. E., Mandle, R. Jr., Kaplan,A. P. 1977. Associationof factor XI and high molecularweightkininogenin humanplasma.J. Clin. Invest. 60:1376-80 14. Ratnoff, O. D., Davie, J. W., Mallet, D. L. 1961. Studies on the action of Hagemanfactor. Evidence that activated Hageman factor in turn activates plasma thromboplastinantecedent. J. Clin. Invest. 40:803-19 15. Cochrane,C. G., Wuepper,K. D. 1971. Thefirst component of the kinin-forming system in human and rabbit plasma.Its relationshipto clotting factor XII (Hageman factor). J. Exp. Med. 134:986-1004 16. Kaplan, A. P., Austen, K. F. 1970. A prealbumin activator of prekallikrein. J. Immunol. 105:802-11 17. Kaplan, A. P., Austen, K. F. 1971. A prealbuminactivator of prekallikrein II. Derivation of activators of prekallikrein from activated Hageman factor with plasmin. J. Exp. Med.138: 696-712 18. Cochrane, C. G., Revak, S. D., Wuepper,K. D. 1973. Activation of Hagemanfactor in solid and fluid phase. J. Exp. Med. 138:1564-83 19. Hathaway,W. E., Belahusen, L. P., Hathaway,H. S. 1965. Evidence of a newplasmacoagulation factor I. Case report, coagulation studies and physiocochemical properties. Blood 26: 521-32

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1998-2002 34. Vander Graaf, F., Tans, G., Bouma, B. N., Griffin, J. H. 1982.Isolation and functional properties of the heavyand light chains of humanplasma kallikrein. J. Biol. Chem.257:14300-305 35. Rosing,J., Tans,G., Griffiti, J. H.1985. Surface-dependentactivation of human factor XII (Hageman factor) by kallikrein andits light chain. Eur. J. Biochem. 151:531-38 36. Webster,M.E., Pierce,J. V.1973.Activation of Hageman factor (factor XII). Identification andrelationship to kallikrein-kinin system.Fed. Proc.32:845 (Abstr.) 37. Schiffman, S., Lee, P. 1974. Preparation, characterization, and activation of a highlypurified factor XI. Evidence that a hitherto unrecognized plasmaactivity participatesin the interaction of factors XI and XII. Br. J. Haematol.27:101-14 38. Griffin, J. H., Cochrane,C. G. 1976. Mechanismsfor the involvement of high molecularweightkininogenin surface-dependent reactions of Hageman factor. Proc.Natl. Acad.Sci. USA73: 2554-58 39. Meier,H. L., Pierce, J. V., Colman, R. W., Kaplan,A. P. 1977. Activationand function of humanHagemanfactor. The role of high molecular weight kininogenand prekallikrein. J. Clin. Invest. 60:18-31 40. Wiggins, R. C., Bouma,B. N., Cochrane, C. G., Griffin, J. H. 1977. Role of high molecularweight kininogenin surface-binding and activation of coagulation factor XIa and prekallikrein. Proc. Natl. Acad.Sci. USA77: 4636-40 41. Silverberg, M., Nicoll, J. E., Kaplan, A. P. 1980. The mechanismby which the light chain of cleaved HMW-kininogen augmentsthe activation of prekallikrein, factor XI, andHageman factor. Thromb.Res. 20:173-89 42. Scott, C. F., Silver, L. D., Schapira, M., Colman,R. W. 1984. Cleavage of humanhigh molecular weight kininogen markedlyenhancesits coagulant activity. Evidencethat this molecule exists as a procofactor.J. Clin. Invest. 73:954-62 43. Bock, P. E., Shore, J. D., Tans, G., Gri~n,J. H. 1985.Protein-proteininteractions in contact activation of blood coagulation. Bindingof high molecular weightkininogenandthe 5-(iodoacetamido) fluorescein-labeled kininogen light chainto prekallikrein,kallikrein, and the separatedkallikrein heavyand

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antigen challengeof allergic individmastcell involvement.J. Clin. lnvest. 76:1374-81 uals. J. Clin. lnvest. 76:191-97 211. Baumgarten,C. R., Nichols, R. C., 221. Togias,A. G., Naclerio, R. M., Peters, Naclerio, R. M., Liehtenstein, L. M., S. P., Nimmagadda,I., Proud, D., Norman,P. S., Proud, D. 1986. PlasKagey-Sobotka,A., Adkinson, N. F. Jr., Norman, P. S., Lichtenstein,L. M. makallikrein during experimentallyinducedallergic rhinitis: Rolein kinin 1986. Local generation of sulfidopeptide leukotrienesuponnasal provoformation and contribution to TAMEcation with cold, dry air. Am. Rev. esterase activity in nasal secretions.J. lmmunol. 137:977-82 Respir. Dis. 133:1133-37 212. Hojima,Y., Cochrane,C. G., Wiggins, 222. Togias, A. G., Naclerio, R. M., Warner, J., Proud, D., Nimmagadda, R. C., Austen, K. F., Stevens, R. L. 1984.In vitro activation of the contact I., Norman,P. S., Lich~enstein,L. M. (Hageman factor) system of plasma 1986. Demonstrationof inhibition of heparin and chondroitin sulfate E. mediatorrelease fromhumanmastcells by azatadinebase: In vivo andin vitro Blood 63:1453-59 213. Baumgarten,C. R., Nichols, R. C., evaluation. J. Am. Med. Assoc. 255: Naclerio, R. M., Proud, D. 1986. Con225-29 centrations of glandular kallikrein in 223. Togias, A. G., Proud, D., Kageyhumannasal secretions increase during Sobotka,A., Norman,P., Lichtenstein, experimentally-induced allergic rhinitis. L., Naclerio, R. 1987. Theeffect of a J. lmmunol.137:1323-28 topical tricyclic antihistamineon the Proud, D., Baumgarten, C. R., response of the nasal mucosato chal214. Naclerio, R. M., Lichtenstein, L. M. lenge withcold, dry air and histamine. J. Allergy Clin. Immunol.79:599-604 1986. The role of kiains in human allergic disease. N. Engl. Reg. Allergy 224. Eggleston, P. A., Kagey-Sobotka,A., Proc. 7:213-18 Schleimer,R. P., Lichtenstein, L. M. 215. Proud, D., Baumgarten, C. R., 1983. Interaction betweenhyperosmolar and IgE-mediated histamine Naclerio, R. M., Ward, P. E. 1987. Kinin metabolismin nasal secretions release frombasophilsand mastcells. during experimentally-induced allergic Am.Rev. Respir. Dis. 130:86-91 rhinitis. J. Imtnunol.138:428-34 225. Silber, G., Naclerio,R., Eggleston,P., 216. Regoli, D., Marceau,F., Barabe, J. Proud,D., Togias,A., Lichtenstein,L. 1978. De novo formation of vascular M., Norman,P. 1985. In vivo release receptors for bradykinin. Can. J. of histamineby hyperosmolarstimuli. J, Allergy Clin. Immunol. 75: 176 Physiol. Pharmacol.56:674-77 217. Naclerio, R. M., Proud,D., Togias, A. (Abstr.) G., Adkinson,N. F. Jr., Meyers,D. A., 226. Naclerio, R. M., Gwaltney,J. M., HenKagey-Sobotka,A., Plant, M., Nordley, J. O., Eggleston,P., Baumgarten, man,P. S., Lichtenstein, L. M.1985. C. R., Lichtenstein, L. M., Proud, D. Inflammatorymediators in late anti1985. Kininsare generatedduring rhigen-inducedrhinitis. N. Engl. J. Med. novirus colds. Clin. Res. 33: 613A 313:65-70 (Abstr.) 218. MacGlashan, D. W.Jr., Schleimer, R. 227. Proud, D., Reynolds, C. J., LaCapra, P., Peters, S. P., Schulman,E. S., S., Kagey-Sobotka,A., Lichtenstein, Adams,G. K. III, Kagey-Sobotka,A., L. M., Naclerio, R. M. 1987. The Newball, H. H., Lichtenstein, L. M. responseto nasal provocationwith bra1983. Comparativestudies of human dykinin.J. Allergy Clin. Immunol.79: basophilsandmastcells. Fed. Proc.42: 254(Abstr.) 2504-9 228. Vavrek,R. J., Stewart, J. M. 1985. 219. Bascom,R., Pipkorn,U., Gleich, G. J., Competitiveantagonists of bradykinin. Lichtenstein, L. M., Naclerio, R. M. Peptides 6:161~4 1986. Effect of systemic steroids on 229. Newball,H. H., Keiser, H. R., Pisano, eosinophils (EOS)and majorbasic proJ. J. 1975. Bradykininand humanairtein (MBP)during nasal antigen chalways.Respir. Physiol. 24:139-46 lenge. J. Allergy Clin. Imrnunol.77 230. Herxheimer,H., Stresemann,E. 1961. (Suppl.): 246(Abstr.) Theeffect of bradykinin aerosol in 220. Togias,A. G., Naclerio, R. M., Proud, guineapigs and man.J. Physiol. London 158:38-39 D., Fish, J. E., Adkinson,N. F. Jr., Kagey-Sobotka, A., Norman,P. S., 231. Varonier,H. S., Panzani,R. 1968.The Lichtenstein, L. M. 1985. Nasal chaleffect of inhalation of bradykininon lengewithcold,dryair results in release healthy and atopic (asthmatic) chilof inflammatorymediators. Possible dren. Int. Arch.Allergy 34:293-96

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232. Fuller, R. W.,Dixon,C. M.S., Dollery, C. T., Barnes,P. J. 1986.Prostaglandin D2potentiates airwayresponsivenessto histamine and methacholine.Am.Rev. Respir. Dis. 133:252-54 233. Orehek,J., Gayrard,P., Smith,A. P., Grimaud,C., Charpin, J. 1977. Airway response to catbachol in normal and asthmatic subjects. Am. Rev. Respir. Dis. 115:937-43 234. Abe, K., Watanabe,N., Kumagai,N., Mouri, T., Seki, T., Yoshinaga, K. 1967. Circulating plasmakinin in patients with bronchial asthma.Experientia 23:626-27 235. Lukjan,H., Hofman,J., Kiersnoroska, B., Bielawiec, M., Chyrek-Borowska, S. 1972. The kinin systemin allergic states. Allerg. Irnmunol.Band.18: 2530 236. Christiansen, S. C., Proud,D., Zuraw, B. L., Cochrane,C. G. 1987.Inhibition of bronchialkallikrein by alpha- 1-proteinase inhibitor (~-I-PI) in asthma. Aller#y Clin. Immunol.79:257(Abstr.) 237. Donaldson,V. H., Rosen,F. S., Bing, D. H. 1977. Role of the second component of complement(C2) and plasrain in kinin release in hereditary angioneurotic adema(H.A.N.E.) plasma. Trans. Assoc. Am. Physicians 40: 174-83 238. Fields, T., Ghebrehiwet, B., Kaplan,A. P. 1983.Kinin formationin hereditary angioedemaplasma: Evidence against kinin derivation from C2 and in support of "spontaneous" formation of bradykinin. J. Allergy Clin. Immunol. 72:54~50 239. Curd,J. G., Yelvington,M., Burridge, N., Stimler,N. P., Gerard,C., Prograis, L. J. Jr., Cochrane,C. G., Muller-Eberhard, H. J. 1983. Generationof bradykinin during incubation of hereditary angioedemaplasma. Mol. lmmunol. 19:1365(Abstr.) 240. Strang, C. J., Auerbach,H. S., Rosen, F. S. 1986. Cls-inducedvascular permeabilityin C2-deficientguineapigs. J. Immunol. 137:631-35 241. Schapira,M., Silver, L. D., Scott, C. F., Schmaier,A.H., Prograis,L. J. Jr., Curd, J. G., Colman,R. W.1983. Prekallikrein activation and high-molecular-weight kininogenconsumption in hereditary angioedema. N. Engl. J. Med. 308:1050-53 242. Talamo,R. C., Haber, E., Austen, K. F. 1969. A radioimmunoassay for bradykininin plasmaandsynovialfluid. J. Lab. Clin, Med. 74:816-27 243. Curd,J. G., Prograis, L. F. Jr., Cochrane, C. G. 1980. Detection of active

kallikrein in inducedblister fluids of hereditary angioedemapatients. J. Exp. Med. 152:742-47 244. Ziccardi, R. J. 1982.Spontaneous activation of the first component of human complement (C1) by an intra molecular autocatalytic mechanism.J. lmrnunol. 128:2500-4 245. Cooper, N. R. 1983. Activation and regulation of the first componentof complement. Fed. Proc. 42: 134 (Abstr.) 246. Ghebrehiwet, B., Silverberg, M., Kaplan, A. P. 1981. Activation of the classical pathway of complementby Hagemanfactor fragment. J. Exp. Med. 153:665-76 247. Ghebrehiwet, B., Randazzo, B. P., Dunn,J. T., Silverberg,M., Kaplan,A. P. 1983. Mechanismof activation of the classical pathwayof complement by Hageman factor fragment. J. Clin. lnvest. 71:1450-56 248. Moskowitz,R. W., Schwartz, H. J., Michel,B., Ratnoff, O. D., Astrup, T. 1970. Generationof kinin-like agents by chrondroitinsulfate, heparin, chitin sulfate, andhumanarticular cartilage: Possible pathophysiologic implications. J. Lab. Clin. Med.76:790-98 249. Silverberg, M., Diehl, S. V. 1985. Autoactivation of humanfactor XII (Hagemanfactor): Effect of heparin and low molecularweight dextran sulfate. Blood 66(Suppl. I. No. 5): 343 (Abstr.) 250. Nagase,H., Cawston,J. E., DeSilva, M., Barrett, A. J. 1982. Identification of plasmakallikrein as an activator of latent collagenase in rheumatoid synovial fluid. Biochem.Biophys.Acta 707:133-42 251. Kellermeyer,R. W., Breckenridge,R. T. 1965. The inflammatoryprocess in acute goutyarthritis. I. Activationof Hageman factor by sodiumurate crystals. J. Lab. Clin. Med.63:307-15 252. Ginsberg,M., Jaques, B., Cochrane,C. G., Griffin, J. H. 1980.Uratecrystaldependentcleavage of Hageman factor in humanplasmaand synovial fluid. J. Lab. Clin. Med. 95:497-506 253. Morrison,D. C., Cochrane,C. G. 1974. Direct evidence for Hagemanfactor (factor XII) activation by bacterial lipopolysaccharides(endotoxins.). Exp. Med. 140:797-811 254. Pettinger, M. A., Young, R. 1970. Endotoxin-inducedkinin (bradykinin) formation: Activation of Hageman factor plasmakallikrein in humanplasma. Life Sci. 9:313-22 255. Kimball,H. R., Melmon, K. L., Wolff,

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KIN1NS AND INFLAMMATION S, M, 1972. Endotoxin-induced kinin production in man. Proc. Soc. Exp. BioL Med. 139:1078-82 256. Mason,J. W,, Kleeberg, U. R., Dolan, P, L., Colman,R. W. 1970. Plasma kallikrein and Hagemanfactor in gramnegative bacteremia. Ann. Int. Med. 73: 545-51 257. Hirsch, E. F., Nagajima, T., Oshima, G., Erdos, E. G., Herman, M. 1974. Kinin-sy.stem responses in sepsis after trauma m man. J. Sur#. Res. 17: 14753 258. Robinson, J. A, Klodynicky, M. L., Loeb, H. S., Recic, M. R., Gunnar, R. M. 1975, Endotoxin, prekallikrein, complement, and system vascular resistances. Sequential measurementin man. Am. J. Med. 59:61-67 259. O’Donnell, T. F., Clowes, G. H. Jr., Talarao, R. C., Colman, R. W. 1976. Kinin activation in the blood of patients with sepsis. Sury. Gynecol. Obstet. 143:539--45 260. Colman, R. W., Edelman, R., Scott, C. F., Gihnan, R. H. 1978. Plasma kallikrein activation and inhibition during typhoid fever. & Clin. Invest. 61: 287261. Alving, B. M., Hojima, Y., Pisano, J. J., Mason, B. L., Buckingham, R. E. Jr., Mozen, M. M., Finlayson, J. S. 1978. Hypotensionassociated with prekallikrein activator (Hagemanfactor fragments) in plasma protein fraction. N. Engl. J. Med. 299:66-70 262. Alving, B. H., Tankersley, D. L., Mason, B, L., Rossi, F., Aronson, D. L., Finlayson, J. S. 1980. Contact activation factors: Contaminants ofimmunoglobulin preparations with coagulant and vasoactive properties. J. Lab. Clin. Med. 96:33zl-46

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263. Mason, J. W., Colman, R. W. 1971. The role of Hageman factor in disseminated intravascular coagulation induced by sepsis, neoplasia, or liver disease. Thromb. Diath. Haemorrh. 26: 325-31 264. Minna, J, D., Robboy, S. J., Colman, R.W. 1974. Disseminatedlntravascular Coagulation in Man, pp. 70-76. Springfield, I11: Charles IL Thomas 265. Carvalho, A, C., Ellman, L. 1976. Activation of the coagulation system in polycythemia vera. Blood 47:669-78 266. Carvalho, A. C., Lees, R. S., Vaillancourt, R. A., Cabral, R. B,, Colman, R. W. 1978. Activation of the kallikrein system in hyperbetalipoproteinemia. J. Lab. Clin. Med. 91:117-22 267. Yamada,J., Herber, P., Pettit, G. W., Wing, D. A., Oster, C. N. 1978. Activation of the kallikrein-kinin system in Rocky Mountain Spotted Fever. Ann. lnt. Med. 88:764~68 268. Honig, G. R., Lindley, A. 1971. Deficiency of Hagemanfactor (factor XII) in patients with the nephrotic syndrome. J. Pediatr, 78:633-37 269. Lange, L. G. III, Carvalho, A., Bagdasarian, A., Lahiri, B., Colman,R. W, 1974. Activation of Hagemanfactor in the nephrotic syndrome. Am. J. Med. 56:56549 270. Wong, P., Colman, R. W., Talamo, R. C., Babior, B. 1972. Kallikrein-bradykinin system in chronic alcoholic liver disease. Ann. Int. Med. 77:205-9 271. Proud, D., Nakafnura, S., Carone, F. A., Herring, P. L., Kawamura, M., Inagami, T., Pisano, J. J. 1984. The kallikrein-kinin system and renin-an. giotension system in rat renal lymph. Kidney Int. 25:880-85

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IMMUNOBIOLOGYOF CR2, THE B LYMPHOCYTE RECEPTOR FOR EPSTEIN-BARR VIRUS AND THE C3d COMPLEMENT FRAGMENT Neil R. Cooper, Margaret D. Moore, and Glen R. Nemerow ResearchInstitute of Scripps Clinic, Departmentof Immunology, 10666North Torrey Pines Road, La Jolla, California 92037 INTRODUCTION The complementsystem is the primary mediator of the biological consequencesof antigen-antibodyreactions. It consists of at least 20 chemically and immunologicallydistinct plasma and cell membraneproteins which acquire the ability to interact with one another, with antibody, and with cell membranes after activation of the system(1, 2). Theseinteractions directly generate the various biologic activities that complement mediates andwhichrangefromlysis of different kindsof cells, bacteria, andviruses to direct mediationof inflammatoryprocesses. In addition, complement is able to enlist the participation of other humoraland cellular effector systemsandto inducehistaminerelease frommastcells, directed migration of leukocytes, phagocytosis, and release of lysosomalconstituents from phagocytes. Complement also possesses immunoregulatoryactions which are, as yet, poorlyunderstood. Withthe exceptionof its membrane disrupting, cytolytic functions, all of the biological activities of the complement system~re mediatedthrough complementreceptors. Complement receptors are cell surface structures that specifically interact with complement protein cleavageproductsproducedduring the activation process(2-4). There are one or moredistinct complement receptors on the surface of mosttypes of circulating cells and 85 0732-0582/88/0410-0085502.00

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on manytissue cells. Interactions of the complementligands with these specific cellular receptors trigger various responses including cellular activation,, secretion of mediators, ingestion, directed migration, or other activities, depending on the receptor engagedand the cell type. Of the complement factors, the third component of complement, C3, plays a central role in the complementreaction sequence as it is activated as a consequenceof triggering of the complementsystem by either of the two activation pathwaysand because it is a key constituent of both of the enzymesthat mediate assemblyof the terminal, cytolytically active, portion of the complementreaction sequence. C3 also possesses multiple biological activities mediated by binding of the six distinct C3 activation/processing fragments to specific C3 receptors on the surface of various types of cells. Twoof these receptors, CR1and CR2, are present on the plasma membraneof lymphocytes (3, 4). CR1, found on most B cells, a minor proportion of T cells, and manyother cell types, is a single chain glycoprotein which exists in several allotypic forms varying between 160 and 250 kd. CR1interacts preferentially with the initial activation/processing products of C3 and C4, C3b and C4b, respectively. The precise functions of this receptor on lymphocytes are not known, but recent evidence supports some type of immunoregulatory role (4-7). CR2is a single glycosylated polypeptide chain with a molecular weight of 145 kd (8-10) which interacts preferentially with the terminal activation/processing fragments of C3, C3dg, and C3d. CR2is present on B lymphocytes and follicular dendritic cells in lymphoidorgans (9, 11). The precise functions of this receptor are not known either, but appear to be immunoregulatory, a point considered in this review. Beginning approximately 12 years ago with work by Jondal, Yefenof, and other workers in George Klein’s laboratory (12, 13), a number studies have suggested that Epstein-Barr virus (EBV), a humanherpesvirus, uses a complementreceptor to attach to, and to infect, B lymphocytes. The EBVreceptor and a complement receptor are coexpressed on multiple B cell lines and co-induced in negative cell lines; the same treatments induce cocapping and costripping from B cell membranes(12-16). These and other shared properties correlate with CR2rather than CR1 expression (17). Epstein-Barr virus, a lymphotropic humanherpes virus, is a significant humanpathogen. After primary infection, which may be inapparent or be manifest as infectious mononucleosis(18, 19), the virus becomeslatent a minor proportion of B lymphocytes and oropharyngeal cells; more than 90%of individuals in the USpopulation carry the virus. It is a candidate humancancer virus, as it is strongly linked to nasopharyngeal carcinoma and Burkitt’s lymphoma,two malignancies, and it is oncogenic in sub-

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humanprimates (20-22). In vitro it transforms humanB lymphocytes, generating immortalpolyclonal lymphoblastoidcell lines (14). Furthermore, EBVis associated with the X-linked lymphoproliferativesyndrome (Duncan’sdisease) (23), and it maycontribute to the development cell neoplasiain certain patients with AIDS (24). Finally, it is associated with several humanautoimmune diseases (25-27). In addition to its ability to transform normalhumanB lymphocytes, EBVis also a polyclonal B cell activator (28-31). In contrast to immune B cell activation, EBV-induced B cell activation does not require the presence of T cells or macrophages (29, 31), and it is not dependent prior immunityto the virus (32, 33). Immune T cells and T cell factors have the ability, however,to modulateB cell activation inducedby EBV and by the other ligands of CR2(5, 25, 34). This reviewcritically examines older as well as recent studies pertaining to the B cell membrane structure(s) to whichC3fragmentsand EBVbind, and it analyzesthe functional consequencesof these interactions. While we emphasizeour views that the CR2complementreceptor initiates EBV activation, infection, and transformation, and that interactions of this receptor with the physiologicC3ligands regulate B cell function, studies suggestingalternative interpretations are also considered.Ourgoal is to provide a conceptual frameworkboth for understanding the status of research concerning CR2and for the design of future experiments to elucidatethe biological functionsof this B cell surfaceglycoprotein. RELATIONSHIP AND CR2

BETWEEN THE EBV RECEPTOR

The EBVReceptor Selective binding of EBVto B lymphocyteshas been demonstratedby a number of techniques--including cyto-adherence with EBV-producing cells (35) and binding of radiolabeled EBV(36)--and by several direct andindirect fluorescenttechniques(I 3, 14, 37). TheEBV receptoris readily cappedon addition of an antibodyto EBV(12), indicating that the receptor is mobile in the plasma membrane.Binding of EBVto B lymphocytesis saturable and, on addition of unlabeled EBV,reversible (36, Figure 1). Thesefeatures, together with the specificity and moderatelyhigh affinity of the interaction, indicate that EBV bindingto B cells is receptormediated. Extracts of Raji cell membranes have been observedto block EBVbinding to fresh Raji cells. Antibodyto such extracts detected a prominent 150 kd band in Raji cell membraneextracts, as shownby Simmonset al (38).

