IL16

IL-16 David M. Center*, Hardy Kornfeld and William W. Cruikshank Pulmonary and Critical Care Division, Department of Medicine, Boston University School of Medicine, 715 Albany Street, R-304, Boston, MA 02118, USA * corresponding author tel: 617-638-4860, fax: 617-536-8093, e-mail: [email protected] DOI: 10.1006/rwcy.2000.03009.

SUMMARY IL-16 is synthesized as a precursor which is processed by caspase 3 into a 121 amino acid bioactive molecule. It has an unusual structure, resembling PDZ proteins and thus represents a rare example of a secreted PDZlike protein. That structure may underlie its ability to autoaggregate to form bioactive multimers which appear to interact directly with CD4 to transduce all signals and functions. In that regard, IL-16 is a potent T cell chemotactic factor with competence growth factor characteristics. It protects against antigen-induced apoptosis and during an IL-16 response T cells are refractory to other antigen-induced activation signals. Thus it could serve to augment CD4+ T cell accumulation by chemoattraction, priming for IL-2 or IL15 proliferation, and preventing antigen-induced cell death. It is a chemotactic factor for all CD4+ leukocytes. It has been found in a number of clinical states including the bronchoalveolage lavage (BAL) and biopsies of patients with atopic asthma, biopsies of patients with Crohn's disease, atopic dermatitis, blister fluid of patients with bullous pemphigoid, and granulomas of patients with sarcoidosis, all diseases characterized by CD4+ (T) cell accumulation. The observations that levels of IL-16 remain high in HIV-1-infected individuals who do not progress to AIDS and that it is a repressor of HIV-1 transcription suggest that it plays an important role in the pathogenesis of the HIV-1 infectious process, and that it may be of value as an adjunct to IL-2 therapy for HIV-1-infected individuals.

BACKGROUND

Discovery Interleukin 16 (IL-16) was the first described and characterized lymphocyte chemotactic factor. The

first published reports (Center and Cruikshank, 1982; Van Epps et al., 1983) named it the lymphocyte chemoattractant factor (LCF) in recognition of this function. In those earliest studies, IL-16 was identified as a secreted product of human peripheral blood mononuclear cells (PBMCs) in response to stimulation with concanavalin A or antigen (purified protein derivative of Mycobacterium tuberculosis, PPD). G-100 Sephadex sizing chromatography of Con A PBMC supernatants revealed an elution pattern consistent with a 55 kDa protein which appeared to be the major lymphocyte chemoattractant generated under these conditions. In subsequent years a large number of other lymphocyte chemotactic factors have been identified following phytohemagglutinin stimulation under similar conditions. Purification of the 55 kDa activity to homogeneity revealed a single 14±17 kDa band on SDS gel electrophoresis, prompting the hypothesis that a monomeric polypeptide autoaggregated into tetramers to create the 55 kDa bioactive form defined by sizing gel filtration chromatography (Cruikshank and Center, 1982).

Alternative names Originally named for its function as a lymphocyte chemotactic factor, IL-16 is also known as lymphocyte chemoattractant factor (LCF). Certain authors (Baier et al., 1995) also believe that it is one of several factors that comprise the CD8+ T cell-derived activity associated with long-term survival in HIV-1infected individuals first described by Walker et al. (1986) named CAF. Clearly, the activity known as CAF is complex and can contain the three chemokines MIP-1, MIP-1 , and RANTES (Cocchi et al., 1995) and IL-16 (Baier et al., 1995; Mackewitz et al., 1996; Zhou et al., 1997; Maciaszek et al., 1997) and probably other factors.

226 David M. Center, Hardy Kornfeld and William W. Cruikshank

Structure In lymphocytes, human IL-16 is generated as a 631 amino acid precursor molecule (Baier et al., 1997) which is enzymatically cleaved between Asp510 and Ser511 to yield a C-terminal 121 amino acid form which comprises all the known bioactivities (Zhang et al., 1998). The 631 amino acid precursor can be identified as a 85 kDa band following SDS-PAGE. The dissociation between predicted size and apparent size on SDS-PAGE may relate to the intrinsic structure of the protein (Muhlhahn et al., 1998). It is also possible that the entire N-terminal sequence has not as yet been identified as the published cDNA (Baier et al., 1997; Keane et al., 1998) for pro-IL-16 continues in frame 50 to the putative start methionine. The size of the mRNA would allow for a slightly larger protein. The structure of IL-16 is unique among secreted cytokines in that the majority of the 121 amino acids comprise a PDZ domain formerly known as Disc-Large homology repeats. PDZ domains are classically associated with intracellular proteins which bind C-terminal peptide motifs of membrane or structural proteins creating adapter or bridging functions between proteins. There is low homology between IL16 and any of the original PDZ family members, mammalian postsynaptic density protein 95 kD (PSD-95), Drosophila disc large tumor suppressor (DIgA), and the mammalian tight junction protein ZO1 (Woods and Bryant, 1995). The homology at the protein level to each is in the range of 33%. IL-16 does contain the conserved GLGF sequence necessary for binding the C-terminal peptides of complementary proteins. However, NMR spectroscopy (Muhlhahn et al., 1998) has defined the IL-16 GLGF-binding cleft as smaller than in most classical PDZ proteins and it appears to be occluded by a tryptophan. Like other PDZ proteins, IL-16 autoaggregates in solution; however the structural requirements for this association are not known. Interestingly, the N-terminus of pro-IL-16 contains two other PDZ domains which are highly conserved between the mouse and human proteins (Keane et al., 1998). There are three PDZ domains within the entire precursor molecule of IL-16 and they are more homologous to each other than to other members of the PDZ family. Thus, NMR spectroscopy of recombinant IL-16 confirms the structure predicted by analysis of the linear amino acid sequence. Comparison of the primary structures of mouse and human IL-16 reveals 82% homology in the Cterminal domain, and surprisingly high (75%) homology for the remaining N-terminus (Keane et al.,