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~"-" x 24

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Peripheral Blood BCeils Figure 1 Binding of ~2~I-EBVto B lymphocytes. Increasing amounts of human B lymphocytes were reacted with a constant amount of ~2SI-EBV(O--O). Nonspeciflc binding, determined in the presence of a 100-fold excess of unlabeled EBV,is also shown(O--~).

The CR2 Receptor Althoughearlier shownto be distinct from other complementreceptors on the basis of functional criteria (39-41), the C3dbinding complement receptor, CR2,wasfirst characterizedas a discrete moleculeby Iida et al (9). Theseinvestigators isolated a 140 kd glycoproteinwith an isoelectric point of 8.2 from B cell membranesby affinity chromatography on Sepharose C3. This protein reacted with a B cell-specific monoclonal antibody, anti-B2, whichhad beenearlier developedby Nadleret al (42). Anti-B2partially inhibited C3drosetting, and this property was further amplified on addition of a polyclonal antibody to mouseimmunoglobulin. Shortly thereafter Weiset al (10) identified a secondB-cell-specificmonoclonal antibody, HB-5, whichwas reactive with a molecule of similar molecular weight; HB-5also inhibited C3d-dependentrosetting in the presence of a second antibody. Furthermore, protein A-containing particles preloaded with HB-5selectively adsorbeda 145-kd protein from labeled B lymphoblastoidcell membraneextracts. Neither anti-B2 nor HB-5interacted with the portion(s) of CR2responsible for binding C3d (9, 10~ 43). Other CR2reactive monoclonalantibodies termed OKB7 (36), AB1, AB2, AB3, and AB5(44), BL13(45), and RFB6(46) have subsequentlyidentified. Of these, only OKB7 effectively inhibits C3drosetting in the absenceof a secondantibody(36, 43, 47); polyclonalanti-CR2 antibodiesalso havethis property(48).

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CR2wasfirst isolated by Barel et al (48) by affinity chromatography Raji cell membraneextracts on C3 Sepharose, but it was erroneously characterized as a C3breceptor. At that time it wasnot knownthat Raji cells have CR2but lack CR1.CR2has also been purified from tonsil B lymphocytesby affinity chromatographyon immobilizedC3dg(49). and others have isolated the 145-kd CR2molecule in high yield and in functionally active form by immunoaffinity chromatographyon monoclonal antibodycolumns(50, 51). In contrast to these studies, Lambriset al (52) isolated a 72 kd glycoprotein (gp72) from the spent culture mediaof Raji cells by affinity chromatographyon C3dagarose. These workers found that rabbit antigp72 blocked C3d dependent rosetting to B lymphocytes and lymphoblastoid cells, but others, whohavealso isolated gp72and generated polyclonalantibody,wereunableto confirmsuch inhibition (53). Digestion of purified CR2and of Raji membraneextracts with trypsin and V-8 protease yielded multiple bands including somewith molecularweights in the 70-75 kd range, but these were not major fragments(43). Thus, the nature of gp72and the possibility that it is either a CR2fragmentor, alternatively, unrelated to CR2has not beenascertained. Relationship Between the EBV Receptor and CR2 Theevidencein favor of identity of these receptors is summarized below. Jondal, Yefenof, Einhorn, and other investigators workingwith George Klein (13-17) found the EBVand C3dreceptors coexpressedon multiple B cell lines. Theseinvestigators(12, 13, 55) also showed that the receptors capped together and independently of other B cell membranemarkers including surface Ig, HLA,and Fc receptors, and that both receptors were proteolytically stripped from the plasmamembrane by the sameenzymes. The receptors were also co-inducedin receptor negative cell lines from patients with undifferentiated lymphomas by treatment with theophylline (54). Their association was further suggestedby the observationthat blocked fusion of EBVwith B cell membranesas assayed by changes in fluorescence polarization (56). Furthermore,rosetting of C3d-coated particles with B cells wasimpairedby prior incubation of the cells with EBV (13, 57), and, in the reverse direction, sequential incubationof cells with C3followed by anti-C3 and anti-immunoglobulinreduced binding of virus (13). Althoughthe type of B cell complement receptor involved someof the earlier studies wasnot known,recent studies with monoclonal antibodies and well-defined reagents have clearly shownthat C3d-dependent rosetting is mediatedvia CR2and that Raji cells possess only CR2 (4, 9, 17,37, 58). Moredirect evidencefor identity of the EBVand C3dreceptors has come

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fromrecent studies of Fingerothet al (37). Theyfoundthat modulation CR2from the surface of B cells with the HB-5antibody, together with antimouseIg, blockedEBVbinding (37). In related studies, Tedderet (59) showedthat the same treatment impaired EBV-dependentB cell activation and Ig production. Protein Abearing S. aureusparticles preloaded with HB-5and incubated with detergent lysates of CR2positive cells were shownby Fingeroth et al (37) to bind radiolabeled EBV. have reported that the anti-CR2 monoclonal antibody OKB7 directly blocksbinding of C3dgmicrospheres,C3dbearing erythrocytes, and radiolabeled EBVto normal humanB lymphocytesand to Raji lymphoblastoid cells in the absenceof a secondantibody(36; Figure2). Consistentwith the

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0.2 0.6 1.0 1.4 1.8 Monoclonal Antibody Figure 2 Effect of monoclonal antibodies on 35S-labeled EBVbinding and infection. (A) Normaltonsillor B (left) or Raji (right) ceils (107) were reacted with various amounts OKB7(0--0) or anti-B2 (O --- O) antibody before the addition of 4000 cpm of labeled EBV.X Axes, monoclonal antibody (#g). (B) B cells (6 x 105) were reacted various amounts of OKB7(0) or anti-B2 (~,) antibody, washed, and then cultured for days in the presence of EBV.Infectivity was measured by the stimulation of DNAsynthesis ([3H]thymidine incorporation) and colony formation.

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inhibition of binding, OKB7 (but not anti-B2) directly blocked infection of normal B cells (36; Figure 2). Polyclonal anti-CR2 has also been found to directly block EBV infection (53). Recently, we reported that purified CR2 binds to purified EBV and C3dg immobilized on nitrocellulose (50; Figure 3). Isolated CR2 did not interact with cytomegalovirus, another human herpesvirus, murine leukemia virus, or with ovalbumin, a protein with physicochemical properties similar to those of C3dg. Specificity was further shown by the ability of OKB7, but not another anti-CR2 monoclonal antibody, to block binding of purified CR2 to both ligands (50; Figure 3). In other studies, we also found that phosphatidylcholine liposomes into which purified CR2 had been incorporated possessed the ability to specifically bind purified EBV to cells expressing EBV antigens on their membrane and to C3d coated erythrocytes (60); this binding was also inhibited by OKB7 monoclonal

Figure 3 Inhibition of receptor binding by anti-receptor MAb. A total of 2 pg of purified C3dg (lanes 1 through 3, upper panel) or ovalbumin (lane 4, upper panel) or 10 ng of EBV (lanes 1 through 3, lower panel) or CMV (lane 4, lower panel) was coated onto nitrocellulose. The blots were then incubated with 40 ng of CR2 alone (lanes 1 and 4; upper and lower panels), with CR2 which had been preincubated with 6 pg of OKB7 MAb (lane 2, upper and lower panels), or with AB-I MAb (lane 3, upper and lower panels). Reactivity was detected by the biotin-avidin system.

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antibody. Finally, study of somatic cell hybrids showed that the genes coding for CR2and the EBVreceptor cosegregate (17). The ability of the OKB7monoclonal antibody to bind to a single 145kd B cell membraneprotein, together with the ability of the purified 145kd B lymphocyte membrane protein to bind specifically to both EBV and C3dg unequivocally demonstrates that the 145-kd molecule is a dual receptor for both ligands. Since OKB7directly blocks EBVbinding to, and infection of, lymphocytes, CR2serves as a B cell receptor mediating both EBVpenetration into the cells and EBVmediated transformation. These data do not, however, unequivocally rule out the existence of either an additional EBVreceptor or another C3dg receptor on the same cells, although it is clear that such receptor(s), if present, can maximallymediate the binding of 10%of the added EBV(36; Figure 2). There are, however, other studies that suggest that the CR2complement receptor and the receptor mediating EBVbinding and transformation are not identical. Several of the results appear to be explainable on the basis of subsequently derived knowledge. One such study (57) used the above mentioned 72-kd B cell membrane as CR2. Gp72 neither inhibits EBV binding to, nor infection of, B cells (57), and antibodyto it does not inhibit EBVbinding (53). It clearly is not the CR2molecule that has been well characterized by manyinvestigators. Thus, the finding that human/mouse somatic cell hybrid clones (made from gp72/EBVreceptor positive human cells and gp72/EBVreceptor negative murine myelomacells as parents) express either gp72 or EBVreceptor function but rarely both properties (57) is not relevant for assessing the identity of CR2and the EBVreceptor. Wells et al (61) concluded that the EBVreceptor and CR2were different structures, on the basis of differential inhibition of virus and C3dbinding to various lymphoblastoid cells by various antisera to complementreceptors. These data were not corrected for the different numbers of EBVreceptors on the various cell types. Other interpretations of this study are also possible, notably that C3d and EBVbinding are properties of different epitopes on the same molecule. Several studies have noted the ability of phagocytic cells to bind EBV (62, 63). Such cells cannot be infected with EBV.The C3d binding structure on neutrophils and monocytesis a structurally different molecule (4, 63, 64). Certain T cell lines, null cells, and undifferentiated T cells bind EBV and express functional C3d receptors (4, 65, 66). In the case of the T cell line Molt-4, this has been found to reflect the presence of antigenically detectable CR2on the cells (8, 37). The nature of the C3d/EBVbinding structure on certain other cell types, which cannot be infected with EBV, remains to be determined. A numberof studies are presently unexplainable on the basis of the 145-

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kd CR2moleculeas the only EBVand C3dreceptor on B cells. The Ramos cell line, whichdoes not contain the EBVgenome(67), bears functional C3dreceptors and EBVreceptors (12, 67) but does not react with antiB2(68). EBVinfection inducedB2expression on these cells (69). These data are not presently explainableon the basis of CR2as a single C3dand EBVreceptor, but it should be kept in mind that anti-B2 only weakly stains mostB cell lines andlow’levelsof reactivity mighthavebeenmissed. Differential expressionof different CR2epitopesis also possible. Somestudies with pre-Bcells also do not readily support the existence of a single receptor for C3dand EBV,but results of several studies in this area are uninterpretable. For example,pre-Bcells from fetal bonemarrow andliver havebeenreported by Tedderet al (8) andby Bofill et al (70) lack the HB-5and RFB6CR2antigens, respectively. However,Hansson et al (71) were able to infect fetal bone marrowcells with EBV,and the derivedcell lines containedlow levels of the B2antigen and rosetted with C3dbearing erythrocytes. Similarly, Katamineet al (72) also derivedsome pre-B cell lines from fetal liver of the sameage by EBVinfection which possessedfunctional C3dreceptors. Hibi et al (73) also wereable to infect pre-B cells from fetal bone marrowwith EBV.B cell lines with pre-B phenotypewhich were generated from bone marrowof patients with Xlinked agammaglobulinemia by EBVinfection by Fu et al (74) contained functionalC3dreceptors. Clearly, the ability to infect pre-Bcells with EBV is in apparent contradiction with the lack of detectable CR2antigen on these cells. It is importantto keepin mind,however,that EBV infectibility and the fluorescent techniquesused to detect CR2antigens are unrelated techniqueswith very different sensitivities. TheEBV-infected cell lines may have represented the progenyof only a few cells or a subpopulationof cells bearing EBVreceptors and possibly CR2whichescapeddetection by immunofluorescence. Alternatively, both Tedderet al (8) and Bofill et (70) havereported the presenceof cells in certain fetal tissues (spleen, lymphnode, and tonsil) of the sameage that were CR2antigen positive; a fewsuch cells, if also present in bonemarrowor liver, could represent the cells that wereinfected by EBV.In addition, since EBVinfection is a single hit process (75, 76), a single EBVvirion and therefore only a few receptors on a pre-Bcell are neededfor infection. Suchlevels could have escaped detection by immunofluorescence.Still other explanations are possible, e.g., (a) the receptors maydiffer on pre-Bcells; (b) pre-Bcells maybe infected in a different mannerby EBV,or (c) there may differential expression of EBVreceptor and CR2epitopes on fetal pre-B cells. Also unexplainableat present is the absenceof EBVreceptors and EBV noninfectibility of B cells from patients with the common, nonsecretory

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form of agammaglobulinemia coupled with the presence of C3dreceptors on their B cells, as assessedby C3d-dependent rosetting (77). In the reverse direction, a single cell line established by EBVinfection of normalperipheral bloodB cells failed to express CR2whetherassayedby reactivity with anti-B2, HB-5,or by C3d-dependent rosetting; but it did express low levels of EBVreceptors (78). Similarly, cell sorting studies showedthat significant proportion of the cells susceptible to transformationby EBV werepresent in the anti-B2negativecell population(79). CELL AND TISSUE DISTRIBUTION EBV RECEPTOR AND CR2

OF THE

EBVwas early demonstratedto selectively infect normal humanB lymphocytesin vitro, and this led to Ig productionandindefinite proliferation (29, 35). EBVprimarily infects mature, high density, resting, GoB lymphocytesbearing complement receptors (80-82). Therelationship between complement and EBVreceptors has been considered in the section above. EBVtransformedB cell lines derived from normalB cells are polyclonal and vary in size, morphology,and expressionof surface markers; all have diploid karyotype(68, 80, 83). Their surface markerpattern resemblesthat of mitogen-activatednormalB cells, and such cell lines often expressIgM and IgDand secrete large amountsof Ig (80, 84). Althoughtransformation of single cells follows single hit kinetics, only a minor proportion of complementreceptor positive lymphocytesbecometransformed (28, 32, 74, 75, 76). EBVinfection is capableof inducingonly limited maturation, suchas inductionof Ig synthesis; it does not lead to Ig generearrangement or Ig class switch, although more pronouncedmaturation mayoccur on occasion (69). Thus, individual cells transformedby EBVsecrete the characteristic of the stage of development wheninfection occurred(25, 72, 75, 85). Burkitt lymphoma-derived cell lines, whetheror not EBVinfected, are monoclonaland never have a diploid karyotype; they also exhibit chromosomaltranslocations involving Ig loci and the c-myc oncogene (80); mostare surface IgDnegative andpoor Ig secretors (68). Mostbut not all bloodand tissue B cells, B lymphoblastoidcell lines, and B cells frompatients with B cell leukemiasand lymphomas react with anti-CR2monoclonalantibodies (8, 42), while bloodand tissue monocytes, NKcells, granulocytes,and normaland activated T cells as well as T cell lines (except Molt-4 and cells frompatients with certain T cell malignancies) do not. Monocytesand neutrophils also contain a C3d-binding molecule(62, 63), but the structure of this moleculeis entirely different from CR2and has been termed CR4(4, 63, 64). A recent report (86)

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suggests that platelets might possess CR2,but the possibility of sufficient B cell contamination to explain these results was not rigorously excluded; others have failed to find CR2on platelets (87). In lymphoidtissues significant differences in the reactivity of various monoclonal anti-CR2 antibodies have been observed. Anti-B2 reacts weaklywith B cells in the peripheral area of primary follicles and in the mantle zone of secondary follicles, while reacting intensely with cells in the germinal centers (88). Other antibodies with likely similar or identical specificity, BL13and RFB6,have a similar pattern of reactivity (45, 46). Anti-B2reacts weaklywith peripheral blood B cells (42, 88, 89). In contrast, HB-5reacts strongly and homogeneously with both germinal center and mantle zone cells in the secondaryfollicle (45); it also reacts strongly with peripheral blood B cells (8). Somecells in T dependent areas are also stained with HB-5but not with BL13(45). In addition, anti-B2, BL13, and RFB6have been found to stain follicular dendritic cells in germinal centers (11, 45, 46, 88). The EBVgenomeis found in epithelial cells taken from patients with nasoepithelial carcinoma (90, 91), although numerous attempts to show EBVreceptors on nasoepithelial cells or to infect the cells with laboratory EBVadapted strains have been unsuccessful (92, 93). Studies in which the membranebarrier was bypassed by implantation of receptor or by other techniques have shown that such cells can be infected with EBV(93). Sixbeyet al (94) wereable to infect primary epithelial explant cultures with EBVpatient isolates but not with laboratory adapted EBVstrains as manifested by induction of EBVDNAand EBVVCAantigens in the cells. Recent studies with two anti-CR2 monoclonal antibodies, HB-5and antiB2, have shown that both antibodies react with humanpharyngeal epithelium in a cell differentiation-dependent manner (95). These findings point to the clear possibility that EBVmaydirectly enter and infect human epithelial cells at particular stages of differentiation in vivo. Further study in this area is needed. CR2 EXPRESSION DEVELOPMENT, DIFFERENTIATION

DURING B CELL ACTIVATION AND

Despite the relatively restricted numberof B cell functions, whichlargely center on antigen presentation, antibody production, and induction of B memorycells, B cells are remarkably heterogeneous in terms of the number and diversity of molecules that can be detected on their membranes.B cell development from stem cells to pre-B cells, early B cells, and mature B

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Mature B Cell

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B Cell Development (Antigen-independent)

Activated B Cells

Plasma Cell

B Cell Differentation ~ ,,--=(Antigen or polyclonal~ activator dependent)

Figure 4 B cell maturation/activation/differentiation sequence. Surface structures are shown./z denotes IgM heavy chain; CR1 and CR2, the complement receptors; Ia, MHC class II antigens; FcR the Fc receptor; Grf, receptors for growth factors; TID, a 35 kd glycoprotein and Y, complete immunoglobulin molecules.

cells, is often considered to be a linear progression (96-99) and is presented as such in Figure 4. The expression of many B cell membranemarkers changes dramatically during B cell maturation, activation, and differentiation. CR2 Expression

Duriny

B Cell

Development

The ability to bind EBV, to rosette with C3d-bearing particles and to react with monoclonal antibodies to CR2have been described as B cell development/differentiation markers restricted to a limited portion of the B cell development sequence (8, 42, 46, 89). However, as noted earlier, various investigators have reported differential expression of these three properties by fetal B cells (8, 70-74). Variable expression of CR2antigens by pre-B cells from various tissues has also been observed (8, 70). Also, adult pre-B cells have been found by Hibi et al (73) to express the CR2 antigens detectable with anti-B2 and HB-5, and to be infectible with EBV, while Campanaet al (46) reported such cells to be negative for CR2 assessed with the RFB6anti-CR2 monoclonal antibody. Clarification of the ontogeny of CR2requires further study. Tosato et al (100) have derived a cell line from normalperipheral B cells by EBVinfection that lacks intracytoplasmic and membraneIg and thus resembles an immature B cell. However, since the cell line contained rearranged immunoglobulin genes, it is more likely derived from a more mature B cell that has undergone ineffective Ig rearrangement or contains a defect in transcription of the Ig genes. Its lack of detectable CR2as assessed with anti-B2 or HB-5is unexplained but could reflect the loss in CR2that occurs on proliferation of activated B cells (see below).

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Alterations in CR2 Expression During B Cell Activation and Differentiation Changesin B cell expression of CR2during B cell activation were first reported by Stashenkoet al (89). Theseworkers assessed reactivity humanperipheral blood B cells with anti-B2 after activation with pokeweedmitogen (PWM).CR2expression transiently increased on day 3 and then disappeared by day 4-5. Activated humanspleen and tonsil B cells lost CR2with the samekinetics but did not exhibit the transient increase observedwith the peripheral B cells. CR2loss wastemporally associated with loss of surface IgD, development of intracytoplasmicIgM, and expression of a plasmacell marker(Figure 4). TheB1 antigen also disappeared 6-7 days after activation with PWM, coincident with the appearance of intracytoplasmic and membrane IgG (C6), Similar loss anti-B2reactivity by splenic B cells followingactivation with anti-Ig has beenshownby Boydet al (101) and by others (8, 88, 89). Markedreduction in B2antigen expression has also been observed18 hours after EBV-induced B cell activation (102), likely reflecting the same process although internalization of the receptor together with EBVwas not excludedin_ these studies. MatureB cells as well as spontaneously activated large B cells bind and internalize EBVand mayproduce Ig althoughthey do not alwaystransform(32, 73, 80, 81). Further study has shownthat while B cells in the resting state (Go)or newlyactivated with mitogen or anti-# (G1) can be transformed with EBV,cells that have entered the S phaseof the cell cycle do not (82, P. Casali, personal communication).Thesestudies indicate that CR2is lost with activation and entry into the cell cycle and proliferation. It has beendocumented with anti-B2 (88, 89) and HB-5(8) that plasmacells lack CR2(Figure

PHYSIOCHEMICAL ANALYSIS STRUCTURE AND ANTIGENIC

OF CR2 EXPRESSION

Biosyntheticapproaches(103) as well as enzymaticstudies (43) haveshown that CR2is derived from a 111-l15-kd nonglycosylated precursor molecule. The fully glycosylated 145-kdCR2glycoprotein contains complex N-linked but not O-linked oligosaccharides (103). Nonglycosylated CR2, high mannose containing CR2, and mature CR2have all been observed to bind to Sepharose-conjugatedC3, indicating that carbohydrate residues are not necessary for CR2function. The presence of oligosaccharides was also found to be unnecessaryfor plasma membrane expression of CR2(103). However,the half-life of nonglycosylatedCR2

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on the plasma membrane was greatly reduced compared to the mature molecule. CR2has been assigned to the CD21antigen cluster of B cell differentiation.(104). Four monoclonal antibodies to CR2(OKBT,HB-5, antiB2, and AB-1) have been found to recognize distinct and nonoverlapping antigenic epitopes on the CR2molecule (43); anti-B2 and HB-5had been earlier found to be nonoverlapping (8). Another anti-CR2 antibody, BL13, has been found to recognize an epitope closely associated with or identical to the B2 epitope, and nonoverlapping with HB-5(45). The RFB6 anti-CR2 monoclonal antibody has a pattern of reactivity with B cells similar to that of anti-B2 (46, 70). Several of these m0noclonalantibodies have been found to have different functional properties. Thus, OKBT,but neither HB-5(36, 37), anti-B2 (36), or AB-1(34), directly blocked EBVbinding to and infection lymphocytes in the absence of a second anti-Ig antibody (Figure 2). addition, OKB7(37) but not HB-5 (10, 34) also directly blocked function; anti-B2 (9, 34), BL-13(45), and AB-1(34, 44) had limited ability to do so. OKB7(34) and AB-1 (34, 44) have also been observed to mitogenic for B lymphocytes in the presence of T cells; anti-B2 had some activity in this regard while HB-5was completely inactive (8, 34). These various data indicate that the 145-kd B cell EBV/C3dreceptor possesses discretely structural domainswith distinct functional correlates. OKBT, HB-5, and AB-1 also fully immunoprecipitate CR2 from lymphoblastoid B cell membraneextracts, while anti-B2 precipitates only a portion of the membraneCR2(43). Also anti-B2 (42, 88, 89), BL-13(45), and RFB6 react only with some B cells in lymph nodes and weakly with peripheral blood B cells, while HB-5(45) reacts strongly with all B cells in tissues and with peripheral blood B cells as noted earlier. These latter observations suggest that differential expression of CR2epitopes mayoccur on someB or subsets of B cells. MOLECULAR THE EBV/C3d

CLONING OF RECEPTOR

THE

cDNA

ENCODING

Weis et al (105) isolated a partial cDNACR2clone from a tonsil B cell ¯ library cloned in 2gt 11. Althoughrelatively little CR2nucleotide or amino acid sequence was presented in the report, several important observations were made. The mRNA-encodingCR2was shown to be approximately 5.0 kb by Northern blot analysis. CR1and CR2were found to be structurally related to the DNAlevel, since a CR1cDNAclone cross-hybridized with

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CR2eDNA at low stringency (105). At the protein level, there is sequence homology, and both show a 60-75 amino acid consensus repeat motif containing6-10 conservedresidues. This structural feature is foundin six other C3binding proteins (106), as well as in several unrelated proteins includingthe interleukin-2receptor (106, 107). Klicksteinet al (108) proposeda CR1modelin whichthe 60-75 aminoacid repeating elements are arranged linearly to form a highly elongatedmembrane protein extending 500 A~fromthe cell surface. This structural modelhas not yet been verified. The genes encodingboth CR1and CR2are located on the long arm of humanchromosome 1 at band 1Q32(109) suggesting that these receptors mayhave arisen from a common ancestral gene through duplication and that there maybe a complement receptor locus on chromosome 1. The other non C3-bindingproteins sharing the 60-75 aminoacid consensus repeat are on other chromosomes (109). Wehave also employed2gtl 1 for the molecular cloning of eDNA encoding the EBV/C3dg receptor (M. D. Moore, N. R. Cooper, G. R. Nemerow,manuscript submitted). ComplementaryDNAwas synthesized from polyadenylatedmRNA from Raji B lymphoblastoidcells. The library wasscreened with a uniquesequence39meroligonucleotide probereported by Weis et al (105) to correspondto a region in the 5’ portion of CR2 cDNA.Withthis probe we isolated two clones designated 2A11and 2F11 whichhybridizedto the probeon three successiveroundsof screening. As shownin Figure 5, both of these clones containedEco Rl-derivedinserts of approximately1.2 kb. As shownin Figure 5, the isolated ,~A11insert hybridized to a 4.7-kb RNAfrom Raji B cells on Northernblots but not from humanHSB-2T cells. Theseproperties are consistent with the size and distribution previously reported for CR2mRNA. Using the 1.2-kb insert, the library was rescreened and two morecDNA clones designated 2E41 and 2Lll were isolated. As shownin Figure 6, the )~E41 clone contained Eco R1derived inserts of approximately1.8, 1.6, and 0.8 kb (4.2 kb total) whilethe 2L11clonecontaineda single insert of 1.2 kb. The 1.8 kb Eco R1fragmentof clone E41and the 1.2 fragmentof clone L11 hybridizedto the hA11insert on Southernblot analysis (Figure 6). Escherichiacoli wereinfected with purified ~,E41phageand inducedto synthesize protein. Plaques containing recombinantprotein were replicated onto nitrocellulose filters and screenedwith either rabbit anti-CR2 or normal rabbit serum. Immune (but not nonimmune serum) reacted with 2E41plaques; other control plaques generatedby irrelevant clones failed to react. Approximately90%of the 4.2-kb 2E41.CR2clone has been sequenced on both strands. A summaryof the restriction mappingand sequence

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Figure 5 Restriction enzyme and Northern blot analysis of ICR2 cDNA clones. IAl1 and IF1 1 DNA was digested with Eco R1, electrophoresed on a 1% agarose gel and stained with ethidium bromide (A). The isolated 1.2-kb insert of I A l was nick translated with "P and used to probe total RNA (20 pg) or poly A + RNA (2 pg) from CR2 positive Raji cells or CR2 negative HSB-2 cells (B).

analysis of the CR2 clones is shown in Figure 7. A major feature of the CR2 sequence is that the entire extracellular domain excluding only the putative signal peptide is comprised of tandem 60-75 amino acid repeat motifs (Figure 7) which share the above noted homology with a number of C3-binding and other proteins. The E41.CR2 cDNA encoding CR2 contains 16 such repeating elements.

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Figure 6 Restriction enzyme and Southern blot analysis of 1CR2 cDNA clones. LE41 and LLl I cDNA clones were digested with Eco R1 and analyzed on a 1.5% agarose gel (left panel). '%-labeled LA11 insert was used to probe a Southern blot of the same gel (right panel). 1-kb ladder and 123-bp ladder markers were run in lanes 1 and 2, respectively.

BIOLOGIC FUNCTIONS OF CR2 B cells serve as effective antigen presenting cells. In addition the B lymphocyte and its end-stage differentiation product, the plasma cell, are the sole producers of antibody molecules. Finally, the B cell also gives rise to the memory cell which is in part responsible for the rapid, heightened appearance of specific antibody on secondary encounter with antigen.

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COOPER, 1~

MOORE & NEMEROW 2k~

I

I

3kb

~

4kb

4.7kb

l 1~

CR2 mRNA

3 Probe AAII, IFII, ALl1

l.Skb

1.6kb

0.Skb i

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~ ,, ,

NH2

COOH

~I f~, J I I I I I I I I I [ I I I I K-~ Signal Figure 7

~E41

CR2 Protein

Transmembran’e Restriction

map of 2CR2 clones

and the deduced CR2 protein.

Such functions of B cells are generally perceived to be dependent on various types of interactions betweenB cells, T cells, and accessory cells together with soluble growthand regulatory factors secreted by these cells, although the molecular mechanismsinvolved have not yet been elucidated. The CR2 receptor on B cells is undoubtedly involved in one or more of the abovelisted B cell functions. It is appropriate at this point to review the studies that documentvarious functional properties of CR2and CR2ligands. CR2is present on B cells only during a restricted portion of the B cell maturation sequence and disappears uponentry of B cells into proliferation; it also disappears with terminal differentiation into plasmacells (8, 42, 46, 89). Thus, it clearly cannot play a role either in the earliest or latest stages of B ¢¢11differentiation. Someinsights into the functions of this receptor can be gleaned from studies with EBV. EBValone does not induce Ig heavy chain gene rearrangement and isotype switching; it also does not induce terminal differentiation since EBVtransformed Ig secreting cells are morphologically and phenotypically different from plasma cells (25, 72, 75, 85). EBVdoes, however, induce some differentiation since EBVinfected cells acquire the ability to secrete significant amountsof Ig (25, 28, 75, 76, 110). Induction of indefinite B cell proliferation is independent of the transforming activity of EBVbecause only a minor proportion of B cells induced by EBVto proliferate go on to form immortal cell lines (32, 73, 110). Furthermore, the nontransforming P3HR1EBVstrain induces

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proliferation without immortalization (25, 111) as does the UV-irradiated transforming MCUV5 EBVstrain (112). These proliferative effects defective EBVrequire T cell factors (25, 111, 112). Other CR2ligands also have the ability to activate or modulate B cells. Thus, Sepharose-bound humanC3d stimulates preactivated immuneB cells to proliferate (5, 113) and soluble C3dinhibits proliferation in the presence of appropriate monocyte-derived growth factors (5). Certain monoclonal antibodies to CR2 and polyclonal anti-CR2 induce B cell proliferation and Ig secretion (34, 44, 114), effects that require T cells or T cell factors. The OKB7 monoclonal antibody has also been found to modestly enhance the generation of Ig secreting cells in a PWM driven system (115). However,these systems are complex, and manyaspects are not understood. Thus, the role is unclear of monocytefactors, indicated as necessary for stimulation of murine B cells by cross-linked C3d(5, 113), and of T cells or T cell factors found be required for proliferation of B cells by nontransforming EBV(25, 11 l, 112) and monoclonal and polyclonal anti-CR2 antibodies (34, 44, 114). This is particularly true since transforming EBValone is a highly efficient T cell-independent B cell activator (29, 31). Also it is not understoodthat murine B cells apparently require preactivation in order to be stimulated by Sepharose-bound C3d (5, 113), but this is not required for human cell activation by transforming EBV(29, 31), nontransforming EBV(25, 111, 112), and antibodies (34, 44, 114). There are other complexities: contrast to their behavior with preactivated murine B cells, Sepharosebound C3d and cross-linked C3d do not stimulate human B cells to proliferate either directly or in the presence of T cell factors; nor do they modulate B cell activation produced by other factors (59; J. F. Bohnsack, G. R. Nemerow,N. R. Cooper, unpublished observations). Also some but not other antigenic epitopes on CR2stimulate B cell activation; thus, OKB7and AB-1activate while anti-B2 and HB-5 do not (8, 34, 44, 59). Furthermore, induction of B cell activation by OKB7is variable since earlier but not later batches of OKB7 have been found stimulatory (34; J. F. Bohnsack, G. R. Nemerow,N. R. Cooper, unpublished observations). Despite these uncertainties, collectively these various studies indicate that under certain circumstances CR2ligands may either produce or modulate B cell activation. Also pointing to the involvement of CR2in a B cell activation pathway is the finding that CR2becomes phosphorylated on activation of human B cells with phorbol ester (116), anti-#, or S. aureus (117). Whetherthe natural ligands, EBVand C3d, or other ligands have the ability to induce phosphorylation is not known. Since CR2does not autophosphorylate (116, 117) and the cytoplasmic tail of CR2is too short itself to serve

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a protein kinase (see above), another protein kinase must be responsible. The CR2receptor is mobile in the plasma membraneof B lymphocytes, as is indicated by the ability to cap EBVon the B cell with anti-EBV(12). This is also true of CR2on addition of HB-5followed by anti-Ig (10). have shownthat CR2mediates internalization of EBVinto normal B cells and B lymphoblastoid cells (118, Figure 8). With normal B cells, this process proceeds via an endocytic pathway and involves the participation of multiple elements of the cytoskeleton (118, 119; G. R. Nemerow,N. Cooper, unpublished observations). In addition, EBVinternalization is calmodulin dependent, indicating also a calcium requirement (119). Calcium is required also for multiple aspects of transmembranesignalling in B lymphocytes (reviewed in 120). These findings lend further support the involvementof this receptor in B cell activation. The natural ligands for CR2are C3 activation/processing fragments. Manystudies over the past 15 years have suggested that complement, and particularly C3, regulates certain immunereactions. There are repeated findings that C3 fragments modulate numerous B and T cell responses in vitro (121-123), and it was suggested early that C3 might mediate T-B cell collaboration since responses to certain T dependent antigens were impaired in the absence of C3 both in vivo (124-126) and in vitro (126). This dependence is not absolute, however, since C3 not essential for many T dependent responses (127). Its role in T dependent antibody responses is thus unclear. Early reports that C3 might provide an essential second signal (in addition to antigen) for T cell-independent B cell triggering (126) were disproved by manystudies showing that antigen and C3 binding to B cells are not sufficient for triggering (128, 129). Early as well as subsequent studies have indicated that C3 facilitates antigen localization in lymphoidfollicles since antigen, antibody, and C3 colocalize there normally (127) but not in C3 depleted animals (130). memory cells also are found in germinal centers (131, 132), and this process is C3 dependent (127, 130, 131). One characteristic feature of germinal centers, which develop in peripheral lymphoid tissues after immunization, is the presence of a unique cell type, the follicular dendritic cell (FDC). These cells possess complementreceptors, including CR2(l l, 45, 46, 88) and retain undegraded antigen-antibody complexes, likely as antigenantibody-complement complexes, on their surface for a long time (133). CR2are also on B cells in the germinal centers and in the mantle zones in secondary lymphoidfollicles (11, 46, 88). These observations suggest that C3 fragments attached to complement receptors on B cells and on FDC may participate in the generation of B memorycells in lymph node germinal centers (132, 134).

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CONCLUSIONS Muchremains to be learned about the biologic functions of CR2. It is important to keep in mind that the natural ligands of this receptor are the terminal processing fragments of C3 activation, C3dg or C3d. These fragments remain covalently bound to complement activators. Most immunecomplexes activate the complementsystem and therefore bear C3 activation/processing fragments. It is not generally appreciated that many and probably most pathogens and substances with pathobiological potential directly activate complement,in the absence of antibody, although antibody can augment the activation (1, 2). Thus, immunecomplexes well as pathogenic agents are likely coated with complementfragments, including C3dg, in vivo and thus have the potential to bind to CR2on FDCand B lymphocytes. It is tempting to speculate, as have Klaus & Humphrey(132, 134), that binding of immunecomplexes and complement activating pathogens to FDCin lymphoid tissues contributes to the generation of B cell memory.This is a particularly appealing hypothesis in view of the C3 dependence of B memorycell induction, the location of FDCin germinal centers in lymphoid organs, and the ability of FDCto retain surface antigen for prolonged periods of time. Surface Ig antigen receptors on B cells can provide the specificity that complementlacks. B cells with the appropriate specificity could potentially react with antigens in the immunecomplex or pathogens which have reacted, via C3dg with CR2on the FDC, as also postulated by Klaus & Humphrey(132, 134). Separate from these considerations, CR2ligands in the presence of T cells or T cell factors either modulate B cell activation or induce limited proliferation and Ig secretion, as noted earlier. Consistent with these functions are the findings that B cell activation is accompaniedby CR2 phosphorylation and that CR2is a participant in an endocytic pathway which is calcium and calmodulin dependent. Phosphorylation plays a role (in a manneras yet incompletely understood) in signal transduction and the regulation of numerousintracellular processes within cells, particularly those involving growth and other differentiated cellular functions; calcium and potentially calmodulin-dependent processes also function in B cell activation. These various findings point to a role for CR2in cross-membrane transmission of B cell surface signals. Furthermore, the ability of CR2ligands, with T cell help, either to activate, or modulate the stimulation of B cells implies that CR2fulfills similar functions in B cell activation as it normally occurs during antigenic triggering of immune responses. NormalB cell activation, initiated by antigen binding to B cell antigen receptors, is dependent upon and regulated by cell-cell contacts and the actions of multiple factors emanating from T cells and accessory

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cells. Immunecomplexes and other complementactivating pathogens with pathobiological potential have the potential to interact simultaneously with antigen receptors and also complementreceptors not only on B cells but also on T cells and accessory cells. Ampleopportunities exist for complementreceptor-mediated regulation not only of B but also of T cell activation. Manylaboratories are using modern biochemical and genetic techniques to explore the molecular features and functional consequences of cognitive interactions between cells and regulatory cytokines of the immunesystem. These studies will undoubtedly also provide insights into the immunoregulatory functions of C3 and of CR2. ACKNOWLEDGMENTS

The authors wish to thank Bonnie Weier for her assistance in preparation of the manuscript, Publication No. 4953-IMM.This research was funded by NIH Public Health Service grants: AI17354, AI25016, CA14692, CA36204, PEWScholars Award for Biomedical Science.

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brane expression and ligand binding. d. Biol. Chem.260:13824-30 104. Reinherz,E. L., Haynes,B. F., Nadler, L. M., Bernstein,J. D., eds. 1986.LeukocytetypingII. In Proeeedin~Ts of the Second International Workshop on Human LeukocyteDifferentiation Antigen. NewYork: Springer-Verlag. 549 PP. 105. Weis,J. J., Fearon,D. T., Klickstein, L. B., Wong,W.W., Richards, S. A., deBruynKops, A., Smith, J. A., Weis, J. H. 1986.Identification of a partial cDNAclone for the C3d/Epstein-Barr virus receptor of humanB lymphocytes: Homologywith the receptor for fragmentsC3band C4bof the third and fourth components of complement. Proc. Natl. Acad. Sci. USA 83:5639-43 106. Reid, K. B. M., Bentley, D. R., Campbell,R. D., Chung,L. P., Sim,R. B., Kristensen, T., Tack, B. F. 1986. Complementsystem proteins which interact with C3bor C4b. Immunol. Today 7:230-34 107. Leonard, W. J., Depper, J. M., Kanehisa,M., Kronke,M., Peffer, N. J., Svetlik, P. B.~Sullivan,M., Greene, W.C. 1985. Structure of the human interleukin-2 receptor gene. Science 230:633-39 108. Klickstein, L. B., Wong,W.W.,Smith, J. A., Weis, J. H., Wilson, J. G., Fearon, D. T. 1987. HumanC3b/C4b receptor (CR1).Demonstrationof long homologousrepeating domains that are composedof the short consensus repeats characteristic of C3/C4binding proteins. J. Exp. Med.165:1095-1112 109. Weis,J. H., Morton,C. C., Bruns, G. A. P., Weis,J. J., Klickstein, L. B., Wong,W.W., Fearon, D. T. 1987. A complement receptor locus: Genes encoding C3b/C4b receptor and C3d/Epstein-Barr virus receptor map to Iq32. J. lmmunol.138:312-15 110. Chan, M. A., Stein, L. D., Dosch, H. M., Sigal, N. H. 1986. Heterogeneityof EBVtransformable human B lymphocytepopulations. J. Immunol.136: 106-12 111. Ho, C., Aman,P., Masucci,M., Klein, E., Klein, G. 1986.Bcell activation by the nontransforming P3HR-1substrain of the Epstein-Barrvirus (EBV). Eur. J. Immunol.16:841-45 112. Hutt-Fletcher, L. M. 1987.Synergistic activationof cells by Epstein-Barr virus andB cell growthfactor. J. Virol. 61: 774-81 113. Melchers, F., Erdel, A., Corbel, C., Leptin, M., Schulz, T., Dierich, M.P.

1986. Cell cycle control of activated, synchronizedmurine B lymphocytes-roles of macrophagesand complement C3. Molec. Immunol.23:1173-76 114. Frade, R., Crevon,M. C., Barel, M., Vazques,A., Krikorian, L., Charriaut, C., Galanaud, P. 1985. Enhancement of humanB cell proliferation by an antibodyto the C3dreceptor, the gp 140 molecule.Eur. J. Immunol.15:73-76 115. Mitler, R. S., Talle, M.A., Carpenter, K., Rao, P. E., Goldstein, G. 1983. Generation and characterization of monoclonalantibodies reactive with humanB lymphocytes. J. Immunol. 131:1754-61 116. Changelian,P. S., Fearon, D. T. 1986. Tissuespecific phosphorylationof complement receptors CR1and CR2. J. Exp. Med. 163:101-15 117. Barel, M., Vazquez,A., Charriaut, C., Aufredou,M. T., Galanaud,P., Frade, R. 1986. gpl40, the C3d/EBV receptor (CR2),is phosphorylateduponin vitro activation of humanperipheral B lymphocytes. FEBSLett. 197:353-56 118. Nemerow, G. R., Cooper, N. R. 1984. Early events in the infection of human B lymphocytesby Epstein-Barr virus: Theinternalization process. Virology 132:186-98 G. R., Cooper, N. R. 1984. 119. Nemerow, Infection of B lymphocytesby a humanherpesvirus,Epstein-Barrvirus, is blocked by calmodulin antagonists. Proc. Natl. Acad. Sci. USA81: 495559 120. Cambier,J. C., Ransom,J. T. 1987. Molecular mechanismsof transmembranesignaling in B lymphocytes.Ann. Rev. Immunol.5:175-99 121. Meuth,J. L., Morgan,E. L., DiScipio, R. G., Hugli, T. E. 1983. Suppression of T lymphocytefunctions by human C3fragments. J. Imrnunol.130: 260511 122. Thoman,M. L., Meuth,J. L., Morgan, E. L., Weigle,W.O., Hugli,T. E. 1984. C3d-K,a kallikrein cleavage fragment of iC3bis a potent inhibitor of cellular proliferation. J. Immunol.133: 262933 123. Hobbs,M.V., Feldbush,T. L., Needleman,B. W., Weiler, J. M. 1982. Inhibition of secondaryin vitro antibody responses by the third componentof complement.J. Immunol.128:1470-75 124. Pepys, M.B. 1972. Role of complement in inductionof the allergic response. Nature 237:157-59 125. Pepys, M.B. 1974. Role of complement in induction of antibodyproductionin vivo. J. Exp. Med.140:126-45

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IMMUNOBIOLOGY OF CR2 126. Dukor, P., Schumann,G., Gisler, R. H., Dierich, M., Konig,W., Hadding, U., Bitter-Saermann, D. 1974. Complement dependentB cell activation by cobra venomfactor and other mitogens. J. Exp. Med.139:337-54 127. Romball, C. G., Ulevitch, R. J., Weigle, W.O. 1980. Role of C3 in the regulationof a splenic PFCresponsein rabbits. J. Immunol.124:151-55 128. Pryjma,J., Humphrey, J. H., Klaus, G. G. 1974. C3 activation and T independentBcell stimulation. Nature252: 505-56 129. Pryjma,J., Humphrey, J. H. 1975. Prolonged C3 depletion by cobra venom factor in thymus-deprived miceandits implication for the role of C3as an essential secondsignal for Bcell triggering. Immunology28:569-76 130. Klaus, G. G., Humphrey,J. H. 1977. Thegeneration of memory cells. Immu-

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nology 33:31-40 131. Thorbecke,G. J., Romano,T. J., Lerman, S. P. 1974. Regulatory mechanismsin proliferation anddifferentiation of lymphoidtissue, with particular referenceto germinalcentredevelopment. Prog. Immunol.3:25-34 132. Klaus, G. G., Humphrey, J. H., Kunkl, A., Dongworth,D. W.1980. The follicular dendriticcell: Its role in antigen presentation in the generation of immunological memory.Immunol. Rev. 53:3-28 133. Mandel,I. E., Phipps,R. P., Abbot,A., Tew,J. G. 1980.Thefollicular dendritic cell: Longterm antigen retention during immunity, lmmunol.Rev. 53: 2959 134. Klaus, G. G. B., Humphrey, J. H. 1986. A re-evaluation of the role of C3in B cell activation. Immunol.Today7: 16365

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Annual Reviews www.annualreviews.org/aronline Ann. Rev. lmmunol. 1988. 6:115-37 Copyright © 1988 by Annual Reviews lnc. All rights reserved

VETO CELLS Annu. Rev. Immunol. 1988.6:115-137. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Pamela J. Fink Departmentof Biology, University of California at San Diego, La Jolla, California 92093 Richard P. Shimonkevitz

and Michael J. Bevan

Departmentof Immunology, ResearchInstitute of Scripps Clinic, 10666North Torrey Pines Road, La Jolla, California 92037 INTRODUCTION One of the main problems confronting an immunesystem designed to eliminate foreign substancesis the establishmentand maintenance of selftolerance. Twoschoolsof thoughtexist on howself-reactivity in the T cell compartmentis avoided. Onemechanism,clonal deletion, proposes that self-tolerance is imposedon immatureT cells in the thymus--that at a certain stage of differentiation, contact with antigen permanentlyinactivates the cell (1, 2). Analternative, nonmutually exclusivesuggestion that anti-idiotypic suppressormechanisms exist to inhibit continually any potential self reactivity. Accordingto this hypothesis, anti-A reactive T cells are held in checkby anti-(anti-A) suppressorT cells (3, 4, 5). self-tolerance in the populationof T cells is apparentlyimposedby clonal deletion of autoreactive lymphocyteswithin the thymus. However,this mechanism of avoiding autoimmune recognition mayalone be insufficient, as autoreactive T cells can be isolated from the population of mature peripheral lymphocytes,and in fact, syngeneiccytolysis can be demonstrated in manyallospecific cytotoxic T lymphocyte(CTL)clones (6). In the following pages weargue for the presence of a mechanism,other than one involvinganti-idiotypic networks,for the continuedmaintenance of self-tolerance in the pool of peripheral lymphocytes.This form of antigen-specific suppressionresults in the functional eliminationof autoreactive peripheral T cells by other lymphoid cells, the mosteffective being 115 07324582/88/0410-0115502.00

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T cells. However,in this interaction, the antigen-specific receptor of the "suppressor cell" is not involved. The cells responsible for maintainingself-tolerance in this waywere first named "veto cells" by Miller and coworkers because they apparently functioned by exercising the power of veto over T cell immuneresponses; a T cell deemed autoreactive by a veto cell was quickly rendered nonfunctional (7). Because vetoing seems to result from a nonimmunogenic, suppressive interaction, it is possible that any cell type expressing antigen (a fibroblast, for example) could serve as a veto cell. However,in vivo, lymphoid cells are muchmore likely to comeinto direct contact with and to present antigen to other T lymphocytesthan are fibroblasts. The in vivo definition of an effective veto cell maytherefore be very different from an in vitro definition. Althoughthe apparent lineage of the veto cell and the duration and severity of the ensuring hyporeactivity differ somewhatwith differing experimental protocols, perhaps a single property defines veto cell-induced tolerance. That property is the unidirectional recognition of the veto cell by the future ~’victim" of that veto cell (8). By recognizing antigen on the surface of a veto cell, a T cell defines itself as autoreactive in the eyes of the veto cell. The veto cell, in the absence of any receptors specific for the autoreactive cell, renders that boundT cell nonfunctional. The specificity of this type of tolerance induction resides only in the cell to be tolerized, and "self" is defined by the antigens expressed on the veto cell surface. This unidirectional recognition event is schematizedin Figure 1. The simplicity and economyof this type of tolerance mechanismshould be readily apparent. There is no requirement for a specialized lineage of cells bearing diverse antigen specific receptors. Tolerance is instead maintained by cells with other immuneroles, including that of antigen specific, majdr histocompatibility complex(MHC)-restricted cytotoxicity. In other words, CTLthat can rid the body of virus infected cells can also function to maintain self-tolerance (9). This system is not only simple, is efficient, ensuring that the autoreactivity frequently generated during the course of a specific immuneresponse will be immediately squelched by the very cells mountingthat specific response (10). Wenow examinethe data pointing to the existence of veto cell-induced tolerance, beginning with the experiments of the main proponents of this mechanism,and following with the reinterpretation of other experiments in light of this modelfor the maintenanceof self-tolerance.