1998). The high degree of homology in the mature C-terminal protein is functionally correlated with cross-species chemotactic function. The bioactive form of mouse IL-16 contains 119 amino acids due to the deletion of Ala571 and Ala572. The entire mouse pro-IL-16 molecule is 624 amino acids with five other deletions from the human. The high homology between precursor mouse and human proteins as well as the high level of intracellular IL-16 expression suggests a potential intracellular function for pro-IL16 although no such function has been identified. The C-terminal bioactive form does not contain any secretory leader sequences associated with classical secretory pathways (Zhang et al., 1998). In this regard it presents a conundrum similar to that of IL-1, although there is absolutely no structural homology between the two cytokines. There is, however, another similarity between IL-1 and IL-16 in that IL-16 is cleaved at Asp510/Ser511 by a member of the interleukin 1-converting enzyme (ICE) family, caspase 3 (Zhang et al., 1998). Considering the intimate relationship between caspase 3 activation and apoptosis it is possible that IL-16 is secreted as a result of programmed cell death; however, there is no evidence for this at the current time. In fact, all evidence indicates that stimulated secretion for all cell types known can occur in the absence of any cytotoxicity. Although the NMR structure of recombinant IL-16 predicts a monomeric form, we have never identified bioactivity associated with monomers. Interestingly, dimers which appear to have substantial bioactivity may elute from sizing HPLC 55 kDa. It is possible, therefore, that the original observations of bioactive LCF at 55 kDa represented an aberrant elution pattern of dimers of IL-16, not tetramers. (Muhlhahn et al., 1998). However, this stoichiometry could not have been predicted from any of the chemical analyses of the natural protein and remains unproven. In fact, dimerization is more consistent with the PDZ structure of IL-16 in that other family members have been shown to utilize regions within the PDZ domain to oligomerize into dimers.

Main activities and pathophysiological roles A variety of target cells for IL-16 stimulation have now been identified. IL-16 was initially described as a chemoattractant with specificity for CD4+ T cells (Berman et al., 1985). It was later determined that through an interaction with CD4, IL-16 is also a potent chemoattractant for all peripheral immune cells expressing CD4, including CD4+ monocytes

IL-16 227 (Cruikshank et al., 1987, 1996a; Center et al., 1996), eosinophils (Rand et al., 1991), and dendritic cells. In vitro studies have indicated that the ED50 (half-maximal effective dose) for recombinant IL-16 is 10-11 M (Rand et al., 1991; Cruikshank et al., 1994), which is consistent with other reported chemoattractants such as RANTES and MCP-1. For lymphocytes, IL-16 demonstrates both chemotactic and chemokinetic activity and has an equal effect on either resting or activated cells (Kornfeld et al., 1985). IL-16 is classified predominantly as a chemotactic factor for both monocytes and eosinophils. For CD4+ T cells, IL-16 is also a competence growth factor. In that regard, stimulation of resting (G0) CD4+ T cells with IL-16 results in cell cycle progression to G1 represented by expression of IL2R (Cruikshank et al., 1987) and IL-2R (Parada et al., 1998) and MHC class II molecules (Cruikshank et al., 1987). Acridine orange staining of IL-16-stimulated cells indicates increased mRNA synthesis but no increased DNA synthesis. Lack of entry into S phase has been confirmed by the lack of [3 H]thymidine incorporation into IL-16-stimulated peripheral blood human CD4+ T lymphocytes (Parada et al., 1998). The addition of either IL-2 or IL-15 to IL-16-primed cells results in an increase in [3 H]thymidine uptake. Thus, while IL-16 stimulation can induce a G0 to G1 transition, it is not sufficient to induce production of IL-2. In long-term human lymphocyte cultures, IL-16 stimulation in combination with exogenous IL-2 results in an increase in CD4+ T cells, with an approximate 1000-fold increase observed over a 6-week period. The resultant cell population is homogeneously CD4+CD25+CD45RO+ (Parada et al., 1998). Stimulation of CD4+ T cells for up to 5 days with IL-16 alone results in the generation and secretion of GMCSF, IL-3 (Parada et al., 1998), IL-6 and TNF (Amiel et al., 1999). It does not appear that IL-16/CD4-mediated growth signals are dependent on coexpression or signaling through the T cell receptor TCR/CD3. Certain CD4+ tumor cells (e.g. SupT1) which do not express TCR/CD3, proliferate in response to IL-16 as indicated by uptake of [3 H]thymidine (Cruikshank et al., 1996c). In addition, the CD4+ monocytoid cell line THP1 synthesizes and responds to IL-16. The addition of anti-IL-16 antibodies to cultures of THP1 cells reduces basal cell proliferation, and the addition of exogenous IL-16 results in enhanced proliferation. In addition, Szabo et al. (1998) have recently demonstrated that IL-16 stimulation of mouse bone marrow cells results in the differentiation of CD4+ pro-B cells into pre-B cells. This IL-16-induced transition is marked by activation of both RAG1 and RAG2 gene expression. In vivo treatment of old or