VETO CELLS CAN INACTIVATE CTL PRECURSORS Experiments directed primarily at studying veto cells have focussed on their ability to suppress specifically any CTLprecursors directed against

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iEl’OCELL

Figure 1 Unidirectional recognition is the basis for veto cell function. Anautoreactive T cell recognizes the self antigens of the veto cell and is in turn suppressed. The antigen specific receptors of the veto cell are not engaged.

them, whetherthose CTLbe alloreactive, recognizingthe veto cell MHC, or MHC restricted, recognizingveto cell-expressed haptenor minorhistocompatibility(H) antigens. Initial experiments all utilized an in vitro system for the induction and suppressionof CTL,while the possible physiological role of veto cell mediatedsuppressionis basedon the morerecent in vivo experiments. In Vitro Experiments BONE MARROW--DERIVED T~CELL COLONIES Miller and his coworkersbegan their studies of veto cell-mediated suppressionwith the discovery that spleen cells from athymic (nude) mice can partially suppress a primary CTLresponse against allogeneic MHC antigens (11). The experimental protocol called for the coculture of lymphnode responder cells with irradiated allogeneic stimulators and graded doses of irradiated nude spleencells syngeneicwith the stimulator cells in a 5-daymixedlymphocyte culture (MLC).In contrast to the situation with nudemice, spleen cells from normal, euthymicmice did not suppress. However,thymocytesand bone marrowcells from euthymic mice were found to suppress a CTL responsedirected against their MHC antigens, and this suppression(unlike that mediatedby nudespleencells) wassensitive to 7 irradiation (11, 12). Non-MHC genes appear to influence the strength of veto cell-mediated suppressionby normalbone marrowcells (13).

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strain D lymph node ceils ÷ ~stroin A spleen cells Assayfor lysis of strain A targets

~ OOo "~ 5d

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strain A bone methylceilulose, marrow cells conditioned medium

strainAmspleencells

strainA spleen cells primedto Am + ~ stra|nAmspleencells Assayfor

~

3d

(’~

Co

CTLClone

~

Lymph node responders

(+) k

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lysls and strain C targets

strain A

(+) S~at~s

}

FixTure 2 In vitro systems for measuring the inactivation of CTLprecursors by veto cells. (a) T cell colonies derived from strain Abone marrowcells can suppress anti-A specific CTL. A spleen cells can suppress anti-A m CTL. (c) A CTL (b) Cultured, minor H bearing, MHC clone expressing irrelevant T cell receptors can suppress the CTLresponse directed against its MHCantigens.

The level of specific suppression in this in vitro system remained unimpressive, however,until it was discovered that this novel type of suppressor cell activity is enriched 30-100-fold on a per cell basis in T cell colonies grown from bone marrow cells from normal mice (see Figure 2a). These

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colony-derived suppressors were found to be resistant to ~ irradiation but sensitive to UVirradiation (14). Data indicated these T cells originate from Thy-1 negative, radiosensitive precursors found in the bone marrow but not in the spleens of normal mice (12). Lymphoidcolonies grown from fetal liver cells from normal mice also demonstrate some suppressive activity, although they cannot be classified as veto cells becauseof the lack of specificity for suppression of only those responses directed against antigens expressed by the fetal liver colonies (15). The results of these experiments can be summarizedas follows: Spleen and bone marrow cells from nude mice, and thymocytes, bone marrow cells, and T cell colonies grown from the bone marrowof normal mice can specifically suppress the primary CTLresponse directed against the MHC antigens expressed by these cells. Such veto cell activity is not exhibited by unmanipulated spleen or lymph node cells from normal mice. However, the experiments described below indicate that activated spleen cells from normal mice can serve as veto cells in vitro. PRECULTURED SPLENICT CELLSUnprimed spleen cells cultured in the absence of any knownexternal stimulation for 2-3 days can inhibit the generation of MHC-restricted CTLspecific for a minor H antigen expressed by those spleen cells (16). The experimental protocol used to assay this suppression is outlined in Figure 2b. Similarly, spleen cells cultured for 2 days in the presence of a T cell mitogen are able to suppress CTL responses directed against them, although with somewhatless specificity (17). Normal splenic responder cells harvested on days 3-5 of MLCcan also suppress the development of allogeneic CTLdirected against their MHCantigens (18). Within the cultured spleen cell population, CDSpositive T cells are largely responsible for this suppression (16). The degree of suppression in some experiments is impressive, being dose to 100%at a veto cell: responder cell ratio of 1 : 1 (16). Nosuppression is seen using either freshly explanted antigen-bearing spleen cells or cultured third party cells bearing minorH antigens other than those expressed by the stimulator cells (16). The suppression therefore showsthe antigen specificity of veto cell function. CLONED VETOCELLSThe in vitro study of veto cells, particularly the mechanism of their function, has profited from the use of a uniform population of potent veto cells. This was madepossible with the discovery that cloned T cells with cytolytic activity directed against irrelevant antigens can efficiently and specifically suppress the generation of CTLdirected against antigens they express (17, 19, 20). As shownin Figure 2c, to best assay this activity, a primary MLCis set up by coculturing strain B responders with equal numbers of irradiated A and C stimulator cells,

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where A, B, and C are mousestrains differing at the MHC.At the end of 5 days, CTLactivity is measured, revealing approximately equal and efficient lysis by the strain B effector cells of target cells bearing A or C antigens. If small numbers of cloned anti-D.specific CTLbearing A antigens are added into this MLCon day 0, at the end of 5 days, the anti-C CTLactivity from the surviving responder cells is undiminished, while the anti-A activity is greatly reduced (17, 19). This suppression is mediated cloned CTLin the absence of any antigen they can recognize and is not only highly specific but extremely potent--a 50- to 100-fold reduction in specific CTLactivity is mediated by veto cells seeded at 1/100 the initial numberof responder cells. In all, 8/8 CTLclones (7 alloreactive and MHCrestricted) exhibited veto activity, indicating that suppression of anti-self responses is one of the normal functions of CTL(17, 20). In good agreement with other work (16), this effect is radiation sensitive and property only of CD8-bearingT cell clones (17). HOWDO VETO CELLS FUNCTIONIN VITRO? The use of the

cultures

described

above--containing two types of stimulator cells and one type of veto cell--eliminates competition for nutrients and overcrowding as possible explanations for howveto cells suppress (17, 19). Furthermore, addition of growth factor-containing supernatants does not override the veto, indicating that veto cells neither prevent production of helper factors nor function merely as highly localized lymphokinesinks (17). Veto cells not function simply as unlabeled competitors for responder cell attention during the CTLassay. This is dear because removingveto cells on the day of assay from a culture by antibody-plus-complement treatment does not abrogate the suppression (17, 20). Furthermore, mixing suppressed and nonsuppressedcultures just before assay does not result in suppression of the latter responders (20). Neither supernatants removedfrom cloned veto cells nor those removedfrom veto cell-suppressed cultures will diminish a CTLresponse (19). Thus, veto cell activity cannot be replaced by soluble factors, indicating that somekind of cell-cell contact is required. Onestudy using timed addition and removalof cloned veto cells from the cultures to be suppressed indicates that veto cells need to be present throughout the culture period, at both early and late timepoints (19). However, other experiments suggest that CTLprecursors are not susceptible to veto cell activity prior to 20 hr of stimulation (14, 20). A resolution to this disagreement is important for determining whether activation of CTLprecursors is a prerequisite for their suppression, a key point whenconsidering intrathymic tolerance induction (see below). Limiting dilution analyses indicate that veto cell suppression appears to operate through a decrease in the number of CTLprecursors and not a

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reduction in the cytotoxicity generated per precursor (12, 14). Reculturing responder cells separated from veto cells after 43 hr with fresh stimulator cells does not reverse their suppression, although only 4 days were allowed for this reversal (14). More long-term culturing of inactivated CTLprecursors in limiting dilution would be required to answer adequately whether or not veto cell suppression of CTLprecursors is reversible, and these experiments have yet to be reported. Separation of suppressed cultures on the basis of CD8expression and addition of these sorted cells to a fresh MLCindicates that veto cells do not induce CD8-positive suppressor cells within the responder population, which then mediate the veto cell function (14). Veto cells function in vitro through the type of unidirectional recognition diagrammedin Figure 1. Thus, cloned CTLexpressing irrelevant receptors are capable of vetoing CTLprecursors that recognize them. Furthermore, only haptenated nude spleen cells can veto hapten-specific CTLin a syngeneic system (21). These in vitro studies on the suppression of CTLresponses by veto cells have given us quite a bit of information on veto cell function. Weknow that veto cell-induced suppression is not due to overcrowding, growth factor deprivation, or cold target competition. Suppression requires both prolonged coculture of veto cell and responder cell populations and cell contact, and it does not operate through a CDS-posifive suppressor induced in the responder population. Suppression is highly specific; the specificity is defined by the antigens expressed on the veto cell surface and the CTLthat recognizes those antigens. Veto cells function directly by decreasing the number of CTLprecursors, and this inactivation is not readily reversed. The question of whether veto cells function by lysing CTLprecursors that recognize them is a more difficult one to answer and is discussed at length below. In Vivo Experiments Before assigning to veto cells any potential role in the maintenance of self-tolerance, it is essential to showconvincingly that veto cells function in vivo. There are two main systems in which suppression of CTLresponses by the veto mechanismhas been demonstrated in vivo, one involving suppression of a class I response (9, 22, 23) and the other, the suppression of an MHCrestricted, minor H antigen-specific response (16, 24-28). Webegin with the class I-specific response because it is analogousto the in vitro systems described above. Suppression of class I-specific CTLis initiated by the injection of splenic T cells intravenously into host mice differing from the injected cells at one or several class I loci. Five or six days later, host PROTOCOLS FOR ASSAYING CTL SUPPRESSION IN VIVO

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spleen cells were cocultured with irradiated stimulator cells bearing class I antigens shared by the injected spleen cells as well as additional class I antigens not sharedby either donoror host cells. After 5 days of culture, CTLactivity was measuredagainst targets bearing these shared or third party antigens. This experimentalprotocol is outlined in Figure 3a. In each of the several antigen combinationstested, a goodCTLresponse was elicited against the third party antigens, while the CTLresponseagainst those antigens shared by the injected spleen cells wassuppressed.As few as 2.5 × 106 injected splenic T cells mediateda significant (20-fold)class antigen-specific suppression (23). Specific unresponsivenesslasted for weeks,and in somecases, recoverywasstill incompleteafter 5 months(9). Suppressionof minorHantigen-specific CTLis inducedby injection of A~’ (minor H antigen-bearing, H-2A) spleen cells into F~(A× B) mice. CTLspecific for the injected minorH antigen in the context of H-2B are efficiently and rapidly induced in such animals by the phenomenon of "cross-priming,"presumablythe result of the processingof the injected antigen and presentation on host F~(A× B) antigen-presenting cells. H-2A-restricted CTLcan be inducedby either cross primingor by direct primingby the injected spleen cells themselves(29). Spleen cells from 7 10

spleen cells

strain AA splenic T cells

~strain AC spleen cells

~ ..~ ~ straln DO mouse

b.

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~ m strain A spleen cells

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Assay lgsis of Amandi~mtargets

FI (AxB) mouse Figure3 In viva induction of veto cell mediated suppression of CTLactivity. (a) Suppression ^ antigens into a strain of an anti-MHCresponse is induced by injecting cells bearing KAD c antigens. B mouse.Host spleen cells are cultured 6 days later with stimulators bearing KAD B and compared Suppression of the K^-specific response is measured on targets bearing KAD c targets. (b) Suppression of an antito a control response against Dc measured on K"D ^minor H specific, MHC restricted response is induced by injecting minor H different MHC (A~) cells into F~(A x B) hosts. Host spleen cells are cultured with minor H-bearing stimulator cells. Suppression of anti-A m CTLactivity is measured relative to the nonsuppressed, cross primed activity measuredon Bm targets.

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F1(A× B) animals injected with as few as 107 viable m spleen c ells w ere cocultured 5q5 days later with irradiated FI(Am x Bm) stimulators, and another 5 days later CTLactivity directed against Am and Bm targets was measured. This revealed a suppression of the anti-A m CTLresponse (Figure 3b). In all the strain combinations tested, a dramatic skewing the ratio of direct versus cross-primed CTLactivity was seen, the result of a haplotype- and antigen-specific suppression of direct primed CTL activity (24). Althoughperhaps more complicated than the class I-specific CTLsuppression described above, this minor H-specific system includes the ideal internal control--an MHCrestricted CTLresponse induced by the injection of the same spleen cells and boosted by the same stimulator cells in the samecultures as those used to induce and boost the suppressed response. One of the distinguishing characteristics of this type of CTLsuppression is that it is both haplotype specific (Am killing is depressed, while Bmkilling is not) and antigen specific (Amkilling is depressed, but A-restricted, haptenspecific cytolysis is unaffected) (24). The extreme specificity of this suppression is in contrast to the generalized immunosuppression that is characteristic of graft-versus-host disease (30) or that is predicted by some anti-idiotypic suppressor hypotheses. Furthermore, the severity and the duration of the CTLhyperactivity induced by the injection of minor H different spleen cells is directly related to the numberof spleen cells injected. Injection of 107 Amspleen cells into F~(A× B) hosts results in a ratio Amto Bmkilling of 1 : 25 on day 4 after injection, tapering off to 1 : 1 at day 10, while injection of 6 x 107 Amcells results in a ratio of < 1:60 even at day 10. This temporarily skewed ratio of direct to cross-primed CTL activity is due to a depressed direct primed response rather than to a heightened cross-primed response for the following reasons. The crossprimed response is induced by F~ antigen presenting cells which should m equally well induce the direct primed response. In addition, irradiated A spleen cells prime both Am and Bm responses equally well, and with the same kinetics as the anti-Bmresponse induced by Am spleen cells. As with the in vitro induced suppression, this type of suppression is not easily reversible; boosting cells in culture for two rounds with irradiated stimulators does not appreciably alter the ratio of Amto Bmkilling (24). And finally, it is more difficult to suppress the Am CTLresponse in FI(A x B) mice already primed to the minor H antigens in question than it is to suppress the response in naive mice (24, 26). CHARACTERISTICS OF THE SPLEEN CELL--INDUCED SUPPRESSION

PROPERTIES OF THE CELLS RESPONSIBLE FOR CTL SUPPRESSION IN VIVO The spleen cells responsible for inducing CTLhyporeactivity in both the class I

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and minor H-specific systems are Ia negative, radiosensitive, nylon wool nonadherent cells that are depleted (though not entirely) by treatment with anti-Thy-1 plus complement(16, 22-25). Within the population of splenic T cells, CD8positive cells are the most potent (by about 10-fold) inducing this suppression, with as few as 3 x 106 CD8positive, Thy-1 positive cells mediating a 20-fold suppression of specific CTLactivity (24). However, even Thy-1 negative spleen cells from nude mice can induce CTLsuppression, although inefficiently (23). The cells responsible for inducing CTLsuppression in vivo are present in low numbers in the thymus and bone marrowof normal mice (24) and are not present in skin grafts (28). Perhaps the most distinctive feature of the cells responsible for this type of CTLsuppression is that they are not required to recognize the cells they are suppressing. This conclusion derives from the following information. Spleen cells preprimed to antigens of the responder animal are not more efficient suppressors, on a per cell basis, than are naive spleen cells (24). Cells from [Am -~ Fl(Amx Bm)]radiation chimeras, that are tolerant of and B MHCantigens, are efficient at suppressing anti-A m CTLactivity from recipient F1(A x B) animals (24). In fact, in a system in which injected spleen cells can be separated at the time of culture from host cells on the basis of Thy-1 expression, it was apparent not only that the host cells are incapable of lysing donor cells, but that the donor veto cells are incapable of recognizing host cell antigens. In other words, there is mutual tolerization in animals injected with class I incompatible cells (31). Most conclusively, F~(Am x A) cells can suppress the anti-A TM CTLfrom A animals to which they are tolerant (24, 26). That foreign spleen cells can efficiently suppress a CTLresponse in animals to which they are tolerant is one of the primary reasons for categorizing this suppression as veto cellinduced. HOWDO VETO CELLS FUNCTION IN VIVO? The

suppression induced by

the

injection of antigen-bearingspleen cells is class I-restricted in the induction phase and cannot be overridden in vitro by growth factor-containing supernatants 06, 18, 23, 25). Both of these findings suggest that the suppression operates at the level of the CTLitself and not indirectly by inactivating helper cells required for the activation of CTL.In the case of the minor H system, such helper cells would need both to recognize minor H antigens in the context of MHC molecules and to be able to distinguish minor H-specific CTLrestricted to H-2A B. from those restricted to H-2 Only such a complicated recognition system could explain the specific inactivation by Am spleen cells of CTLspecific for minor H antigens plus H-2g. In fact, limiting dilution analyses of spleen cells from suppressed

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versus nonsuppressed animals indicate a greater than 200-fold reduction in the numberof CTLprecursors specific for the injected antigen, with no detectable change in the numberof CTLprecursors specific for third party antigens (23). Thus, spleen cell-induced suppression operates at the level of CTLprecursors in vivo just as it does in vitro, causing a functional clonal deletion of specific CTL. Is this reduction in CTLprecursor frequency the result of an active suppression operating in vitro? Limiting dilution analyses of CTLprecursor frequencies demonstrate single hit kinetics, implying that if such suppressors exist, they are present in vast excess relative to the numberof target CTLprecursors (23). Removalof donor spleen cells at the time of culture does not relieve the suppression assayed 5 days later (31). Experiments in which spleen cells from suppressed animals were mixed in various ratios with spleen cells from normal animals revealed no suppression of the CTLresponse from the latter spleen cell population (24, 28). Tolerance could not be transferred by spleen cells from suppressed mice injected 14 days previously with minor H different spleen cells (28). The transfer of tolerance by spleen cells from mice injected 4 days previously with a massive dose (108 cells) of minor H different spleen cells probably due to carryover of donor veto cells (8, 26, 32). Thus, all evidence points to the absence of an active mechanismoperating to maintain suppression of CTLprecursors in vitro. The fact that tolerant spleen cells can suppress makesit unlikely there are active suppressors in the spleen cell inoculumother than veto cells. Cells recognizing the idiotype of a CTLreceptor specific for a particular antigen in the context of a given MHCmolecule would be primed by the injection of immunocompetent, receptor-bearing cells (3), and priming has no effect on veto cell frequency(24). If the spleen cell inoculumwere merely responsible for the induction of host suppressor cells, these suppressors would have to have a short lifespan in vivo, would have to be incapable of suppressing in vitro, and wouldhave to be induced passively by antigen and yet distinguish CTLof different restriction specificities. The weakness of the evidence in favor of a suppressor loop and the more compelling arguments against its likelihood have led us to abandon this explanation for in vivo CTLsuppression. One final explanation exists for spleen cell-induced suppression of CTL in vivo that does not involve veto cells. This is the sequestration of these activated CTLand their subsequent exclusion from the cell population taken for the in vitro assay. Several experiments have addressed this possibility, all with negative results. Suppression of the appropriate CTL response is apparent in lymph node cells from normal and splenectomized mice, indicating that the activated precursors are absent from both spleen

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and lymph node, and that the spleen is not required for their specific inactivation (24). Spleen cells inoculated by any of several routes (intravenous, intraperitoneal, subcutaneous, and in the footpad) seem to suppress equally well (24, 28). The activated CTLprecursors are not simply more fragile than naive cells and lost during the process of makinga spleen cell suspension, because diced splenic fragments show the same level of antigen specific suppression as do spleen cell suspensions (24). In summary,the specific CTLsuppression induced in vivo by the injection of antigen-bearing spleen cells bears all the hallmarks of the veto cell-induced suppression previously defined in vitro. It is induced most efficiently by T cells or by cells in the T cell lineage; it is inducedthrough the unidirectional recognition of the inoculated cells by the host and therefore operates directly at the level of the CTLprecursor. It is specific both for antigen and MHC restriction specificity. For all these reasons, we believe that veto cells do operate in vivo to suppress CTLresponses directed at antigens they express, and they can thereby function to help maintain self-tolerance. Do Veto Cells

Kill

the Cells

They Suppress?

The question of whether veto cells suppress primarily by cytolysis is an obvious one. It is raised by the difficulty in reversing suppression and the relative efficiency of suppression mediated by CTLclones and CD8-posifive T cells, relative to helper T cell clones and CD8negative T cells. Previous work suggests that unidirectional recognition leads to unidirectional killing among CTL populations. Thus, when an A anti-B CTL recognizes a B anti-C CTL,the strain B T cell does not lyse the strain A cell (33, 34). More recent work has demonstrated that backwards killing can occur once a T cell receptor is triggered by its appropriate target. In the above example, the strain B CTLclone would lyse the recognizing strain A CTLonly in the presence of strain C target cells (35). The current understanding of the directionality of the killing apparatus makes this result difficult to comprehend(36), although it does fit with our observations that CTLclones are more efficient veto cells whencultured in the presence of monoclonal antibodies directed against their receptors (19). Whatis clear is that CTLclones can serve as veto cells in cultures in the absence of any antigens they specifically recognize (17, 19, 20). These clones are, furthermore, inefficient at lectin-mediated lysis of nontumor cell targets, and this makesit unlikely that veto cells lyse CTLprecursors glued to them by virtue of the CTLreceptor (19). Cloned veto cells able to suppress the generation ofa CTLresponse are unable to kill those same responder cell populations harvested at 17 hr or on day 5 of MLC(19). In other experiments a surprisingly high percentage of cells is apparently

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VETO CELLS 127 lysed after 48 hr of MLC (20). It is unknown what this assay was measuring, since this percentageundoubtedlyexceedsthe number of cells specifically respondingto the veto cell alloantigens.Alsoarguingagainst cytolysis as the common meansfor veto cell function is the observation that human helper T cell clones can still proliferate in responseto exogenousgrowth factors at a time whenthey are whollyunresponsiveto antigen (37-39). At least in this system,whichmayor maynot involveveto cells (see below), suppressiondoes not function through respondercell death. In experimentsthat were once considered conclusive evidencc against a requirementfor cytolysis by veto cells, anti-idiotypic antibodies that recognize the T cell receptors of a cloned CTLwere shownto block cytotoxicityby that clone, but not its ability to veto (19). Theseantibodies werecoculturedwith responders, stimulators, and clonedveto cells during the course of a 5-day MLC. Thegeneration of the appropriate CTLactivity was efficiently inhibited, even though antibody-containingsupernatants taken at the end of 5 days completelyblocked cytolysis by the cloned CTL/vetocell both of its specific target and of nonspecifictargets in a lectin mediatedassay (19). Theseresults are equivocal, however,in light of the experimentsdemonstratingbackwardkilling by CTLwhosereceptors are occupied(see above). In other attempts to dissociate CTLactivity fromveto cell function, no correlation wasseen betweenveto activity and cytotoxic activity among CTLclones whoseefficiency of target cell lysis varied with growth(20). Subdonesof CTLthat wereinvariant in cytotoxicity activity wereseen to vary withtheir ability to veto (20). Finally, respondercells fromautologous MLC exhibit veto activity with no demonstrableCTLactivity (18). A11of these data suggest that cytolysis is unlikely to be the major mechanism of veto cell-induced suppression. Is the deletion of anti-veto CTLprecursorsdueto receptor paralysis, a block in the differentiation to effector cell, or someother mechanism? ARE VETO CELLS TOLERANCE?

INVOLVED

IN

ALLOGRAFT

If veto cell-inducedsuppressionis to be acceptedas a meansof maintaining self tolerance, it maybe implicatedin assays morephysiologicallyrelevant than the in vitro assay of CTLfunction. Wenowreinterpret experiments, designed and executed without veto cells in mind, in light of the data alreadypresented.Weadmitthat our selection is not all inclusive, andthat numerousfindings cannot be explained by veto cell-mediated suppressors alone. "This drawbackis, however,hardly peculiar to the veto cell model.

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In each of the experimentsdescribedbelow, allograft tolerance is induced in immunodeficienthosts (either neonatal or immunocompromised adult animals)and assayedby the reduction in the graft-versus-host reaction or by the enhancedretention of allogeneic skin or bonemarrowgrafts.

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Immunocompromised Adult

Hosts

Adult strain Amice, treated with antilymphocyteserumto removerecirculating T cells, acceptstrain B skin grafted immediately after the injection of strain B bonemarrowcells (40). This enhancedskin graft survival correlated with an antigen-specific reduction in proliferation and CTL activity measured in vitro. Inductionof this in vitro hyporeactivityrequires the presenceof donortype T cells in vivo (40), in obvioussupportof veto cell involvement. In other systemsusing immunodeficient adults, adult strain A miceare given several split doses of lymphoidirradiation followedby an injection of FI(Ax B) bonemarrowcells (41). In these animals, there is long-term survival of strain B skin grafted 1 day after the bonemarrowinjection. Theability of cells differing at minorHantigens to mounta graft-versushost reaction in irradiated hosts is diminishedby prior injection of the donorwith spleen cells bearing the minorHantigens of the host (42, 43). Similarly, as few as 2.5 × 105 cells from a strain ACTLclone bearing irrelevant receptors coinjectedinto irradiated Astrain micewith strain B lymphoid cells protects those recipients fromthe onset of graft-versus-host diseasedueto the B anti-Aresponse(18). Finally, there is evidencefor the prolonged survival of allogeneic and xenogeneicbone marrowchimeras whenirradiated host animalsare coinjected with syngeneicand allogeneic or xenogeneic bone marrowcells (44). This experimental protocol depicted in Figure 4a. In these animals, anti-host activity in the foreign cell inoculummaybe eliminated by veto cells amongthe coinjected host type bonemarrowcells. In each of these cases, lymphoidcells are knownto induce the specific suppressionof immunological activity specific for the antigens of those lymphoidcells, whetherthat activity is measuredas skin or bone marrow graft injection or graft-versus-host reaction. Future work mayreveal whetherthese donor cells are T cells and whether they are required to respondto the host antigens--characteristics that are critical for determiningwhetherthey can be categorizedas veto cells. Neonatal Hosts A large body of workhas demonstratedthat animals can be tolerized to alloantigens by injection, at the time of birth, of lymphoidcells bearing

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strainB skin graft 950 red ! 2 weeks

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strain

145 days

acceptance of

A mouse 5x10 strain A + l.SxloTstrainB bone marrow stem cells

strain

B graft

strainC skin graft

acceptanceof strainB graft

4xlo7~ (Axe)

strainB skin graft

bone marrow cells 6-0 weeks

neonatal strain A rat

~ strain C skin yraft

LN, or spleen cells

~0 r~

acceptanceof strain B graft

adult strainA rat strainB skln graft

Figure 4 Veto cell induction of allograft tolerance. (a) Allogeneic radiation bone marrow chimeras showbetter survival and tolerance of strain B skin grafts only upon coinjection of irradiated strain A hosts with bone marrowcells (a source of veto cells) from strains Aand B. (b) The induction of neonatal tolerance, as assayed by the survival of allogeneic skin grafts, maybe mediated by F~ veto cells in the tolerizing bone marrowinocnlum. The transfer of tolerance to irradiated adult animals requires injection of F~cells from the spleen, lymph node, or thoracic duct of tolerant donors.

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those alloantigens (4, 45-50). Thus,strain Arats injected within 24 hr birth with 4 x 107 bone marrowcells from FI(A x B) donors will not reject strain B-skin grafted 6-8 weekslater. This allograft tolerance is radiosensitive (45), antigen specific (third party skin grafted at the same time is rejected), and transferrable. As diagrammed in Figure 4b, as few as 3 x 107 lymphoidcells fromA strain rats tolerized to B alloantigens can transfer the suppressionof B skin graft rejection to irradiated, naive adult Astrain rats (4, 46-48).For our story, the interest lies in whichcells can adoptivelytransfer this tolerance. Neonatallytolerized animalsretain a characteristic lowlevel chimerism in their lymphoid tissues; thoracic duct, bone marrow,lymphnode, spleen, and peripheral blood lymphocytesall harbor an approximatelyuniform2-3%donor type cells. The presence or absenceof chimericF~cells correlates well with the fate of the test skin graft, even in adoptive hosts (47). Removalof chimeric cells from the inoculumwith alloantisera abolishes the transfer of tolerance (47). Toleranceis permanentlyabolished evenin primaryhosts treated in vivo with donor specific antibodies to removechimeric cells (50). These chimeric cells do not simply serve as a source of antigen because thoracic duct lymphocytes transfer tolerance efficiently, while bonemarrowcells do not, although the percentage chimerismin these two populations is identical (47). Evenmoreto the point, elimination of chimeric T but not B cells eliminates the transfer of tolerance (48), while treatment with anti-CD8 plus complementdiminishesbut does not abolish its transfer (4). These results showthat the transfer of neonatally induced tolerance to skin allografts is mediatedby long lived, rapidly recirculating donor type T cells that are radiation sensitive andmainlyCD8positive (45-48). What,if anything, do these tolerogenic donor T cells recognizein the host environment?Accordingto one hypothesis, the cells transferring allograft tolerance bear anti-idiotypic receptors, specifically recognizing all Astrain cells bearinganti-strain B receptors(48). Thereis somedirect evidenceagainst this hypothesis,in experimentsusingthe transfer of allo¯ graft tolerance to the male specific antigen H-Yas a measure for suppression. As in the system described above, both the induction and transfer of tolerance to H-Yin the mouseare mediated by donor T cells and not B cells or macrophages.Toleranceis also conferred, albeit somewhat inefficiently, by an H-Y-bearingCTLclone expressingirrelevant allospecific receptors (49). In this case, no anti-idiotypic interaction possible. Invokingveto cells as the mediatorsof allograft tolerance not only takes into account most of the available data, it is a simplifying hypothesis,alleviating the needfor a specializedcell type andunifyingthe findings fromdiverse systemsmeasuringthe antigen-specific suppression of immunocompetence.

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VETOCELLS

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ARE VETO CELLS INVOLVED CELL TOLERANCE? "

131

IN T HELPER

In Vivo Experiments Thefirst clues that veto cells mayin somecases suppresshelper T cells were observations madeperipherally in the course of other experiments. Neonatallytolerized rats not only accept allogeneic skin grafts, they make no alloantibodies (47), and they showa diminishedproliferative capacity in response to the appropriate allogeneic stimulators (40, 41, 43, 47). Moredirectly, the proliferative response to lactate dehydrogenaseB in nonresponderstrains of miceis suppressedby the apparent recognition by the helper cell of antigenandI-A on the surface of the suppressoreffector T cell (51). Wewouldlike to suggest (25) that the suppressoreffector cell is a veto cell that bears class II-like moleculeswhichpicks up soluble antigen, and is thereby recognizedby the class II-restrict~d,. antigenspecific helper cell. Thehelper cell is then inactivatedbyits unidirectional recognition of the suppressor cell, succumbingto a veto cell-induced tolerance. Similar meansof suppressionmayoperate in mice that are low respondersto other soluble antigens such as hen egg lysozyme(52). In Vitro Experiments Clonesof humanT cells specific for definedpeptides of influenza hemagglutinin can be tolerized in vitro, in the absenceof other cell types, by their exposureto high levels of antigen for 6-16 hours at 37°C.Tolerance is measuredby the absenceof proliferation in response to immunogenic dosesof peptide pulsedpresentingcells, and it lasts for over 7 days after the removalof antigen and the addition of exogenousgrowthfactors (3739). Toleranceinductionis antigen specific and class II restricted (human T cells expressclass II antigens). Although the mechanism of this tolerance induction is unknown,it is associated with both a loss of the T cell receptor-associated T3 proteins fromthe cell surface and a loss in the ability to bind to monolayersof antigen pulsed presenting cells (39). Importantly,this in vitro-induced tolerance does not result in the death of the suppressedhelper T cells, as their proliferation in the presenceof growthfactors alone is undiminished (37). Thus,T cells specific for class II plus hemagglutininundergoa class II-restricted inactivation in the presence of antigen. At high doses, perhaps enoughantigen binds (either specifically or nonspecifically) to these clonedT cells that someof the peptide associates with class II antigens. This complexcould then be recognizedby neighboringcells that in turn are inactivated. In other words, wethink these experimentsdemonstratethat cloned humanhelper T ceils can serve in vitro as veto cells for class II-restricted responses.Although

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the amountof peptide addedto induce tolerance in this systemsurpasses normalphysiological levels of soluble antigens, wepropose an in vivo modelin whichantigens endogenous to the tolerizing cell are presentedin the context of class II moleculesand serve as the ligand recognizedby the T cell to be suppressed.Clearly, the cells functioningas veto cells for class II-restricted responsesmustthemselvesbear the relevant class II determinants(see below). DO VETO CELLS TOLERANCE?

INDUCE

INTRATHYMIC

Recentexperimentson the ability of fetal thymuslobes to inducetolerance have suggested that bone marrowderived elements in the thymusare much more effective at inducing tolerance to MHC antigens than are thymic e.pithelial or mesenchymal elements(53-55). For example,whenfetal thymuslobes are depleted of macrophages,dendritic cells, and T cell precursors by culturing in deoxyguanosine,the resulting "stromal" lobes retain the ability to attract T cell precursors, to supporttheir maturation, andto select their restriction specificity (56), but they lose the ability inducetolerance to their ownclass I and class II antigens. Thus,a strain A nude mousethat has been grafted with a strain B "stromal" thymus contains T cells whichmaturedin the B thymusbut whichcan respondto B alloantigens. In contrast to this, unmanipulated thymusgrafts do induce tolerance. Thesestudies haveled to the assumptionthat macrophages and dendritic cells in particular are responsiblefor tolerance induction in an untreated thymus. However,these data are compatible with the notion that cells committedto the T lineage also operate in the induction of self-tolerance in a regenerating immunesystem. In recent experimentsaddressing this possibility, Thy-1positive CD4/CD8 negative ("double negative") T stem cells wereisolated fromthe thymusand injected directly into the thymus lobes of lethally irradiated, bone marrow-protectedallogeneic mice. In the situation in whichthe host and T stemcell donormiceexpressedclass I and class II antigen differences, tolerance to the class I antigen was observed, both amongthymocytesand mature T cells, for up to 8 weeks post irradiation and transfer. However,no tolerance wasinducedto donor type class II antigens, whichmacrophages and dendritic cells express but T cells do not. Theselection of Thy-1positive doublenegative thymocytes as the tolerogen and the resulting "split tolerance" suggeststrongly that T cells (and not a minor non-T cell contaminantin the inoculum)are responsible for inducing or maintainingclass I-specific tolerance (57). That is, a subpopulationof immaturethymocytesor their descendantscan

Annual Reviews VETO CELLS

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serve as veto cells in vivo. Whileit is likely that someof the tolerance is imposed on the regenerating host cells in the thymus by the donor stem cells differentiating simultaneously, it is also conceivable that mature T cells derived from the donor F1 cells recirculate in the periphery and back to the thymus to maintain tolerance (58).

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PERSPECTIVES For the most part, veto cells have been studied in artificial in vitro or in vivo systems in which CTLresponses to alloantigenic differences expressed by the veto cell have been suppressed. Responses against major and minor H differences have been analyzed. This artificial system leads to the proposal that veto cells may be key players in normal immunostasis by inducing and/or maintaining tolerance to self antigens. But what about the potential risks of the existence of a cell that can efficiently suppress an immuneresponse directed against its own antigens? In the systems we have studied, and in particular, in the highly efficient suppression of anti-minor H CTLresponses in vivo (24), the most effective veto cells were CD8positive mature T cells. Analogously, CD8positive CTLclones but not CD4positive helper clones suppressed CTLresponses in vitro (17, 19, 20). If a rather small dose of CD8positive T cells can suppress the generation of a CTLresponse when injected into a host competent to recognize dozens of minor H antigens on the cell surface, then we wouldassume that virally induced antigens expressed by veto cells wouldalso be able to eliminate the anti-viral CTLresponse. For this effect to comeinto play requires that the veto cells becomeselectively infected with the virus. For the effect to be long lasting requires that the immunogenic routes of antigen processing and presentation to CTLare bypassed. Weknow this because in the suppression of the CTLresponse to minor H antigens, the normal route of antigen presentation eventually wins out and the suppression is overcome(24). Whenthe antigenic difference is one that cannotbe effectively presented by the host (i.e. an allogeneic class I difference) then the veto suppression maypersist for a long period (9). If a virus were tropic for T cells and did not damagethe host cell in way that would encourage conventional presentation, T cell and B cell induction wouldbe avoided. The infected T cell, expressing viral antigens on its surface, would function as a veto cell and be immunosuppressive for viral specific T cells, the very lymphocytesimplicated in viral clearance in vivo(59). Another worry is the potential for T cell receptors to becomeloaded with antigen (for exampleclass I molecules complexedwith soluble anti-

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gen). Could these exogenous antigens be presented by T cells to other antigen specific T cells as "self" and thereby result in vetoing? To test this alarming possibility, allospecific CTL clones were "loaded" with antigen in vitro and tested for suppression of CTLresponses directed against true endogenous antigens and against these exogenously acquired antigens. Only those responses directed against endogenous antigens were vetoed (17). This argument is undoubtedly a quantitative one, but it does demonstrate that an "average" CTL clone with an "average" number of receptors is unlikely to take up enough antigen, even in vitro, for antigenloaded receptors to serve as ligands for veto cell function. A final limitation of the veto cell model for the maintenance of selftolerance is that it excludes any antigens not expressed by veto cells. Thus in the mouse, where T cells (the most efficient veto cells) do not express class II molecules, class II-restricted responses would not be subject to the same sort of control. In fact, induction of allograft tolerance to class II antigens has been shown to be qualitatively different from that for class I antigens (60~2). Tolerance to these non-T cell expressed antigens may maintained either by inefficient vetoing by other lymphoid and nonlymphoid cells that do express these antigens, or perhaps by some alternative mechanism for deleting autoaggressive cells among the peripheral population. ACKNOWLEDGMENTS This work was supported AI19499 and AI19335.

by US Public

Health

Service

grant

numbers

Literature Cited 1. Good,M. F., Pyke, K. W., Nossal, G. J. V. 1983. Functional clonal deletion of cytotoxic T lymphocyteprecursors in chimeric thymusproducedin vitro from embryonic Anlagen. Proc. NatL Acad. Sci. USA80:3045 2. Kappler, J. W., Roehm,N., Marrack,P. 1987. T cell tolerance by clonal elimination in the thymus.Cell 49:273 3. Bellgrau, D., Wilson,D. B. 1978. Immunological studies of T cell receptors I: Specificallyinducedresistance to graftversus-host disease in rats mediatedby host T cell immunity to alloreactive parental T cells. J. Exp. Med.148:103 4. Roser, B. J., Herbert, J., Godden,U. 1983. The role of suppressor cells in transplantation. Transplant. Proc. 15: 698

5. Cunningham, A. J. 1976. Self tolerance maintained by active suppressor mechanisms. Transplantation31: 23 6. Claesson, M. H., Miller, R. G. 1985. Functionalheterogeneityin allospecific cytotoxic T lymphocyte clones. II: Developmentof syngeneic cytotoxicity in the absenceof specificantigenicstimulation. J. Immunol.134:684 7. Miller, R. G. 1980. Lymphoid suppressor cells. In Strategies of lmmuneRegulation, ed. E. E. Sercarz, A.J. Cunningham, p. 507. NewYork: Academic 8. Rammensee, H. G., Bevan, M. J., Fink, P. J. 1985.Antigenspecific suppression of T cell responses--the veto concept. Immunol. Today 6:41 9. Rammensee, H. G., Fink, P. J., Bevan, M. J. 1985. The veto concept: an eco-

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VETO CELLS 135 nomic system for maintaining self tolerance of cytotoxic T lymphocytes. Transplant.Proc. 17:689 10. Miller, R. G. 1986. The veto phenomenon and T cell regulation. Immunol. Today 7:112 11. Miller, R. G., Derry, H. 1979. A cell population in nu/nu spleen can prevent generation of cytotoxic lymphocytesby normalspleencells against self antigens of the nu/nu spleen. J. Immunol.122: 1502 12. Muraoka,S., Miller, R. G. 1980. Cells in bone marrowand in T cell colonies grownfrom bone marrowcan supprcss generation of cytotoxic T lymphocytes directed against their self antigens. J. Exp. Med. 152:54 13. Hurme,M. 1986. Genetic variation in the in vitro veto activity of bonemarrow cells. Scand.J. Immunol.23:389 14. Muraoka,S., Ehrnan,D. L., Miller, R. G. 1984.Irreversibleinactivationof activated cytotoxic T lymphocyteprecursor cells by "anti-self" suppressor cells present in murin¢ bone marrowT cell colonies. Eur. J. Immunol.14:1010 15. Muraoka,S., Miller, R. G. 1983. Cells in murine fetal liver and in lymphoid colonies grownfromfetal liver can suppress generation of cytotoxic T lymphocytesdirectedagainst their self antigens. J. Immunol.131: 45 16. Rammensee, H. G., Nagy,Z. A., Klein, J. 1982. Suppressionof cell mediated lymphocytoxicityagainst minorhistocompatibilityantigens mediatedby LytI + Lyt-2÷ T cells of stimulatorstrain origin. Eur. J. Immunol.12:930 17. Fink, P. J., Rammensee, H. G., Bevan, M.J. 1984.Clonedcytolytic T cells can suppress primary cytotoxic responses directed against them. J. ImmunoL 133: 1775 18. Claesson, M.H, Ropke,C. 1986. Antiself suppressive(veto) activity of respondercells in mixedlymphocytecultures. Curr. Top. MicrobioLlmtnunol. 126:213 19. Fink, P. J., Rammensee, H. G., Benedetto, J. D., Staerz, U. D., I.~francois, L., Bevan,M. J. 1984. Studies on the mechanismof suppression of primary cytotoxic responsesby clonedcytotoxic T lymphocytes. J. ImmunoL133:1769 20. Claesson, M. H., Miller, R. G. 1984. Functionalheterogeneityin allospecific cytotoxic T lymphocyteclones I. CTL clones expressstrong anti-self suppressive activity. J. Exp. Med.160:1702 21. Miller, R. G. 1980. An immunological suppressorcell inactivating cytotoxic T lymphocyteprecursor cells recognizing

it. Nature287:544 22. Miller, R. G., Phillips, R. A. 1976. Reductionof the in vitro cytotoxic lymphocyte response produced by in vivo exposureto semiallogeneic cells: r~ruitmentor active suppression?J. Immunol. 117:1913 23. Rammensee, H. G., Fink, P. J., Bevan, M.J. 1984. Functional clonal deletion of class I-specific cytotoxic T lymphocytesby veto calls that expressantigen. J. lmmunoL133:2390 24. Fink, P. J., Weissman, I. L., Bevan,M. J. 1983. Haplotypespecific suppression of cytotoxic T cell inductionby antigen inappropriatelypresentedon T cells. J. Exp. Med. 157:141 H. G., Juretic, A., Nagy, 25. Rammensee, Z. A., Klein, J. 1984.ClassI restricted interaction between suppressor and cytolytic cells in the responseto minor histocompatibilityantigens. J. lmmunol. 132:668 26. Gascoigne,N. R. J., Crispe, I. N. 1984. Suppression of the cytotoxic T cell responseto minoralloantigens in vivo: Linked recognition by suppressor T cells. Eur. J. Immunol.14:2t0 27. Ishikawa, H., Suzuki, H., Hino, T., Kubota,E., Saito, K. 1985.In vivo priming of mouseCTLpr~ursors directed to product of a newlydefined minor H-42 locus is undera novelcontrol of class II MHC gene. J. Immunol.135:3681 28. Ishikawa, H., Hino, T., Kato, H., Suzuki, H., Saito, K. I986. Cytotoxic T lymphocyteresponse to minor H-42a alloantigen in H-42bmice: clonal inactivation of the precursor cytotoxic T lymphoeytes by veto like spleencells that express the H-42aantigen. J. Immunol. 137:2080 29. Bevan, M. J. 1976. Minor H antigens introducedon H-2different stimulating cells cross react at the cytotoxicT cell level duringin vivopriming.J. Irnmunol. 117:2233 30. Shearer, G. M., Polisson, R. P. 1980. Mutualrecognition of parental and F~ lymphocytes. Selective abrogation of cytotoxic potential ofF~ lymphocytes by parental lymphocytes.J. Exp. Med.151: 20 31. Rammensee, H. G., Bevan, M. J. 1987. Mutualtolerization of histoineompatible lymphocytes.Eur. J. Immunol.17: 893 32. Crispe, I. N., Owens,T. 1985. Veto in vivo? Immunol.Today 6:40 33. Kuppers,R. C., Henney,C. S. 1976.Evidencefor direct linkage betweenantigen recognitionandlytic expressionin effector T cells. J. Exp. Med.143:684