nude mice with recombinant human IL-16 results in expansion of pre-B cells (RAG1+ and RAG2+) detected in the bone marrow, essentially eliminating the B cell defect in these mice. Like other ligands for CD4 (i.e. anti-CD4 antibodies and HIV-1 gp120) stimulation of resting CD4+ T cells by IL-16 results in transient inhibition of cell activation induced through TCR/CD3 (Bank and Chess, 1985; Chirmule et al., 1990). IL-16 stimulation is sufficient to inhibit a mixed lymphocyte reaction (MLR) when incubated with the responder cell population between 24 hours before and up to 4 hours after addition of stimulator cells (Theodore et al., 1996). The effect is dose dependent with maximal effectiveness seen at 10ÿ9 M, but significant inhibition is still present at 10ÿ11 M. Similarly, IL-16 stimulation inhibits anti-CD3 or specific antigen-induced activation in a dose-dependent fashion when added prior to TCR activation (Cruikshank et al., 1996b). It has not been determined whether the inhibitory effect for IL-16 is due to generation of a negative signal or by steric inhibition of the TCR/CD3/CD4 complex required for T cell activation. We have, however, identified that IL-16 stimulation prevents subsequent TCR-induced IL-2R expression (Cruikshank et al., 1996b), IL-2 production and CD95 (Fas) expression. In addition, there is a direct positive correlation between the inhibitory activity of IL-16 on subsequent TCR stimulation and cells that demonstrate IL-16-induced migratory activity (Cruikshank et al., 1996b). The role of IL-16 in vivo remains to be elucidated; however, one hypothesis is that it is capable of contributing at least in part to a general antigen-independent nonclonal recruitment and priming of CD4+ cells in an inflammatory process. While the recruited cells would be responsive to cytokine stimulation, they would be refractory to antigen-specific activation. The effect would be to increase the number of cells recruited to an inflammatory focus and to increase further the number of viable cells by simultaneously reducing the susceptibility of those cells to antigen-specific induced cell death. Relationship of IL-16 to HIV-1 infection One of the more recently described functions for IL16 is as a suppressor of human immunodeficiency virus (HIV-1) (Baier et al., 1995) and simian immunodeficiency virus (SIV) infection (Lee et al., 1999). Initially, Baier et al. (1995) and later Mackewicz et al. (1996) reported that IL-16, at a concentration of 1± 5 mg/mL, could suppress approximately 40% of viral replication. This activity of IL-16 may comprise one of many activities derived from CD8+ T cells initially termed CAF by Walker et al. (1986). However, unlike

228 David M. Center, Hardy Kornfeld and William W. Cruikshank the chemokines, the inhibitory effect of IL-16 is not as a result of steric inhibition of viral binding. Rather, the inhibitory effect of IL-16 appears to be at the level of transcriptional regulation (Baier et al., 1995; Viglianti et al., 1997; Zhou et al., 1997). The mechanism was best defined by Maciaszek et al. (1997) who reported that in transient transfection studies with HIV-1 LTR-reporter gene constructs, IL-16 pretreatment repressed either PMA- or Tat-stimulated HIV-1 promoter activity by 60-fold. This effect of IL-16 required sequences within the core enhancer, but was not simply due to downregulation of the binding activity of transcription factors such as NFB. Data thus far suggest that IL-16 stimulation results in activation of a transcriptional repressor which functions through sequences within or immediately adjacent to the core enhancer. Zhou et al. (1997) confirmed this finding and further demonstrated that CD4+ T cell lines transfected to express the bioactive portion of IL-16 were resistant to HIV-1 infection. Studies by Idziorek et al. (1998) and Lee et al. (1999) suggest that IL-16 is capable of inhibiting both T tropic and M tropic isolates of HIV and that some antiviral effects are observed even if IL-16 is added after establishment of infection. Serum IL-16 levels have been shown to remain at normal levels in HIV-1-infected long-term nonprogressors as compared with those that progressed to AIDS in whom levels dropped significantly (Amiel et al., 1999). In addition, IL-16 levels rise dramatically in severely immunodeficient HIV-1-infected individuals following antiviral treatment with indinavir (Bisset et al., 1997). All of the IL-16-mediated functions noted to date appear to have an absolute requirement for cell surface expression of CD4. In that regard, IL-16 induces phosphorylation of CD4-associated p56lck (Ryan et al., 1995) with subsequent rises in intracellular Ca2‡ and inositol trisphosphate (IP3) (Cruikshank et al., 1991). The chemotactic responses in T cells, however, are not dependent upon the kinase activity of p56lck as herbimycin does not inhibit IL-16induced chemotaxis and cell lines stably transfected with a CD4-lck chimeric molecule which lacks the kinase (SH1) domain still migrate normally to IL-16 (Ryan et al., 1995). Rather the association of p56lck with CD4 is essential to provide an adapter function. PKC activation, however, is essential for the chemotactic activity as evidenced by inhibition of IL-16induced migration by a variety of PKC inhibitors (Parada et al., 1996). IL-16 has effects on CD4+ monocytes and macrophages that are distinct from its chemotactic activity. IL-16 treated peripheral blood monocytes increase expression of MHC class II (HLA-DR) in 24±48 hours by a mechanism that is independent of IFN