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34. Kuppers, R, C., Henney,C. S. 1977. Studies on the mechanism of lymphocyte mediatedcytolysis. IX. Relationships betweenantigen recognition and lytic expressionin killer T cells. J. Immunol. 118:71 35. Lanzavecchia, A. 1986. Is the T cell receptor involved in T cell killing? Nature 319:778 36. Kupfer, A., Swain, S. L., Janeway,C. A., Singer,S. J. 1986.Thespecificdirect interaction of helper T cells and antigen presentingBcells. Proe.NatLAead.Sci. USA 83:6080 37. Lamb,J. R., Skidmore, B. J., Green, N., Chiller, J. M., Feldmann,M. 1983. Inductionof tolerancein influenzavirusimmuneT lymphocyteclones with synthetic peptides of influenza hemagglutinin. J. Exp. Med.157:1434 38. Lamb,J. R., Feldmann,M. 1984. Essential requirement for major histocompatibility complexrecognition in T cell tolerance induction. Nature308:72 39. Feldmann,M., Zanders, E. D., Lamb, J. R. 1985. Tolerancein T cell clones. Immunol. Today 6:58 40. Maki, T., Gottschalk, R., Wood,M. L., Monaco,A. P. 1981. Specific unresponsiveness to skin allografts in anti-lymphocyteserum-treated, marrow injected mice: participation of donor marrow-derivedsuppressor T cells. J. lrnmunoL127:1433 41. Slavin, S., Strober,S., Fuks,Z., Kaplan, H. S. 1977.Inductionof specific tissue transplantation tolerance using fractionated total lymphoidirradiation in adult mice: long-termsurvival of allogeneic bonemarrowand skin grafts. J. Exp. Med. 146:34 42. Halle-Pannenko,O., Pritchard, L. L., Rappaport, H. 1983. Alloimmunization-activatedsuppressorcelts. I. Abrogation of lethal graft-versus-hostreaction directed against non-H-2antigens. Transplantation36:60 43. Pritchard, L. L., Halle-Pannenko, O. 1983. Alloimmunization-activatedsuppressorcells. II. In vitro activity of suppressor cells implicated in the abrogation of lethal graft-versus-host reaction. Transplantation36:310 44. Ildstad, $. T., Sachs,D. H. 1984.Reconstitution with syngeneieplus allogeneic or xenogeneic bone marrowleads to specific acceptanceof allografts or xenografts. Nature307:168 45. Holan, V., Hasek,M., Chutna, J. 1978. Radiosensitivity of suppressorcells in neonatallytolerant rats. Transplantation 25:27 46. Roser, B., Dorsch,S. 1979.Thecellular

basis of transplantationtolerancein the rat. Immunol.Rev. 46:55 47. Dorsch, S., Roser, B. 1982. Suppressor cells in transplantation tolerance. I. Analysisof the suppressorstatus of neonatally and adoptively tolerized rats. Transplantation33:518 48. Dorsch,S., Roser, B. 1982.II. Identification and probable modeof action of chimeric suppressor T cells. Transplantation 33:525 49. Weissman,I. L., Jerabek, L., Greenspan, S. 1984. Tolerance and the H-¥ antigen: requirementfor male T cells, but not B cells, to inducetolerance in neonatal female mice. Transplantation 37:3 50. Lubaroff, D. M., Silvers, W.K. 1970. Theabolition of tolerance of skin homografts in rats withisoantiserum.J. bnmunoL 104:1236 51. Baxevanis,C. N., Ishii, N., Nagy,Z. A., Klein, J. 1982. H-2-controlled suppression of T cell response to lactate dehydrogenaseB. Characterization of the lactate dehydrogenaseB suppressor pathway. J. Exp. Med.156:822 52. Araneo, B. A., Yowell, R. L. 1985. MHC-linked immune response suppression mediatedby T cells bearing IA-encodeddeterminants. J. Immunol. 135:73 53. von Boehmer, H., Hafen, K. 1986. Minorbut not major histocompatibility antigens of the thymusepithelium tolerize precursors of cytolytic T cells. Nature 320:626 54. Jenkinson,E. J., Jhittay, R., Kingston, R., Owen,J. J. T; 1985. Studies on the role of the thymieenvironmentin the induction of tolerance to MHC antigens. Transplantation39:331 55. Schuurman,H. J., Vaessen, L. M. B., Vos,J. G., Hertogh,A., Geertzema,J. G. N., Brandt, C. J. W.M., Rozing, J. 1986. Implantation of cultured thymic fragmentsin congenitally athymicnude rats: ignorance of thymic epithelium haplotype in generation of alloreactivity. J. ImmunoL 137:2440 56. Lo, D., Sprent,J. 1986.Identity of cells that imprint H-2restricted T cell specificity in the thymus.Nature319:672 57. Shimonkevitz,R. P., Bevan,M.J. 1987. Tolerogenie interactions amongthymocytes. Manuscriptsubmitted 58. Fink, P. J., Bevan,M. J., Weissman, I. L. 1984. Thymic cytotoxic T lymphocytes are primed in vivo to minor histocompatibility antigens. J. Exp. Med. 159:436 59. Brenan,M.1983. T lymphocytesin viral clearance, lmmunoLToday 4:319

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comparisonof the neonatal tolerance60. Streilein, J. W., Gruchalla,R. S. 1981. Analysisofneonatallyinducedtolerance inducing capacities of H-2class I and of H-2alloantigens. I. Adoptivetransclass II antigens. J. Immunol.131:1670 fer indicates that tolerance of class I 62. McCarthy,S. A., Bach, F. H. 1983. The and class II antigens is maintainedby cellular mechanismof maintenanceof distinct mechanisms.Immunogenetics neonatally induced tolerance to H-2 12:161 class I antigens. J. Immunol.131:1676 61. McCarthy,S. A., Bach, F. H. 1983. A

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ANTIGENIC VARIATION IN LENTIVIRAL DISEASES Janice E. Clements,’~ Susan L. Gdovin,~f Ronald C. Montelaro* and Opendra Narayan~f ~" The Johns HopkinsUniversity School of Medicine, Departmentof Neurology,600 N. WolfeStreet, Baltimore, Maryland21205, and * Departmentof Biochemistry,Louisiana State University and Louisiana Agricultural ExperimentStation, Baton Rouge,Louisiana 70803 INTRODUCTION Virus infections in animals involve complexvirus-host interactions in whichvarious schemesof replication of the agent are pitted against a multitude of host-defense mechanisms.The immunesystem is the most effective of the antiviral host defensesandusuallysucceedsin eliminating pathogensafter brief periods of infection. Failure to cure infection indicates a shift in favor of the virus, and this involves either somenovel mechanism of viral replication that eludes the immune systemor a defect in the immune system, often virus induced, that interferes with antiviral functions. This review examinesone modeof viral persistence in which normalreplication of the agent can occur in the functionally competent immuneenvironment.The basic mechanism of this process is mutation of the virus genomewith attendant alteration in surface antigens permitting escape from immunecontrol. Thelentiviruses are a uniquegroupof agentsthat elude the host defense systemsand that not only persist indefinitely in the infected animalbut replicate continuously, often in the face of competentimmune responses (1-3). Theseagents are nononcogenic retroviruses that infect cells of the immunesystem. Theyare host species-specific and are transmitted horizontally fromindividual to individual in bodyfluids. Theviruses belong to a taxonomicgroup that includes visna-maedivirus of sheep, caprine arthritis encephalitis virus (CAEV) of goats, equineinfectious anemiavirus 139 0732-0582/88/0410-0139502.00

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(EIAV) of horses, human immunodeficiency virus in humans (HIV), simian immunodeficiency virus in non-humanprimates (SIV) (4, 5, Within this group of viral infections, host responses to visna-maedi, CAEV and EIA viruses have been the most thoroughly investigated, and in this report we summarize various strategies used by these agents to escape immunological control.

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ANTIGENIC

DRIFT

AND PERSISTENCE

Antigenic drift or change in the antigenicity of the surface glycoprotein is a well-established mechanism among parasites for escaping immune elimination (7). It is the principal meansof persistence of protozoans such as trypanosomes,plasmodia, and several species of bacteria. Recent studies suggest that this phenomenonmay also be important in persistence of several viruses. The best-known exampleof viral antigenic drift is among influenza viruses where variants with minor antigenic changes in the hemagglutinin and/or the neuraminidase glycoproteins are selected during sequential epidemics by populations immuneto preceding strains of virus (8). Recent studies showthat variant strains of rabies and measles virus can replicate in the presence of antibody to other strains of the samevirus, suggesting antigenic variation (9, 10). The best modelfor viral drift in single animal, however,comesfrom studies of infections with lentiviruses. The primary examples are equine infectious anemia (EIA) (l 1-13) ovine visna (14, 15). Horses infected with equine infectious anemia virus develop sequential episodes of acute hemolytic crises during persistent infection. The animals produce neutralizing antibody to the virus after the first episode, but succeeding disease crises are associated with mutant viruses that are not neutralized by the preexisting antibody. This is reminiscent of protozoan diseases. Visna virus of sheep also causes persistent infection, although episodic disease does not occur. However, the agent undergoesantigenic drift in the animal during persistent infection in a way similar to EIA in horses. Such antigenic drift by visna virus could be duplicated in cell culture systems using plaque-purified virus for inoculation and monospecific antibody for selection (15, 16). These viruses provide ideal modelsfor studying the viral genetic mechanismsof antigenic variation. In addition to antigenic drift the caprine virus, CAEV,escapes from immuneelimination by molecular masking of the viral epitopes responsible for neutralization. This permits the virus to persist in an immunocompetent animal. However, when neutralizing antibodies are produced experimentally, analysis of virus stocks with these antibodies reveals an extremely high rate of antigenic variation.

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LENTIVIRUSES Lentiviruses are a subgroupof the retrovirus family. Their viral genome is a positive stranded RNAmolecule(9500 bases) whichcontains three genes that encodethe major structural proteins of the virus 07). The env gene encodes the viral glycoproteins (external and transmembrane proteins) whichcomprisethe envelopecoat of the virus (17). Thegag gene encodesthe viral core proteins and a viral RNA binding protein. The pol gene codes for a complexof enzymesinvolvedin reverse transcription of the viral RNAinto proviral DNA and integration of the proviral DNA; these include the RNA-dependent DNA polymerase(reverse transcriptase, RNaseH, DNA endonuclease-integrase). The genes are organized on the RNA genome(5’-3’)gag-pol-env. Lentiviruses have a genetic organization similar to the other retroviruses with the addition of small open reading frames (ORF)betweenthe po! and env genes (17, 18). These ORFsprobably code for proteins that play roles in the comp}exregulation of gene expressionof these viruses. The envelope gene of visna virus and CAEV encodesan outer membrane protein of 135 kd whichbecomeshighly glycosylated and whichis the target for virus neutralizing antibodies (19, 20). The outer membrane protein of EIAVis 90 kd, highlyglycosylated,and the target for antibodies that neutralize the virus (21-23). All three viruses have transmembrane proteins whichare glycosylated and hydrophobic, and whichapparently span the membrane of the virus serving as an anchor for the outer membrane protein. The outer membrane proteins of the lentiviruses are substantially larger than other retrovirus envelopeglycoproteinsand appear to be more highly glycosylated. These common features maycontribute to their ability to accumulate mutationsyet maintaina functional structure on the surfaceof the virus. Anotheractivity of the outer membrane glycoprotein is to induce cell fusion. Bothvisna virus and CAEV causeextensive fusion of infected cells, andvisna virus can causecell fusion fromoutsidethe cell at multiplicities of infection of four virions per cell (23, 24). This fusion by visna virus can be blocked by monospecificantibody to gp135, the outer membrane glycoprotein. Thus, the outer membrane glycoproteins of the lentivirus contain epitopes responsible for virus neutralization, and probablyfor virus-cell receptor interaction andvirus-inducedcell fusion. Thelife cycle of lentiviruses providesan obviousmechanism for eluding host immunedefenses: Viral RNAis transcribed by RTaseinto proviral DNAwhichintegrates into the host cell genomeand can be eliminated only by the deathof the cell (26). However, since the target cells for these viruses are end stage cells of the monocyte-macrophage lineage (27, 28),

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integration of the proviral DNA provides only temporarysurvival of the viral genome.The virus must replicate and disseminate to other target cells in order to persist for the life of the animal.Since virions budding fromcells are potentially accessibleto neutralizingantibodies,lentiviruses have evolved multiple strategies to evade the humoralimmunityof the immunocompetent host. First, epitopes responsiblefor virus neutralization are not recognizedby the immune systemof the host. This results in the failure of the host to produceneutralizing antibodies while it producesa plethora of non-neutralizingantibody against the gp135as well as other viral proteins. Thelack of neutralizingantibodiesallowsthe virus to persist andreplicate in the animal.Second,neutralizing antibodies are inducedin infected animalsby someviruses. However, these antibodies are inefficient in virus neutralization, requiring a prolongedincubation with virus to accomplish efficient neutralization, and are of relatively low titer compared to neutralizing antibodies directed against other viruses. Virus can spread fromcell to cell beforeneutralization occurs; againthis results in a persistent infection. Third, lentiviruses sustain frequent point mutations throughouttheir genomeduring replication; this is due to an error-prone viral RTasewhichlacks editing functions. Point mutationsin the env gene of a variant virus that alters the neutralization epitope provide a strong selective advantage for growth in an immuneanimal. The resulting nonneutralized virus can replicate and spread in the immunehost. Thus, antigenic variation provides a mechanism for the virus to persist in the immune host. TheCAEviruses use the first two strategies while visna and EIAviruses use the secondand third. IMMUNE RESPONSE

TO CAEV

Goats infected naturally or experimentallywith CAEV becomepersistently infected andproduceantibodiesthat bind to all the viral proteins (29, 30). However, these antibodiesare ineffective in neutralizing the virus (29-31). Thelack of neutralizingantibodiesin infected goatsis difficult to overcome; even experiments in which adjuvants were used together with purified and disrupted virus did not succeedin eliciting a neutralizing antibody response. However,recent studies havebeensuccessful in eliciting a neutralizing antibody response to CAEV whenanimals are inoculated with virus in conjunction with large amountsof inactivated Mycobacterium tuberculosis (30). Studies on the neutralizing antibody haveshownthat they are of extremelynarrowspecificity. The serumneutralizes only the virus used for inoculation, no strains of visna virus or other strains of CAEV are recognized. This includes CAEviruses isolated from goats from the sameherd from whichthis neutralized virus had been derived. This

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ANTIGENS IN LENTIVIRAL DISEASES 143 suggests these viruses are not only poor antigens for the induction of neutralizing antibodiesbut that various strains bear distinct neutralization epitopes. Since CAEV does not normallyinduce neutralizing antibodies, it is not possibleto determinewhetherdifferent antigenic types of virus are present in different animalsor whetherthere are antigenicdifferencesin the viruses obtained from the sameanimal. Thus, the neutralizing sera obtained by injection of inactivated M. tuberculosis and virus were used to determine if antigenically distinct viruses werepresent in stocks of CAEV. It was foundthat 1 in 1 × 104parental virus wasnot neutralizedby the antibodies (32, 33). This is an extremelyhigh rate of spontaneousantigenic mutation since these variants arose in stocks withoutthe selective pressure of neutralizing antibody.Theoccurrenceof unselectedantigenic variants in visna virus stocks is at least 100 times lower(34). This high rate of mutation mayprovide an explanationfor the antigenic variability of CAEV in nature without the obviousselective pressure of neutralizing antibodies. Further evidenceof this high variability comesfrommolecularanalysis of three cloned strains of CAEV, all of whichhave widely divergent restriction endonucleasemaps(20, 35-38). PERSISTENT INFECTION AND ANTIGENIC VARIATION OF VISNA VIRUS Sheepinfected naturally or experimentallywith visna virus becomepersistently infected with the virus despite the productionof neutralizing antibodies3-4 weeksafter infection(14, 39-41 ). Visnavirus canbe isolated fromthe circulating blood monocytesof these animalsat all times after infection (41-43). Early studies on persistent infection in sheephad shown that virus isolated from the animals late in the infection could not be neutralized by serumantibodies (44). These results suggested that the presenceof neutralizing antibodyis not sufficient to clear the virus infection, that virus mayundergomutation during persistent infection, and that virus variants no longer neutralized by the antibodies mayhave selective growthadvantage. Twoaspects of this antigenic variation seen in persistent infection should be emphasized:(a) Variant viruses do not appearto replace the infecting strain of virus, and(b) antigenicvariation of visna virus in sheepcan only be detected in 25 %of animals.Tworecent studies concludedfromthese observationsthat antigenic variation is rare and thus could account neither for viral persistence nor for continued replication of the virus in immune animals(45, 46). However,that variant viruses do not replace the infecting strain should not be surprising since these viruses are retroviruses that integrate into the host genome.In

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addition, studies of persistently infected sheep showthat monocyteprecursors in the bonemarrowcontain viral nucleic acid providinga reservoir of the infecting strain (as well as other strains) in stemcells in the bone marrow(43). That with pooledpolyclonalsera antigenic variation is found in one quarter of all infected animalssuggests that this is a biologically significant phenomenon. In addition, monoclonalantibodies can detect subtle antigenic changesin "parental" type virus that are not detected by pooledserum(47). MOLECULAR STUDIES OF VISNA VIRUS

OF ANTIGENIC

VARIANTS

Todeterminethe molecularbasis of antigenic variation westudied antigenicvariants of visna virus fromtwoinfected sheepas well as visna virus mutants selected in culture in the presence of early immunesera. Two sheep (1 and 4) were inoculated with plaque-purified virus. At various intervals during a 3-year period, blood from the animals providedserum for measuringneutralizing antibody,and white bloodcells that werecocultivated with sheep fibroblasts for virus isolation (15, 16). Theearliest antibodies producedby the sheep neutralized the parental virus (strain 1514)used for inoculation but riot viruses isolated later frombloodleukocytes (sheep viruses LV1and LV4). The antibody responses broadened with time, and sera obtainedlate in the infection neutralized the variant viruses (Table 1). However,the last serumsamplefrom each sheep could still distinguish the parental andvariant viruses, emphasizing the antigenic difference of the agents. To determinewhether the variant viruses may havepreexistedin the virus suspensionused for inoculation of the animal, weexamineda concentratedpreparation of plaque-purifiedvisna virus for the presence of variants. Astock of parental virus containing 1 7x 10 plaque-formingunits (pfu) was mixedwith early immunesera and examined for non-neutralizablevariants. After 6 hr of incubationat 37°C,followedby overnightincubationat 4°C, all survivingviruses hadthe parental phenotype,i.e. less than 1 infectious virus in 1 x 107 of parental virus was antigenicallydistinct (33, 34). In contrast to CAEV, therefore, visna virus has a low rate of spontaneousantigenic mutation. The variant viruses in the animal must therefore have developed under selective pressure by antibodiesduringthe persistent infection. To test the ability of immune sera fromthese animalsto select variants in tissue culture, weinoculatedcell cultures with 103pfu of plaque-purified virus 1514 and maintained the cultures with mediumcontaining early immunesera from the two animals (16, 47). Viruses developingin these dultures (tissue culture variants AD1-1 and AD4-1)were no longer neu-

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145

Table 1 Genetic changes in antigenic variants ofvisna virus strain 1514, isolated from two persistently infected sheep and afrom tissue culture

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Virus

Neutralization Early Late

Genetic changes

1514

++++

++++

LV1-2 LVI-7 LV 1-4 L¥ 1-3 LV1-6 LV1-5 LV1 - 1

++++ -------

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

AB AB AB ABC ABC ABC ABCDEF

LV4-2 LV4-3 LV4-1

+ + + + ---

+ + + + + + ++

A EF A EF A C EF

ADI-1 AD4-1

---

÷ ÷ + +

AB AB

E E

a TheLVIviruses wereisolated fromsheep1 and the LV4viruses fromsheep 4. The tissue culture derived virus variants ADI-Iand AD4-1were selected with early immunesera from sheep 1 and 4, respectively.Thegeneticchangesare thoselocatedin the 3’ regionof the genome.

tralized by the sera used to select them and were thus antigenic variants similar to those arising in the animal. These in vitro-derived viruses were comparedwith those variants obtained from the animals during persistent infection (Table 1). Viral antigenic drift has been postulated to result from point mutations in the viral genome, which produce changes in the outer membraneglycoprotein, gp135. As a first step in investigating the molecular basis for the altered antigenicity of the naturally occurring variants, as well as of the tissue culture-derived mutants, the genomic RNAsof these viruses were compared by RNaseTl-resistant oligonucleotide fingerprinting (34, 48). Eighty unique RNAseTl-resistant oligonucleotides were identified, and their locations were mappedon the viral RNA.Sequence analysis was performed (a) to determine whether the unique oligonucleotides in the different viruses were the same, and (b) to determine the differences between altered oligonucleotides. These experiments showedthat the antigenic variants were closely related to the parental virus and differed from it by only a few changes localized in the env region of the viral genome.