as anti-IFN antibodies do not inhibit this regulatory function (Cruikshank et al., 1987). Moreover, IL-16 activates the SAP kinase (stress-activated protein kinase) pathway in macrophages. Specifically, the IL-16-induced phosphorylation of SEK-1 results in activation of the SAP kinase p46 and p54 protein and to the phosphorylation of c-Jun and p38 MAP kinase (mitogen-activated protein kinase). However, unlike other ligands that induce this pathway, IL-16 does not induce apoptosis (Krautwald, 1998). The ultimate downstream effects of activation of these signaling pathways have not been determined. Of note, monocytes do not express p56lck, the src family kinase required for all IL-16-induced functions in T cells. In monocytes it is likely that lyn acts as the initial kinase link as we have recently demonstrated that lyn is directly associated with CD4 in p56lcknegative THP1 cells and is regulated following binding with anti-CD4 antibodies and IL-16. The only known effects of IL-16 on CD4+ eosinophils is as a chemotactic factor. Of note, it does not prolong longevity of eosinophils in culture, does not induce MHC class II molecule expression, and is not associated with peroxide generation or other phagocytic or microbicidal functions. As eosinophils lack p56lck it is not known how the chemotactic signal is transduced. Like monocytes, eosinophils do express lyn but no direct experiments have been done to show a link between lyn and CD4 in eosinophils.

GENE AND GENE REGULATION

Accession numbers Mouse: AF006001 Human: M90391

Chromosome location Mouse: 7 (39 cM); human: 15q26.1.

Regulatory sites and corresponding transcription factors The structure of the human IL-16 is currently believed to consist of eight exons and seven introns. However, the transcriptional start site for IL-16 has not been conclusively established. Human and mouse genomic clones of IL-16 have been sequenced in areas that are presumed to represent the 50 flanking region of IL-16. A putative promoter (P30 ) has been identified

IL-16 229 immediately upstream of the published IL-16 open reading frame, and a second putative promoter (P50 ) has been identified 2 kb further upstream. An additional short exon lies immediately downstream of P50 . P30 is a TATA-less promoter. Putative consensus sites in P30 include a CAP site, a CAT box, an E2 box, and potential binding sites for Ets-1, c-myb, and AP-1. The P30 promoter is constitutively active and PMA-inducible in lymphoid, monocytoid, and fibroblast cell lines. P50 contains a TATA box. Putative consensus sites in P50 also include a CAT box, an E2 box, and two GC boxes, and a glucocorticoid response element, as well as potential binding sites for STAT1, p53, mycmax, PEA-3, PPAR, and NF-Y. The P50 promoter is constitutively active and PMA-inducible in lymphoid, monocytoid, and fibroblast cell lines. Compared with P30 , P50 appears to be more active in lymphoid cells and monocytoid cells, and less active in fibroblasts. The activation of P50 by PMA is inhibited by dexamethasone, while dexamethasone has no effect on PMA-activated P30 activity.

Ser511 to yield a C-terminal 121 amino acid form which contains all the known bioactivities (Zhang et al., 1998). The 631 amino acid precursor can be identified as a 85 kDa band following SDS-PAGE. The dissociation between predicted size and apparent size on SDS-PAGE may relate to the intrinsic structure of the protein (Muhlhahn et al., 1998). It is also possible that the entire N-terminal sequence has not as yet been identified as the published cDNA (Baier et al., 1997; Keane et al., 1998) for pro-IL-16 continues inframe 50 to the putative start methionine. The size of the mRNA would allow for a slightly larger protein.

Important homologies

See Keane et al. (1998).

The structure of IL-16 is unique among secreted cytokines in that the majority of the 121 amino acids comprise a PDZ domain formerly known as DiscLarge homology repeats. PDZ domains are classically associated with intracellular proteins which bind Cterminal peptide motifs of membrane or structural proteins, creating adapter or bridging functions between proteins. There is low homology between IL-16 and any of the original PDZ family members, mammalian postsynaptic density protein 95 kDa (PSD-95), Drosophila disc large tumor suppressor (DIgA), and the mammalian tight junction protein ZO1. The homology at the protein level to each is in the range of 33%. IL-16 does contain the conserved GLGF sequence necessary for binding the C-terminal peptides of complementary proteins. However, NMR spectroscopy (Muhlhahn et al., 1998) has defined the IL-16 GLGF binding cleft as smaller than in most classical PDZ proteins and it appears to be occluded by a tryptophan. IL-16 autoaggregates in solution, however the structural requirements for this association are not known. Interestingly, the N-terminus of pro-IL-16 contains two other PDZ domains which are highly conserved between the mouse and human proteins (Keane et al., 1998). There are three PDZ domains within the entire precursor molecule of IL16 and they are more homologous to each other than to other members of the PDZ family. Thus, NMR spectroscopy of recombinant IL-16 confirms the structure predicted by analysis of the linear amino acid sequence.

Description of protein

Posttranslational modifications

In lymphocytes, human IL-16 is generated as a 631 amino acid precursor molecule (Baier et al., 1997) which is enzymatically cleaved between Asp510 and

As noted above, IL-16 is synthesized as a precursor molecule which is cleaved at Asp510 by caspase 3 to yield a 121 amino acid active molecule. All evidence

Cells and tissues that express the gene Multiple-tissue northern analyses suggest that organs of lymphoid origin all express IL-16 mRNA. In addition, mRNA is expressed to a lesser extent in pancreas, lung, and a larger size message is observed in brain. CD4+ T cells, CD8+ T cells (Laberge et al., 1996), mast cells (Rumsaeng et al., 1997), eosinophils (Lim et al., 1996), and airway epithelial cells (Laberge et al., 1997a) express the gene, as described in detail in other sections of this chapter.

PROTEIN

Accession numbers M90391, AF006001

Sequence

230 David M. Center, Hardy Kornfeld and William W. Cruikshank suggests that this 121 amino acid protein aggregates (without requirement for disulfide bonds) into multimers of dimeric or tetrameric form that comprise the bioactive secreted interleukin. No details are available about further modifications of the molecule, but recombinant protein produced in E. coli is fully functional without subsequent modifications.

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce IL-16 is synthesized by a variety of cell types, however it was first identified as a CD8+ lymphocyte cell product (Center et al., 1983a; Berman et al., 1985). CD8+ T cells release IL-16 in response to stimulation by mitogens, antigens, or vasoactive amines such as histamine (Center et al., 1983a; Laberge et al., 1995) and serotonin (Laberge et al., 1996). While data are accumulating on the mechanism by which IL-16 is synthesized and processed, the mechanism by which IL-16 is secreted has not been elucidated. The predicted amino acid sequence for precursor or secreted IL-16 does not contain a signal peptide required for transport into the endoplasmic reticulum. It is possible that IL-16 is cleaved and secreted in a fashion similar to IL-1 in which processing and secretion is facilitated by caspase 1 (Hogquist et al., 1991). The mechanism of IL-16 processing and secretion is however regulated separately, at least in the CD8+ T cell. Cell lysates generated from unstimulated CD8+ T cells contain preformed IL-16 bioactivity (Laberge et al., 1996). CD8+ T cells express constitutive IL16 message and contain large amounts of the precursor IL-16 molecule. In addition, resting CD8+ T cells express detectable levels of activated caspase 3 enzyme (Zhang et al., 1998). Thus, it is presumed that in CD8 cells, constitutive mRNA is associated with synthesis of pro-IL-16, a small percentage of which is processed by low levels of active caspase 3. Bioactive IL-16 is stored in an unknown compartment and is secreted by unknown mechanisms in response to vasoactive amines or stimulation via the TCR complex. In the last regard, histamine type 2 receptors and serotonin type 2 receptors mediate the secretory process (Center et al., 1983a; Laberge et al., 1995, 1996). The kinetics of release of IL-16 following stimulation of CD8+ T cells with either histamine or serotonin is 1±4 hours. The release of IL-16 induced by vasoactive amine stimulation is not affected by