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Further analysis showedthat manyof the changes were commonamong the variants, reflecting the samepoint mutation. This suggestedthat the changesaccumulatedin the genomesof the variant viruses and that antigenic drift occurred in a progressive manner(Table 1). Oligonucleotide changesoutside the env gene were rare, and no one changewas observed in morethan one virus. This suggested that these mutations were random and had no selective advantage for the virus carrying them. However, neutralizing antibody in the immuneanimal appeared to have a strong selection for mutationalchangesin the env geneof visna virus, and these changeswere maintainedand passed to progenyvirus. Theprogressivenature of antigenic mutationof visna virus in sheep is illustrated by the changesdetected in the viruses isolated fromsheep 1. Virus LV1-2,isolated from this sheep, wasof parental serotype; however, it was found to differ from the parental virus by two oligonucleotides located in the env gene (Table 1 summarizesthe genetic changes and serologic characterization of the variants from two sheep). This change wasfoundin all subsequentantigenic variants fromthis animal, suggesting that all the variants descendedfromthis virus and that it had a selective advantageover the parental virus. Theoriginal serological classification of the variants (Table1) is consistent with the cumulativechangesobserved in the env gene. Whenvariants from a second infected animal (sheep 4) were examined, it was surprising to find that the same oligonucleotide changes were detected and that these changes reflected the same point mutations, as determinedby sequence analysis. These common changes in the variants fromthe two animalssuggestthat the selective pressure for the phenotypic changein the viral antigenicity requires very specific alterations in the viral genome,since mutations probably occurred randomly.The selected mutationspresumablyspecifically alter the site of interaction of the neutralizing antibodymoleculewith the viral glycoprotein. This mightinvolve three or four epitopes on the protein, each containing a small numberof aminoacids. Further support for this highly specific selection of particular point mutationscomesfrom RNaseTi fingerprint analysis of antigenic variants isolated in tissue culture. Early immune sera fromsheep 1 and 4 wasused to select variants in tissue culture, AD1-1 and AD4-1.Theoligonucleotide changesdetected in these viruses werea subset of the changesfoundin the antigenic variants isolated fromthe persistently infected sheep(Table1). Thus,the samepoint mutationalchangeswereselected in vitro and altered the antigenic phenotypeof these tissue culture-derivedvariants. To determinethe exact number,position, and potential biological role of particular mutationsin the antigenic variants of visna virus, clones of

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147

a numberof the variants from sheep 1 have been obtained, and the completenucleotide sequenceof the enveloperegion has been determined. In LVI-1, the most distant variant, the envelopegene contains only 11 nucleotide changes from the parent strain 1514. Previous RNaseT~ oligonucleotidemapping,whichonly resolves 10-15%of the genetic information, detected 6 changes. Therefore a larger numberof changes were expected. Despite the dramatic difference in the antigenic phenotypeof LVI-1fromthe parental phenotype,it differs fromthe parental virus by only a small numberof point mutations. Further only 7 of the 11 base changesresulted in changesin aminoacids in the envelopeproteins, and only six of these aminoacids are in the outer membrane protein. At the carboxyl terminus of the outer membrane protein, there is a cluster of three aminoacid changes, and two of these represent changes from unchargedaminoacids in the parental strain to chargedaminoacids in the variant LVI-I. Thus, this site mayrepresent a region of the outer membrane protein that is important in recognition by the immuneresponse. It is interesting that noneof the aminoacid changesaltered potential glycosylationsites in the envelopeproteins. Thus,relatively few changes in the amino acid composition of the gp135 appear to have profound effects on the antigeniccharacterof the protein. Studies using monoclonalantibodies against the gp135of visna virus strain 1514further demonstratedthat small numbersof base changesin the variants hadprofoundeffects on the topological arrangement of epitopes in this protein (47). Thesestudies suggested that point mutations in the env gene result in the exposureof epitopes previously present in the glycoprotein, rather than creation of newantigenic sites. Themonoclonal antibodies prepared against gp135 defined five partially overlapping epitopes which comprise two domains. One domainis called an overt domainbecause it is exposedon the native virus; the other domainis covert and does not appear to be accessible to antibodyon native virus. All five epitopes defined by the monoclonal antibodies werealtered in the antigenic variants from sheep 1. Nosimilar changeswere detected in the core protein p27 whensimilar analyses were done with anti-p27 monoclonal antibodies. Binding of the highest avidity MAbsto epitopes gp135a, gp135b, and gp 135c, whichare exposedon virus strain 1514,decreasedduringantigenic drift; these three epitopeswereobscuredor lost on the variants (Figure1). However,whenthe LV1viruses were treated with nonionic detergents or reducing agents, they then regained MAb-binding abilities against these three epitopes. This suggests that these surface epitopes weresometimes blockedor buried rather than lost during antigenic variation. Bindingof lower-avidity MAbsto epitopes gp135d and gp135c, whichare covert or

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ET

AL

1.6 1.2 0.8 0.4

G8

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0.6 0,4 0.2

~J O~ 0,6

02

0.2

0.8

0,8

0.6

0.6

0,4

0.4

0.2

0.2 i~ 9P 1§14 LV LV LV LV LV LV 135 I-2 I-4 i-3 I-5 I-6 I-I

Qp 1514 LV LV LV LV LV LV 135 I-2 I-4 I-3 I-5 I-6 I-I Figurel

Topographical

MAb binding

The LV1 viruses divergence

are

from strain

against

epitop~s

epitope

might

density

changes

to purified

(b)

in visna

gp 135 during

gp 135 (strain ordered

along

1514), the

abscissa

1514.

The five

gp135 epitopes

and (e)

displayed

two distinct

be subdivided

at 492 nm; S.D.,

(subscripts

standard

antigenic

strain

1 and

1514, from

left

drift,

or the to

right

are marked (a) ELISA patterns, 2).

shown by differential

six LV1 antigenic

Abbreviations:

by increasing through

(e)

genetic

(boxes);

which suggested O.D.

variants.

that

492 nm, optical

deviation.

less accessible on strain 1514, increased during antigenic drift; these two epitopes were reciprocally exposed or unblocked on the variants (Figure 1). Treatment of strain 1514 with reducing agents increased MAbbinding to covert epitope gp135e three- to five-fold. This suggests that disulfide bonding on gp135 may diminish MAbbinding to epitope gp135~ on strain 1514; enhanced MAbbinding to this epitope on the LV1 viruses may be

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due to conformational rearrangements of gp135 which involve altered disulfide linkages. MAbsagainst epitope gpl35~1 bound the LV1viruses better than they boundstrain 1514, just as they had boundgpl35 purified from strain 1514better than they boundthe virus itself. This suggests that during antigenic variation, the covert gp135~1 epitope may have been exposed on the LV1viruses in a conformation resembling that caused by detergent treatment and purification of gp 135 from the envelope of strain 1514. By ELI SA, MAbsagainst epitopes gp135e through gp 135d clearly differentiated strain 1514 from its closest progeny, LV1-2, whereas the antibodies in early- or late-post infectious immunesheep sera had been unable to distinguish the two viruses in neutralization tests. Paradoxically, by ELISA, the MAbsdid not separate LV1-2 from LVI-1, LV1-3, or LV14, whereasserum neutralization tests had separated them. It is of interest that the anti-gp 135 MAbswe used did not neutralize visna virus. Thusthe five epitopes on gp135 recognized by our MAbsmay be distinct from the epitopes responsible for neutralization. This suggests that antigenic drift is associated with complex rearrangements of both neutralizing and nonneutralizing epitopes on the visna virus envelope glycoprotein. These data suggest that topographical flip-flop or refolding of the tertiary structure of gp135 may phenotypically amplify genetic changes in variable regions of the env gene. Topographical flip-flop of gp135 might further explain the symmetrical losses and gains of MAb-bindingsites on the viral glycoprotein during antigenic drift. Three epitopes exposed on strain 1514 (gp135a, gp135b, gp135c) were lost or obscured on the LV1 viruses, while two covert epitopes on strain 1514(gp 135~ and gp 135¢) were reciprocally exposed or unblocked on the LV1mutants. Topographical permutability may dramatically increase the amount of information stored on the envelope glycoprotein and mayallow visna virus to sequester biologically active epitopes from the immunesystem. Wehad earlier observed that neutralizing antiserum from a sheep infected with visna virus strain 1514 did not neutralize the mutant LV1-1virus that was isolated later from the same sheep (15). However,whenanother sheep was hyperimmunizedwith nonionic detergent-disrupted strain 1514, the animal developed antibodies that neutralized both strain 1514 and the LVI-1 virus. This suggests that strain 1514 contains the neutralization epitope(s) of LV1-1, but that this site on its envelopeglycoprotein is conformationally concealed or blocked until it is exposedto the immunesystem by detergent solubilization. Such exposure of conformationally hidden neutralization epitopes on strain 1514 gp135 strikingly resembles the manner in which covert epitopes gp135d and gp135~ were topographically exposed or unblocked on the LV1viruses during antigenic drift in this study. Cor-

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relation of the genetic analysis of the antigenic variants with the monoclonal antibody studies suggests that a small numberof changes in the aminoacid compositionof visna virus gp135has a profoundeffect on the structure of the virus envelope. The mechanismby whichthese changes alter the structure appears to be changingthe epitopes exposedon the surface rather than directly changingan epitopeor creating a newantigenic site. CLINICAL COURSE AND IMMUNE RESPONSES DURING PERSISTENT EIAV INFECTION Theclinical responsesof horses followingnatural or experimentalinfection by EIAVcan be dividedinto characteristic stages (1, 50). AcuteEIA(fever and hemorrhages)is typically associated with the first exposureto virus andappears to correlate with massivevirus replication in and destruction of macrophages. In the initial stages of EIAthe animalsare seronegative, but maybecomeseropositive, developing neutralizing antibody, within 16-30 days post infection. The earliest and predominant antibody responsesare directed against the EIAVenvelopeglycoproteins, gp90and gp45. Minorantibody responses can then be detected against the major internal proteins of the virus, whichare 10-20times moreabundantin the virus than the viral glycoproteins. The moreclassical symptoms of EIA(anemia, weight loss, edema,etc) are observedlater during recurring cycles of viremia and illness which appearat irregular intervals separated by weeksor months.This stage of disease is called chronic EIA.Horses with chronic EIAare seropositive, and their T-lymphocytesare responsive to purified, inactivated EIAV. There is no evidence of depression of humoral or cellular immune responses. However, the magnitude of humoral immuneresponses to EIAVcan vary markedly during the course of chronic EIA, and cellfree EIAVis frequently found in the blood as infectious antibody-virion complexes(51). Thefrequencyand severity of clinical episodes decline with time, and the chronic stage of disease usually ends within the first year after infection, after an averageof 6-8 disease episodes. Duringthis stage of disease the virus can be efficiently transmitted by wholeblood transfers from infected to naive horses via insect vectors (52) or humans (hypodermic needles, scalpels, etc). At this point, the infected horse enters the inapparent stage of EIA during whichthere are no clinical symptomsor detectable viremia. The horse continues to harbor EIAV,however,as evidenced by the fact that disease can be efficiently transmittedby experimentalwholebloodtransfers to naive horses. In addition, it has beendemonstratedthat recrudescence

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of chronic EIAcan be induced in someinapparently infected horses by administration of dexamethasone or by stress, even in animals that have beenfree of febrile episodesfor years (53). Thus, the periodic nature of EIAoffers a uniquely dynamicmodelfor examininglentivirus persistence in the presence of fully competenthost immuneresponses. Moreover,EIAV-infectedhorses represent a system in whichthe infected animal routinely establishes control of lentivirus replication, presumablyresulting from cumulativeimmune responses generated during the first year post infection. Clarifying the immune status by whichthe horse eventuallycontrols EIAVreplication, evenin the face of relatively rapid antigenicvariations of the virus, shouldprovidevaluable insights into strategies for the immunologicmanagement of lentivirus infections. NATURE OF ANTIGENIC VARIATION PERSISTENT EIAV INFECTION

DURING

The uniqueperiodic nature of chronic EIAcan evidently be attributed to the sequential evolution of antigenic variants of EIAVthat temporarily escape established humoraland cellular immuneresponses. The initial evidence for EIAVantigenic variation is based on comparative neutralization assays employingvirus isolates and immunesera recovered fromchronically infected horses duringvarious stages of disease (54, 55). Morerecently a detailed characterization of EIAVantigenic variation has been madepossible by the developmentof an animal model system in whichShetland ponies inoculated with a standard strain of EIAVreproducibly develop chronic EIA, during which time EIAVisolates can be efficiently recoveredin the sequential cycles of disease (11). Apanel over 20 of these virus isolates has been characterized by a variety of immunologic and biochemicalproceduresto ascertain the nature of antigenic variation in EIAV(12, 56-59). Themajor conclusions of this survey can be demonstratedby examining the properties of a panel of 13 virus isolates recovered during febrile episodes in three experimentallyinfected ponies (Table 2). Eachvirus isolate wasinitially examined in neutralization assays usingserumsamples collected during sequential afebrile periods in the infected ponies. The results of these assays reveal several importantproperties of persistent EIAVinfections. First, within each group of virus isolates froma single pony, each isolate can only be inactivated by serumsamplestaken after the clinical episodeof origin; noneof the virus isolates can be neutralized by serumsamplestaken prior to the disease cycle of origin. Second,crossneutralization assays indicate that each isolate is antigenically distinct,

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CLEMENTS ET AL Table 2 Characterization of EIAVisolates obtained during sequentialclinicalepisodesin threeponiesexperiencing chronicEIA inducedbyexperimental infection witha standardvirus inoculum (11). Theglycoprotein epitopeswereidentifiedin eachvirusisolated basedon reactivity in immunoblots with a panel of monocional antibodies(60)

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EIAV Isolates Pony91:P3.1-1 P3.1-2 P3.1-3 P3.1-4 Pony127:P3.2-1 P3.2-2 P3.2-3 P3.2-4 P3.2-5 Pony135:P3.3-1 P3.3-2 P3.3-3 P3.3-4

Isolationday post inoculation 12 32 58 198 13 41 99 137 158 16 46 75 107

Epitopes gp90 gp45 ABCD ABCDE ABF AE AD ABCD ABCDF AE AE ACD ABCDF ABCDF A

A A A A AB AB AB AB AB A AB AB AB

although an occasional low level of cross reactivity can be detected in the neutralization assays. The antigenic uniqueness of most isolates can also be demonstrated by assaying the reactivity in Western blots using a panel of monoclonal antibodies specific for gp90 and gp45, respectively. The panel of 16 gp90-specific monoclonalantibodies define four distinct, but overlapping, epitopes (designated A, B/F, C/D, and E), two of which (C/D and E) are involved in virus neutralization. The panel of eight gp45-specific monoclonal antibodies defines two distinct, non-overlapping epitopes (designated A and B), neither of which is involved in virus neutralization. Thus, these antigenic analyses indicate that a distinct antigenic variant is associated with each febrile episode in an experimentally infected animal and that the evolution of a new predominant strain of virus can take as little as two weeks or as long as several months. There is no detectable common pattern of variant evolution in the parallel infections in different ponies. Thus, EIAVantigenic variation differs from that observed in visna virus in that the former is evidently more rapid and morerandom(i.e. less selected upon). The biochemical nature of antigenic variation amongthe panel of EIAV isolates has been examined both by peptide and glycopeptide mapping procedures to compare viral proteins and by oligonucleotide fingerprint

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analysis to comparegenomicRNA(12, 56-59). The peptide/glycopeptide variations are specific for the viral envelopeglycoproteins(gpg0and gp45); no structural alterations are observedamongthe internal viral proteins of the respective virus isolates. Thecombinationof peptide and glycopeptide mappingindicates that each of the 13 virus isolates contain structurally unique gp90 and gp45 components, as no two maps were identical in pairwise comparisons.Moreover,analysis of the variant peptides detected amongsequential virus isolates fromeach ponyreveals that peptide alterations appearingin oneisolate are not necessarily retained in the next or successivevariants in that pony.Similarly,the oligonucleotidefingerprints differentiate amongall of the virus isolates and onceagain demonstratea noncumulativepattern of variation amongsequential isolates. Pairwise comparisons of oligonucleotidefingerprints typically identify 3-4 variant oligonucleotidesout of a total of over 50 oligonucleotidesresolved for each genomicRNA.The degree of protein and genomicvariation detected by these proceduressuggests that the antigenic variation observedin EIAV is the result of randompoint mutations in the viral RNA,someof which result in altered glycoproteinaminoacid sequences.Alterations in peptide mapsand oligonucleotide fingerprints are not detected during passage of the virus isolates in tissue culture, indicating the requirementof immune selection in generating newpredominantvariants in horses. In these aspects, EIAVantigenic variation closely resembles variation in visna virus. However,the apparently noncumulative and completely random nature of EIAVvariation is in markedcontrast to the cumulative,common patterns of visna virus variation describedabove. To relate the observedantigenic and structural variations directly to nucleotide sequencevariation in EIAV,the env gene sequences of four virus isolates obtained from pony91 weredeterminedand compared(59). All but one difference in nucleotide sequenceamongthe four isolates resulted fromsingle base substitutions; a single three-base insertion was observedin isolate P3.2-5. Figure 2 summarizesthe deducedaminoacid sequencefor the gp90and gp45components of each virus isolate. Overall, the nucleotide divergencebetweenany pair of virus isolates ranges from 16-37 bases out of a total of 2583bases in the env gene. Approximately 75%of all nucleotidesubstitutions result in aminoacid replacements,and aminoacid divergencerangesfrom11-29residues out of 860 total. In light of the extent of variation observedin peptide mapping,the relatively low level of nucleotide (0.6-1.4%) and aminoacid (1.3-3.4%) divergence somewhatsurprising. However,the majority of all amino acid substitutions are nonconservative,andlikely to induce changesin structure and in antigenic properties. Variation is not evenly distributed throughout the eno gene. Twoto

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leader

< > gp90

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P3.2-1 HVSI AFYGG l pGGI STP I TQOSEKSKCEEHTMFQPYCYI~NSHAE~KEARDQEH#LKEESKEEKRANDMA(I GHFLLCLAGTTGG I L~EGL~QH t t ~~ t P3.2-2 ~ E P3.2-3 P3.2-5 I ~

P3,2-I I AQFGKCILWSH I GH~ l F~ILGAS I I KY I VMFLLI YLLL]TSSPK I LRALWI(VTSGAGSSGGRYLKKKFHHKHASREDTkl)QAQHH I HLAGVTGGSGDKYYKQ C P3.2-2 S Y t P3.2-3 ~ S N C P3.2-5 S Y t P3,2"1 LF KYSRND~NGE$EEYN~PKS~KS~EAF~ES~SEKTKGE]SQPGAA[NEHK~GGNNPH~G~LDLE~EGGN~Y~CC~KAr~EGTLA~PCCGFPLW ~~ tt tt 1" ~_ R P3.2-2 P3.2-3 ~ P3.2-5 T ~ P3.2-IWGLVIIVGRIAGYGLRGLAV[ I R ] CI RGLNLI FE I I RKHLDYI GRALNPGTSHVSMPQYV P3.2-~ P3.Z-3 P3.2-5 F

Figure 2 Deducedamino acid sequences for the env genes of EIAVisolates from pony 91. The complete sequence is shownfor isolate P3.2-1 while amino acid acids begins with the initiator methionine. Regions corresponding to the leader peptide, the highly glycosylated outer membraneglycoprotein, gp90, and the proposed transmembrane glycoprotein, gp45, are indicated. Cysteine residues are indicated by arrows (T T). Potential N-linked glycosylation sites are highlighted by shaded boxes. Within gp90, conserved regions are indicated by lines above the aminoacid sequence, and the hypervariable region is indicated by asterisks (*). The proposed precursor cleavage site (RHKR)is designated by an arrow (,~). Putative membranespanning regions with gp45 are indicated with square brackets ([ ]).

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three times as manyamino acid substitutions occur in the exterior gp90 as do in the transmembrane gp45. Further, within gp90, two conserved and one hypervariable region can be identified. The first conserved region within gp90 includes the N-terminal 21 amino acid leader peptide and the following 1 l0 aminoacids, while the second conserved region encompasses aminoacid residues 370 to 445. In these regions, there are few substitutions, and all potential N-linked glycosylation sites and cysteine residues are absolutely conserved amongthe four isolates. In contrast, a variable region of gp90 spans amino acid residues 143 and 366, in which pairs of virus isolates differ by 3% to 9% in amino acid sequences. A hypervariable region of 35 amino acids (residues 306-338) is located within the gp90 variable region. Within this hypervariable region, amino acid divergence between any two isolates ranges from 3%to 20%. EIAVgp90 is highly glycosylated and contains numerouspotential N-linked glycosylation sites (N-X-S/T) within the variable region of the protein. Since about 40% all aminoacid substitutions in the env gene involve the loss or gain of an asparagine (N) residue, it is perhaps not surprising that the number glycosylation sites within the variable region of gp90range from six to ten in the different virus isolates. In contrast to gp90, EIAVgp45 is more highly conserved among the four virus isolates, with amino acid substitutions between isolate pairs ranging from 0.7% to 2% (3-9/415 residues). Within gp45, the amino terminal half is more highly conserved than the carboxyl terminal half of the molecule, including two hydrophobic (putative transmembrane) regions (residues 448-473 and 617-636) and four N-linked glycosylation sites that are conserved amongall isolates. The variant sequence data described above indicate that the antigenic differences amongthe virus isolates examinedcan be correlated with relatively minor differences in aminoacid sequence, most ofwhiohare localized to a relatively small region of gp90. It is not yet possible to correlate epitopes defined by monoclonalantibodies with specific peptide sequences. However,it is interesting to note that limited proteolytic digestions of EIAVgp90 produce fragments of about 12,000 tool wt which react with all of the neutralizing and most of the non-neutralizing monoclonalantibodies (Montelaro, unpublished). Thus, it is tempting to speculate that the antigenic sites maybe localized in the variable/hypervariable region of gp90. The sequence data also permit estimation of the mutation rate of the EIAVenv gene during persistent infection. This calculation uses the formula R = D/2T (61), where R is the number of nucleotide substitutions per site per year, D is the divergence calculated from the proportion of nucleotide differences between pairs of isolates, and T is the divergence time represented here as the time betweenthe isolation of the first isolate

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(P3.2-1) and the later isolates. Usingthese parameters,mutationrates 1 × 10-1, 2 × 10-2, and 1 × 10-2 were obtained for isolates P3.2-2, P3.2-3, and P3.2-5, respectively. Thesemutation rates are about 10-fold higher than those observedfor HIV(62) and 1000-foldgreater than observedfor other RNA viruses (63).

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CONCLUSIONS Antigenicvariation of the lentiviruses as exemplifiedby visna virus and EIAVposes a formidable problem to the host immunesystem as well as to those designingvaccinesagainst these agents. It is clear fromthe studies described in this review that immunizationwith the undenaturedenvelope protein of any onelentivirus will not protect against the rapidly emerging variants that occur during infection with these viruses. The studies describedhere suggestthat there are hypervariableregions in the envelope genes of these viruses. The molecular mechanismof this high rate of variability in a small genetic region mayprovide clues for genetically engineering novel immunogenswhich would provide a broad range of neutralizingantibodiesto lentiviruses. Indeedmutationappears to be only one aspect of the complexantigenic phenotypeof the envelopeproteins of lentivirus, the three dimensional structure of the outer membraneprotein also determines recognition of epitopes by the host immunesystem. Thus, studies on the structure and organizationof the envelopeproteins will be importantfor the design of effective vaccinesagainstlentiviruses. ACKNOWLEDGMENTS

This workwassupportedby grants fromthe National Institutes of Health NS21916, NS16145, NS07000and CA38851. Wewould like to thank Linda Kelly for preparation of the manuscript. Literature Cited I. Cheevers,W.P., McGuire,T. C. 1985. cytopathic retroviruses (HTLV-III)from Equineinfectious anemiavirus. Immupatients with AIDSand pre-AIDS.Scinopathogenesis and persistence. Rev. ence 224:497-500 4. Gonda,M. A., Wong-Staal,F., Gallo, Infect. Dis. 7:83-88 2. Narayan, O., Cork, L. C. 1985. LenR. C., Clements, J. E., Narayan, O., tiviral diseases of sheep and goats. Gilden, R. V. 1985. Sequencehomology Chronic pneumonia,leukoencephalitis and morphologicsimilarity of HTLV-III and visnavirus, a pathogeniclentivirus. and arthritis. Rev. Infect. Dis. 7: 8998 Science 227:173-77 5. Gonda,M. A., Braun, M. J., Clements, 3. Popovic, M., Sarngadharan, M. G., Read,E., Gallo, R. C. 1984.Detection, J. E., Pyper, J. M., Casey,J. W.,Wongisolation and continuousproduction of Staal, F., Gallo, R. C., Gilden, R. V.