transcription or translation inhibitors, indicating that the vasoactive amines are functioning primarily as secretagogues. The time course for IL-16 release, however, is not consistent with granule extrusion, and suggests some other mechanism for secretion. Unlike IL-1 (Vannier and Dinarello, 1993), histamine stimulation does not result in increased IL-16 message stability. Whether IL-16 is secreted from the suppressor or cytotoxic CD8+ T cell subset or from both subsets is unclear at present. However, since IL-16 has immunosuppressive bioactivities, a functional classification would be that IL-16 is generated at least in part by suppressor T cells. In CD4+ T cells the circumstances of synthesis, processing, and release appear quite different from CD8 cells (Wu et al., 1999). Over 95% of CD4+ T cells express constitutive IL-16 message and promolecule. The message is not substantially regulated by TCR stimulation, nor is stability altered by costimulation via CD28. However, no bioactive IL-16 is stored in CD4+ T cells, probably because CD4+ T cells do not contain active caspase 3 unless they are stimulated. Following activation, IL-16 is processed and secreted within 24±48 hours; however, no new IL16 mRNA is identified, nor new pro-IL-16 until 96± 168 hours. Vasoactive amines appear to play no role in secretion of IL-16 from CD4+ T cells. Three other immune cell types also have been shown to generate IL-16. Eosinophils obtained from either normal or hypereosinophilic donors express IL16 message and protein following isolation and culture in the presence of GM-CSF (Lim et al., 1996). Pro-IL-16 is found preformed in bone marrowcultured human mast cells as well as in the human mast cell line HMC-1 as demonstrated by intracytoplasmic staining, immunocytochemistry, and western analysis. Interestingly, in these cells C5a and PMA plus ionomycin stimulation results in 6- to 10-fold increases in IL-16 mRNA and increased secretion of bioactive IL-16 in supernatants as confirmed by bioassay in the presence of neutralizing IL-16 antibodies and ELISA. The time course was 2±4 hours, similar to that observed in CD8 cells stimulated with serotonin. The majority of the secreted lymphocyte chemotactic activity detected under these conditions appeared to be IL-16 (Rumsaeng et al., 1997). Last, dendritic cells derived from PBMCs after 6 days of culture in the presence of IL-4 and GM-CSF express IL-16 mRNA and secrete bioactive IL-16 protein (Kaser et al., 1999). Of interest, the majority of the lymphocyte chemotactic activity found in 6-day dendritic cell supernatants appeared to be IL-16 by neutralizing antibody studies. Several nonimmune cell types are also capable of generating IL-16. Bellini et al. (1993) identified IL-16

IL-16 231 in cell supernatants of cultured primary airway epithelial cells, obtained from asthmatic individuals, stimulated in culture with histamine. IL-16 was not detected in cultures of histamine-stimulated primary epithelial cells obtained from normals. The mechanism of histamine stimulation on the epithelial cells is unclear at present. Unlike lymphocytes however, Arima et al. (1995) reported an increase in IL-16 message in an epithelial cell line stimulated with histamine, suggesting that histamine is inducing de novo IL-16 protein production. Our work has demonstrated the presence of both IL-16 protein and message in airway epithelium obtained from chronic asthmatics (Laberge et al., 1997b). While large numbers of T cells were present in the mucosal biopsies, the majority of the IL-16 staining was observed in the epithelium, indicating that airway epithelium is probably the primary source of IL-16 in asthmatic inflammation. The amount of IL-16 message and protein detected in the epithelium demonstrated a positive correlation with the numbers of infiltrating CD4+ lymphocytes. Mucosal biopsies obtained from normals or atopic nonasthmatic individuals showed only infrequent isolated pockets of immunoreactive IL-16 and IL-16 mRNA. Recent studies have demonstrated that human fibroblasts from several different tissue sources are capable of generating bioactive IL-16 when stimulated with a variety of cytokines such as IL-1, IL1 , and TNF (T. Smith, unpublished observation). Unlike most of the immune cells, but similar to airway epithelial cells, fibroblasts do not contain detectable constitutive pro-IL-16 protein. Pro-molecule must be generated, processed and then bioactive IL16 is released following cell activation. Fibroblast generation of IL-16 is also similar to the epithelial cell in that the magnitude of synthesis is far greater on a cell per cell basis than for immune cell generation, indicating that both the epithelium and fibroblasts are potentially major sources of IL-16 in vivo. Multiple tissue northern analyses suggest that under normal circumstances IL-16 is expressed predominantly in lymphoid organs (spleen, lymph nodes, thymus) with some expression in brain and pancreas (Chupp et al., 1998). As noted above, and detailed below, in inflammatory states the lung epithelium is a major source of IL-16 as is the gut epithelium in Crohn's disease and retro-orbital fibroblasts in Graves' ophthalmopathy. Of course, since IL-16 is expressed in all CD8+ T cells and CD4+ T cells, the presence of these cells in any immune or inflammatory tissue reaction would predict the presence of IL-16. The functions of IL-16 in brain (in which an alternate higher size message is also present) or in pancreatic exocrine epithelium are unknown.

Eliciting and inhibitory stimuli CD8+ T cells secrete IL-16 in response to stimulation with the vasoactive amines histamine and serotonin. For histamine, H2-type receptors are required based upon inhibition of secretion by H2 blockers, but not H1 antagonists. Similarly, H2 synthetic agonists induce secretion. Histamine has no effect on the secretion of IL-16 from CD4+ T cells (Center et al., 1983a; Laberge et al., 1995). The neurotransmitter and secretagog serotonin has similar effects on CD8+ T cells via S2 receptors, but no effect on CD4+ T cells (Laberge et al., 1996). The time course for secretion of preformed IL-16 from CD8+ T cells by histamine and serotonin is 2±4 hours, suggesting a more complex secretory pathway than simple extrusion of intracytoplasmic granule contents. In fact, the secretion of IL-16 does not correspond temporally with granzyme secretion nor can IL-16 be localized within granules by immunocytochemistry. It is more diffusely present within the cytoplasm without any clearcut compartmentalization noted to date. Both T cell subsets can be induced to release IL-16 in response to Con A, anti-CD3 antibodies, and antigen although there is little evidence in CD8+ T cells for new protein synthesis. Of interest, the preformed IL-16 is released from CD8+ T cells within 6±12 hours following anti-CD3 stimulation. The reasons for the differences in the time course of release following CD3 stimulation versus serotonin or histamine of (presumably) the same IL-16 pool are unknown. In CD4+ T cells, however, abundant pro-IL-16 is present without any processed stored preformed IL-16. Stimulation with anti-CD3 results in activation of caspase 3 from its inactive precursor molecule, consistent with the time course of processing pro-IL-16 into IL-16. In this circumstance, costimulation via CD28 accelerates IL-16 secretion temporally related to the presence of active caspase 3 (Wu et al., 1999). TGF and IL-1 induced mouse pulmonary epithelial cells to initiate pro-IL-16 transcription and translation and cleavage to IL-16 (Keane et al., 1998). Serotonin accelerates the secretory process but by itself has no effect on transcription or translation. TNF, IL-1, and IL-1 have similar effects on fibroblasts. They all initiate transcription and translation and are sufficient stimuli to induce processing and secretion of IL-16. There are no known effects of histamine and serotonin on IL-16 release from fibroblasts. As noted above, PMA and ionomycin or C5a induce mast cell pro-IL-16 mRNA, synthesis, processing, and secretion of bioactive IL-16 (Rumsaeng

232 David M. Center, Hardy Kornfeld and William W. Cruikshank et al., 1997). The synthesis, processing and secretion of IL-16 is less well studied in eosinophils. In vitro, in the presence of GM-CSF required for cell viability, constitutive IL-16 mRNA and pro-IL-16 are present and bioactive IL-16 can be identified in the supernatants. There are no identified regulators of any of these processes in eosinophils (Lim et al., 1996). The only study that has addressed inhibition of IL16 mRNA or protein expression was performed in vivo. In that study, Laberge et al. (1997b) treated individuals with atopic rhinitis with inhaled corticosteroids in one nostril, controlled by the inhaler vehicle in the other for 3±6 weeks. Baseline IL-16 mRNA and protein staining in the nasal epithelium was decreased in the corticosteroid-treated side. Furthermore, antigen challenge increased expression of both mRNA and protein in the control-treated side; a response that was markedly blunted on the corticosteroidtreated nasal mucosa.

Exogenous and endogenous modulators In this regard, the only known potential endogenous modulator might be corticosteroids. The effects of cytokines like MIF on the IL-16 corticosteroid response have not been studied. There are, however, a number of exogenous inhibitors of IL-16 function. The first described such modulator was Fab fragments of anti-OKT4 antibody. The monomeric Fab fragments appear somewhat inert themselves, but inhibit IL-16-induced chemotaxis and IL-2R expression on T cells (Cruikshank et al., 1987). Moreover, this inhibitory effect is observed regardless of the CD4+ target cell as anti-OKT4 Fab inhibit IL-16induced eosinophil (Rand et al., 1991) and monocyte (Cruikshank et al., 1987) chemotaxis also. Antibodies to epitopes of CD4 that are involved in MHC binding or HIV-1 interaction located in the D1D2 domain of CD4 tend not to be inhibitory for IL-16-induced functions. As expected, rsCD4 inhibits all known functions of IL-16; rsD1D2 lacks IL-16-neutralizing activity while rsD3D4 retains this activity. We subsequently developed polyclonal antibodies against rIL-16 and found antisera that inhibited all IL-16 functions. In addition, when we made anti-rhIL-16 monoclonal antibodies, our original screening procedure was based upon inhibition of IL-16-induced chemotaxis and IL-2R expression. The epitopes of the two monoclonals identified (14.1 and 17.1) both map to the C-terminal 16 amino acids. Monoclonal 14.1 has been used in vivo in a mouse model of OA-induced pulmonary inflammation

and airway hyperreactivity. Intraperitoneal injection of antibody prior to the antigen challenge phase inhibits the airway hyperreactivity and the inflammatory response (Hessel et al., 1998). Keane et al. (1998) recently demonstrated that a hydrophilic hexadecameric peptide derived from the C-terminus of IL-16 is also inhibitory for IL-16induced chemotaxis (and induction of IL-2R expression). While the peptide was derived based upon the human sequence, it demonstrated species crossneutralization for chemotaxis for mouse T cells. This was predicted based upon the high degree of interspecies homology; however, this finding also implies that there must be some significant homology between human and mouse IL-16 receptor complexes. These studies also corroborate the epitope mapping of the neutralizing monoclonals.