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ANTIGENSIN LENTIVIRALDISEASES 1986. HTLV-IIIshares sequence homology with a family of pathogeniclentiviruses. Proc.Natl. Acad.Sci. USA83: 4007-11 6. Murphey-Corb, M., Martin, L., Rangan, S., Baskin, G., Gormus, B., Wolf, R., Andes, A., West, M., Montelaro, R. 1986. Isolation of an HTLV-III-related retrovirus from macaqueswith simian AIDSand its possible origin in asymptomaticmangabeys.Nature 321: 435-37 7. Bloom,B. R. 1979. Gamesparasites play: howparasites evade immune surveillance. Nature279:21-26 8. Webster, R. G., Laver, W.G., Air, G. M., Schild, G. C. 1982. Molecularmechanismsof variation in influenza virus. Nature 296:115-21 9. Wiktor, T. J., Koprowski,H. 1980. Antigenic variants of rabies virus. J. Exp. Med. 152:99-112 10. Ter Muelen,V., Loftier, S., Carter, M. J., Stepbenson,J. R. 1981. Antigenic characterization of measles and SSPE virus haemaglutinin by monoclonal antibodies. J. Gen.Virol. 57:357~4 11. Kono,Y., Kobayashi,K., Fukunaga,Y. 1973.Antigenicdrift of equineinfectious anemia virus in chronically infected horses. Arch. Virusforchung41:1-10 12. Orrego,A., Issel, C. J., Montelaro,R. C., Adams,W.V. 1982. Virulence and in vitro growthof a cell-adaptedstrain of equineinfectious anemiavirus after serial passagein ponies.Am.J. Vet. Res. 43:1556-60 13. Montelaro,R. C., Parekh, B., Orrego, A., Issel, C. J. 1984.Antigenicvariation during persistent infection by equine infectious anemiavirus, a retrovirus. J. Biol. Chem.259:10539-44 14. Narayan,O., Griffin, D. E., Chase,J. 1977.Antigenicshift of visna virus in persistently infectedsheep.Science197: 376-78 15. Narayan,O., Griffin, D. E., Clements, J. E. 1978.Virus mutationduring "slow infection." Temporaldevelopmentand characterization of mutants of visna virus recovered from sheep. J. Gen. Virol. 41:343-52 16. Narayan,O., Clements,J. E., Griffin, D. E., Wolinsky,J. S. 1981. Neutralizing antibody spectrumdeterminesthe antigenic profiles of emergingmutants of visna virus. Infect. Immunol.32: 104550 17. Sonigo, P., Alizon, M., Staskus, K., Klatzman, D., Cole, S., Danos, O., Retzel, E., Tiollais, P., Haase,A., WainHobson,S. 1985. Nucleotide sequence of the visna lentivirus: Relationshipto

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the AIDSvirus. Cell 42:369-82 18. Davis,J. L., Molineaux, S., Clements,J. E. 1987. Visnavirus exhibits a complex transcriptional pattern: Oneaspect of geneexpressionshared withthe acquired immunodeficiency syndromeretrovirus. J. Virol. 61:1325-31 19. Vigne,R., Filippi, P., Querat,G., Sauze, N., Vitu, C., Russo,P., DeLori,P. 1982. Precursor polypeptides to structural proteins of visna virus. J. Virol. 42: 1046-56 20. Pyper, J. M., Clements, J. E., Molineaux, S. M., Narayan,O. 1984. Genetic variation amo.n~%lentiviruse~: Homologybetween vlsna virus a~d capfine arthritis-encephalitis virus is confined to the 5’ gag-polregionanda small portion of the env gene. J. Virol. 51: 713-21 21. Montelaro,R. C., Lohrey, N., Parekh, B., Blakeney,E. W., Issel, C. J. 1982. Isolation and comparativebiochemical properties of the major, internal polypeptides of equine infectious anemia virus. J. Virol. 42:1029-38 22. Montelaro,R. C., West, M., Issel, C. J. 1983.Isolation of equine infectious anemiavirus glycoproteins.Lectinaffinity chromatography proceduresfor high avidity glycoproteins. J. Virol. Methods 6:337-46 23. Montelaro,R. C., West,M., Issel, C. J. 1984. Antigenicreactivity of the major glycoproteinof equineinfectious anemia virus, a retrovirus. Virology136:368-74 24. Harter, D. H., Choppin,P. W.1967.Cell fusing activity of visna virus particles. Virology31: 279 25. Narayan,O., Clements,J. E., Strandberg, J. D., Cork, L. C., Griffin, D. E. 1980. Biologiccharacterization of the virus causingleukoencephalitisarthritis in goats. J. Gen.Virol. 50:69-79 26. Haase, A. T., Varmus,H. E. 1987. Demonstration ofa DNA provirusin the lytic growthof visna. NatureNewBiol. 245: 237-39 27. Narayan,O., Wolinsky,J. S., Clements, J. E., Strandberg,J. D., Griffin, D. E., Cork,L. C. 1982.Slowvirus replication: the role of macrophagesin the persistence and expressionof visna viruses of sheep and goats. J. Gen. Virol. 59: 345-56 28. Narayan, O., Kennedy-Stoskopf, S., Sheffer, D., Griffin, D. E., Clements,J. E. 1983.Activationof caprinearthritisencephalitis virus expression during maturation of monocytes to macrophages. Infect. Immun.41:67-73 29. Crawford, T. B., Adams,D. S. 1981. Caprinearthritis encephalitis: clinical

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features and presence of antibody in selected goat populations. J. Am.Vet. Med. Assoc. 178:713-19 30. Narayan,O., Sheffer,D., Griffin, D. E., Clemems,J. E., Hess, J. 1984. Lackof neutralizingantibodiesto caprinearthritis-encephalitislentivirus in persistently infected goats can be overcome by immunizationwith inactivated Mycobacteriumtuberculosis.J. ViroL49: 34955 31. Klevjer-Anderson,P., McGuire,T. C. 1982. Neutralizingantibodyresponse of rabbits and goats to caprine arthritisencephalitis virus. Infect. lmrnun.38: 45541 32. Narayan,O., Clements,J. E., KennedyStoskopf, S., Royal,W.1986. Antigenic variation in ovinecaprine lentiviruses. In AntigenicVariationin Infectious Diseases, ed. T. H. Birkbeck,C. W.Penn, pp. 25-40. Oxford:IRL Press 33. Narayan,O., Clements,J. E., KennedyStoskopf, S., Royal, W. 1987. Mechanismsof escape of visna lentiviruses from immunologicalcontrol. In Antigenic Variation: Molecularand Genetic Mechanisms of RelapsingDisease, ed. J. M. Cruze, pp. 60-76. Basel: S. Karger Publ. In press 34. Clements, J. E., D’Antonio, N., Narayan, O. 1982. Genomicchanges associated with antigenic variation of visna virus. II. Common nucleotide changesdetected in variants fromindependent isolations. J. Mol. BioL 158: 415-34 35. Roberson, S. M., McGuire, T. C., Klevjer-Anderson, P., Gorham,J. R., Cheevers,W.P. 1982. Caprinearthritisencephalitisvirus is distinct fromvisna and progressive pneumoniaviruses as measured by genome sequence homology. J. Virol. 44:755-58 36. Chiu,I. M., Yaniv,A., Dahlberg,J. E., Gazit, A., Skuntz,S. F., Tronick,S. R., Aaronson, S. A. 1985. Nucleotide sequenceevidence for relationship of AIDSretrovirus to lentiviruses. Nature 317:366-68 37. Pyper, J. M., Clements, J. E., Gonda, M. A., Narayan, O. 1986. Sequence homology between cloned CAEVand visna virus,58:665-70 twoneurotropiclentiviruses. J. Virol. 38. Yaniv,A., Dahlberg,J. E., Tronick,S. R., Chiu, I. M., Aaronson,S. A. 1985. Molecularcloning of integrated caprine arthritis encephalitisvirus. Virolo#y145: 340-45 39. Narayan,O., Griffin, D. E., Silverstein, A. M. 1977. Slowvirus infection: Replication and mechanism of persistence of

visnavirus in sheep.J. Infect. Dis. 135: 800-6 40. Griffin, D. E., Narayan,O., Adams,R. J. 1978.Early immune responsein visna, a slowviral diseaseof sheep.J. Infect. Dis. 138:340-50 41. Narayan,O., Wolinsky,J. S., Clements, J. E., Strandberg,J. D., Griffin, D. E., Cork,L. C. 1982.Slowvirus replication: The role of macrophagesin the persistence andexpressionof visnavirus of sheepand goats. J. Gen.Virol. 59: 34556 42. Gendelman,H. E., Narayan, O., Kennedy-Stoskopf,S., Kennedy,P. G. E., Ghotbi, Z., Clements,J. E., Stanley, J., Pezeshkpour, G. 1986. Tropismof sheep lentiviruses for monocytes:Susceptibility to infection and virus gene expression increase during maturation of monocytesto macrophages.J. ViroL 58:67-74 43. Gendelman,H. E., Narayan, O., Molineaux, S., Clements,J. E., Ghotbi, Z. 1985.Slowpersistent replication of lentiviruses: Role of tissue macrophages and macrophage-precursors in bone marrow.Proc. Natl. Acad. Sci. USA82: 7086-90 44. Gudnadottir, M. 1974. Visna-maediin sheep. Prog. Med.Virol. 18:336-49 45. Thormar, H., Barshatzky, M. R., Kozlowski,P. B. 1983. The emergence of antigenicvariation is a rare event in long term visna64:1427-32 virus infection in vivo. J. Gen. Virol. 46. Lutley,R., Petursson,G., Palsson,P. A., Georgsson,G., Klein, J., Nathanson,N. 1983.Antigenicdrift in visna: virus variation duringlong term infection of Icelandic sheep. J. Gen. Virol. 64: 143340 47. Stanley, J., Bhaduri, L. M., Narayan, O., Clements,J. E. 1987. Topographical rearrangementsof visna virus envelope glycoproteinduring antigenic drift. J. Virol. 61:1019-28 48. Dubois-Dalcq, M., Narayan, O., Griffin, D. E. 1979.Cell surfacechanges associated with mutationof visna virus in antibody treated cell cultures. VirolotTy 92:353~56 49. Clements, J. E., Petersen, F. S., Narayan, O., Haseltine, W. S. 1980. Genomicchanges associated with antigenicvariation of visna virus duringpersistent infection. Proc.Natl. Acad.Sci. USA77:4454-58 50. Issel, C., Coggins,L. 1979.Equineinfectious anemia: Current knowledge. J. Am. Vet. Med.Assoc. 174:727-33 51. McGuire,T., Crawford,T., Henson,J. 1972.EIA:Detectionof infectious virus-

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tious anemiavirus during parallel perantibody complexesin the serum. Immusistent infections. J. Virol. 61:1266-70 nol. Commun. 1:545-51 52. Issel, C., Foil, L. 1984.Studieson EIAV 59. Payne, S., Fang, F. D., Lui, C. P., transmission by insects. J. Am. Vet. Dhruva,B., Rwambo, P., Issel, C., Montelaro, R. 1987.Antigenicvariation and Assoc. 184:293-97 lentivirus persistence: variations in 53. Kono,Y., Hirasawa,K., Fukunaga,Y., Taniguchi, T. 1976. Recrudescenceof envelope gene sequences during EIAV EIA by treatment with immunosupinfection resemblechangesreported for pressive drugs. Natl. Inst. Anim.Health sequential isolates of HIV.Virology,In press Q. 16:8-15 54. Kono,Y., Kobayaski,K., Fukunaga,Y. 60. Hussain,K., Issel, C., Schnorr,K., West, 1973.Antigenicdrift of equineinfectious M., Rwambo,P., Montelaro, R. 1987. anemiavirus in chronically infected Epitope mappingof the envelope proteins of EIAV. J. Virol. In press horses. Arch. Virol. 41:1-10 S. 1985. Rates 55. Kono,Y., Kobayaski,K., Fukunaga,Y. 61. Gojobori,T., Yokoyama, 1971. Serological comparisons among of evolutionof the retroviral oncogene strains of EIAV.Arch. Virol. 34:202-8 of Moloneymurine sarcomavirus and 56. Payne,S., Parekh,B., Montelaro,R. C., of its cellular homologues.Proc. Nail Acad. Sci. USA82:4198-4201 Issel, C. J. 1984. Genomic alterations associated with persistent infections by 62. Hahn, B., Shaw, G., Taylor, M., equine infectious anemiavirus. J. Gen. Redfield, R., Markham, P., Salahuddin, ViroL 65:1395-99 S., Wong-Staal, F., Gallo,R., Parks, E., Salinovich, O., Payne, S., Montelaro, 57. Parks, W.1986. Genetic variation in HTLV-III/LAV over time in patients R., Hussain,K., Issel, C., Schnorr,K. 1986. Rapid emergenceof novel antiwith AIDSor risk for AIDS.Science genic and genetic variations of equine 232:1548-53 nfectiousanemiavirus duringpersistent 63. Holland,J., Spindler,F., Horodyski,E., infection. J. Virol. 57:71-80 Grabau,E., Nichol, S., VanDePol, S. 58. Payne, S., Salinovich, O., Nauman, S., 1982. Rapid evolution of RNA genomes.Science 215:1577-85 Issel, C., Montelaro,R. 1987. Course andextent of variation of equineinfec-

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Ann. Rev. lmmunol. 1988. 6 ." 161-95 Copyright © 1988 by Annual Reviews Inc. All rights reserved

STRUCTURE, ORGANIZATION, AND REGULATION OF THE COMPLEMENT GENES R. Duncan Campbell, S. K. Alex Law, Kenneth B. M. Reid, and Robert B. Sire Medical Research Council ImmunochemistryUnit, Department of Biochemistry, University of Oxford, South Parks Road, OxfordOX13QU, United Kingdom

INTRODUCTION cDNA and genomicclones are nowavailable for nearly 30 glycoproteins whichcomprisethe components,regulatory proteins, and receptors associated with the complement system(Figure 1, Table1). Thesecloningstudies have provided information which emphasizes the mannerin which the membersof the complementsystem maybe divided into families of structurally andfunctionally related proteins manyof whichare codedfor on the samechromosome and are closely linked. This review is divided into six mainsections in order to illustrate these structural andgenetic relationships: (a) the Clqr2szcomplexand Cl-inhibitor; (b) C2, factor C4, and21-hydroxylase,the class III genesof the majorhistocompatibility complex;(c) C5and the thioester containing proteins C3and C4; (d) regulators of complementactivation, (the RCAlinkage group) on human chromosome1 which includes the plasma proteins factor H and C4bbinding protein (C4bp)and the membrane molecules complementreceptor type 1 (CR1),complement receptor type 2 (CR2),decay accelerating factor (DAF)and probably membranecofactor protein (MCP);(e) complement receptor type 3 (CR3) as a memberof the cell adhesion glycoprotein family; and (f) the componentsof the terminal attack complexC5b-C9. 161 0732-0582/88/0410-0161502.00

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ACTIVATION AND CONTROL OF THE COMPLEMENT SYSTEM Themainfeatures of activation andcontrol of the systemare outlined below. Moreextensive background informationis given in other recent reviews(1, 2, 3). Thereare twodistinct routesof complement activation, the classical andalternative pathways(Figure1), with the majorcomponentC3(1.3 mg/ml)playinga central role in each. TheC3and12 other plasmaglycoproteinsformthe 13 components of the system(Figure 1). Controlis mediatedpartly by 7 plasmaproteins andpartly by various (about15) membrane boundproteins andreceptors; Table1. Theclassical pathwayis consideredto be triggered primarily by the interaction of subcomponent Clq of the C1complexwith the Fc regions oflgG, or IgM, in immunecomplexes.Alternative pathwayactivation can be mediated by a widerangeof nonimmunoglobulin activators such as fungi, yeast, lipopolysaccharides,etc, althoughcomplexesof IgG, IgA, andIgE may also cause activation. Theearly activation steps, or controlof activated components, involve the splitting of only one or twopeptidebondsby one

ENZYMECOMPLEXES WHICHACTIVATEC3 ANDC5

EARLYACTINGCOMPONENTS

TERMINAL COMPONENTS

CLASSICAL / PATHWAY - - - -~J ACTIVATOR ~ C 4-~--~

C 4b .~.

C 4bC 2--~---~

C4b2a

C3 C3a

ca(H~oI;’~ ALTERNATIVE J~ PATHWAY ~J ACTIVATOR - - Yl C3(H20),~,~)

~

~i

_

~ Surface C3b+ Site +CS--]J---~ - CSb~C5b-9 ~C3a

/

Ba

¯ Denotes proenzyme form ° Denotes activatedform

Figure 1 An outline of the activation of the classical and alternative pathways is shown. The system is down-regulated, as outlined in Table 1, mainly by: Cl-Inh (for activated Clr and Cls); C4bp and factor I (for C4b); factors H and I (for C3b); anaphylatoxin inactivator (for C3a, C4a, C5a); S-protein (for C5b-9); CR1 and factor ] (for C3b and C4b); DAF(for C3b and C4b); MCPand factor I (for C3b). Properdin, unlike the other control proteins has an enhancing effect on activation via its stabilization of the C3bBbcomplex.

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of six highly specific serine proteases (Figure 1) or by a carboxypeptidase B-like enzyme(Table 1). In classical pathwayactivation, the Clq (460 kd) molecule,whichhas no enzymicactivity, interacts via its six globular headgroupswith aggregated Fc regions of immunecomplexes.This binding appears to allow a conformational change to take place within the Clq Clr2Cls2 complex allowing autoactivation of the proenzymeform of Clr (83 kd) which turn activates proenzyme Cls (83 kd). Thethree chainC4molecule(~, 95 /3, 70kd; y, 33 kd) is split byCis at a single point in its 0¢ chainto yield the anaphylatoxin C4a(9 kd) and the large C4bfragment. The freshly activated C4bhas the capacity to bind covalently to hydroxylor amino groups via a reactive acyl group of a glutamyl residue derived from a thioester bondin its ~’ chain. This feature, whichis shared with C3b, allows these large activation fragmentsof C4and C3to bind to a variety of surfaces by ester or amidebonds and is probably of someimportance in the generation of C3 and C5 convertases and also in clearance mechanisms. Thethird serine protease of the classical pathway,proenzyme C2 (102 kd), associates (probably via its N-terminal C2bdomain)with in a Mg2+-dependent fashion andthen is split by Cis to yield a noncatalytic chain C2b(30 kd) and a catalytic chain C2a(70 kd). Theresulting C4b2a complexis the C3convertase of the classical pathway,splitting C3(~, 115kd;/3, 75 kd) at onepoint in its 0¢ chainto yield the anaphylatoxin C3a (9 kd) and the large fragmentC3b. There are two serine proteases in the alternative pathway,proenzyme factor B (90 kd) and factor D (24 kd) (whichhas only beenfound in activated formin plasma). In activation of the alternative pathway,factor B associates (via its N-terminal Ba portion) with C3b, or C3(H20) C3b-likeformof C3), in a Mg2 ÷_dependent interaction whichallowsfactor Dto split B into its N-terminal,noncatalytic, Ba chain (30 kd) andits terminal, catalytic, Bb chain (60 kd), thus yielding the C3convertase C3bBb.Efficient activators of the alternative pathwayare consideredto possesssites for C3bwhereit is protectedfromcontrol by protease factor I and its cofactors. Associationof C5(0¢, 115 kd;/3 75 kd) with surface boundC3ballows the splitting of C5by the C3/C5convertases to yield the anaphylatoxinand chemotacticfactor C5a(9 kd) and large fragment C5bwhich, withoutfurther proteolysis, initiates the self-assemblyof the C(5b-8)C9n(n = 1-18) complexinvolved in membranelysis. Control the activated complementfragments and complexproteases is mediated partly by the intrinsic decayof the C3/C5convertases,partly by interaction with a variety of control proteins, and partly by specific degradationby proteases(Table1).

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THE C1 COMPLEXAND CI-INHIBITOR The Clq moleculeis composedof six globular "heads," each joined by a collagen-likeconnectingstrand to a fibril-like central portion. Eachof the three types of chain (6A, 61~, and 6C) found in the moleculecontain terminal collagen-like sequences, and these combineto form the triple helixes whichmakeup the connectingstrands andfibril-like central portion (2). Each of the "heads" is composedof the C-terminal regions of one A-, one B-, and one C-chain. The Clr2Cls2 Ca2÷-dependentcomplexis consideredto interact with the collagen-like connectingstrands of Clq in such a mannerthat the catalytic domainsof Clr and Cls are closely associatedwithin the "cage"of collagen-like strands (2). Cl-Inhinteracts reversibly with the native unactivatedC 1 complexpreventingspontaneous activation of Clr within the complex.This inhibitory effect is overcome by efficient activators of the classical pathway,such as immune complexes, while activation by nonimmune substances such as DNAis impaired (2). Over-activationof the systemis prevented by the rapid removalof activated Clr and Cls from the C1 complexby Cl-Inh.

Clq A cDNA clone for the B chain of humanClq was isolated from a liver library (4). SubsequentNorthernblot analysis indicated that high levels of Clq B-chain mRNA are found in cultured monocyteswhile relatively low amountsare seen in liver, and no evidencefor significant synthesis couldbe seen in a variety of other tissues. Thefinding of cDNA clones for the A chain of humanClq in a monocytecDNA library (5) along with the demonstration of the synthesis, by cultured monocytesand macrophages,of a C 1 q moleculeapparentlyidentical to serumC1 q (6, 7) together indicate that macrophagesmaybe a major source of serumClq. The Clq B-chaingene is approximately2.6 kb long (4) and has been assigned chromosome 1 p, as has the A-chaingene (5). The B-chaingene contains a single intron of 1.1 kb locatedwithinthe codonfor glycineB-36,precisely the position wherethe triple helical chains of Clq appear to bend when viewedin the electron microscope.Thus,it is of someinterest to see if the A- and C-chaingeneswill also havean intron at the equivalentposition. Twotypes of C1q deficiency havebeendescribed, both of whichlead to the developmentof immune-complex related disease. In one type, a nonfunctional formof Clqwhichhas antigenic activity is found;in the second type no functionalor antigenicactivity is seen. In those with the first type of deficiency, the B chain appears normalas judged by genomicSouthern blotting. The sequenceof the B-chaingeneisolated fromthe library of a patient having the second type of deficiency showedthe presence of a

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single base changewhichgenerateda termination codonwithin the globular region of the chain at residue B-150.Thus,in this particular case the deficiency appears to be a consequenceof inability to synthesize the B chain or to secrete a moleculewith a truncated formof the B chain.

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Clr and cDNA clones for the Clr and Cls proenzymeshave been isolated from a humanliver cDNAlibrary, and their amino acid sequences show 40% overall homology. In the activated form of both enzymes, an A- or noncatalytic heavychain of approximately55 kd originates fromthe amino terminal end of the proenzyme.It contains an LDLreceptor type B domain and two different pairs of internal repeating sequences,followedby a Cterminal B chain of approximately27 kd whichcontains the active site characteristic of serine proteases (8, 9, 10). Thefirst set of repeating homologyunits (occupying approximately positions 10-80 and 185-255) in the A chains does not appear to have counterparts in other known protein sequencesand thus maybe involved in the activation and control of specificity of Clr and Cls. The