RECEPTOR UTILIZATION All existing evidence suggests IL-16 requires the expression of CD4 as part (or all) of a receptor complex for both chemoattractant and growth factor functions. First, all IL-16-induced functions are specifically inhibited by monomeric Fab of anti-OKT4 monoclonal antibodies. Second, the chemotactic activity of IL-16 for CD4+ monocytes and eosinophils is proportional to the amount of cell surface CD4 expressed (Cruikshank et al., 1987). Third, IL16-induced IL-2R expression, rises in intracellular Ca2‡ and inositol trisphosphate, and autophosphorylation of p56lck are all observed following transfection of human CD4s which have preserved intracytoplasmic tails capable of interacting with p56lck (Cruikshank et al., 1991; Ryan et al., 1995). CD4 mutants that do not bind lck or CD4-lck chimeras that lack the SH3 domain are unresponsive. Again, all these responses are inhibitable by Fab of anti-OKT4 monoclonal antibodies and by rsCD4. A direct association has been demonstrated as rIL-16 physically binds in solution to rsCD4 and rIL-16 can be purified by affinity chromatography utilizing rsCD4 bound to insoluble matrix (Cruikshank et al., 1994). However, the most compelling evidence is that transfection of human CD4 into L3T4±, IL-16 nonresponsive mouse T cell hybridoma cells imparts IL16 responsiveness (Cruikshank et al., 1991; Ryan et al., 1995), and that IL-16 can displace anti-CD4 antibody binding on primary T cells (Theodore et al., 1996). There is substantial additional information about the receptor-mediated signals required for the

IL-16 233 IL-16-induced chemotactic response. As noted above, cell surface expression of CD4 is required, and the CD4 must associate with the src kinase family member p56lck. The kinase enzymatic activity of p56lck is not required for the chemotactic signal in that herbimycin A does not inhibit IL-16induced chemotaxis and mouse CD4±T cells transfected with CD4±lck chimeric constructs that lack the kinase (SH1) domain of p56lck migrate normally in response to IL-16. Constructs with chimeric p56lck molecules that lack the SH3 domain lose chemotactic responsiveness, implying the requirement for this adapter portion of p56lck (Ryan et al., 1995). The chemotactic response of CD4+ T cells does require kinase enzymatic activity of a protein kinase C (PKC) isoform. All inhibitors of PKC completely eliminate IL-16-induced motile responses in T cells (Parada et al., 1996). A potential downstream event from the SH3 adapter region of p56lck is the activation of phosphatidylinositol 3-kinase (PI-3 kinase). This is likely for two reasons: wartmannin inhibits IL-16-induced chemotaxis and IL-16 activation in CD4+ T cells results in induction of PI-3 kinase activity. The evidence for the requirement of association of CD4 with p56lck for the IL-16-induced motile response in T cells leaves open the question as to how eosinophils and monocytes respond to IL-16 through their cell surface expressed CD4. In that regard, we have recently demonstrated that the src family member lyn associates with CD4 in the monocytoid cell line THP1, a p56lck-negative cell. Lyn is modulated following IL-16 (and anti-CD4) stimulation. Unlike CD4+ T cells, herbimycin A does inhibit IL-16-induced chemotaxis in monocytes and THP1 cells, implying a completely different kinase cascade for the CD4-mediated chemotactic response in monocytes. How this information relates to the demonstration that IL-16 induces phosphorylation of members of the stress-activated protein kinase family in monocytes is unclear at the current time (Krautwald, 1998). Furthermore, there is no information about IL-16/CD4 chemotactic signaling in eosinophils or dendritic cells. Of interest is the cross-species activity that IL-16 shows for CD4+ cells. This implies homology not only in the functional domains of IL-16 but also some common regions on CD4 that mediate the IL16-induced signal. In that regard, the region of CD4 in the D4 domain corresponding to amino acids 328± 415 contains the highest intraspecies homology. Recent peptide studies have localized the CD4 signaling domain for IL-16 to a six-residue region in the proximal portion of D4.

IN VITRO ACTIVITIES

In vitro findings Six in vitro activities have been identified for IL-16 (detailed in Center et al., 1997). First, it is a chemotactic factor for CD4+ T cells, monocytes, eosinophils, and dendritic cells. Second, in normal human and mouse CD4+ T cells it induces a shift from G0 to G1 associated with the expression of IL-2R and ILR , and in humans, MHC class II molecules (i.e. HLA-DR). These phenomena require 8±24 hours of incubation with IL-16. This shift is not associated with IL-2 secretion itself; however, the cells are rendered competent to respond to IL-2 and IL-15. In that regard, subsequent IL-16/IL-2 stimulation of PBMCs results in selective expansion of CD4+ T cells, the majority of which are CD45ROCD25+. Increases in [3 H]thymidine incorporation and actual numbers of CD4+ T cells are observed under these conditions. No specific TH subtype appears to be induced. However, the major cytokines that appear as a result of IL-16 stimulation alone are GM-CSF, IL-3, TNF, and IL-6 (Parada et al., 1998; Amiel et al., 1999). Third, preincubation of T cells results in transient lack of responsiveness to antigen as demonstrated by decreased cell proliferation following either anti-CD3 stimulation (Cruikshank et al., 1996b) or during a mixed lymphocyte reaction (MLR) (Theodore et al., 1996). These responses are not rescued by adding exogenous IL-2 as preincubation with IL-16 (one hour) inhibits anti-CD3-induced IL-2R expression, thus providing at least one explanation for the lack of IL-2 responsiveness. In human monocytes, IL-16 activation is associated with upregulation of HLA-DR (Cruikshank et al., 1987). This phenomenon is independent of secretion or activity of IFN as anti-IFN antibodies have no effect, while anti-CD4 Fab completely abolish the IL16-induce HLA-DR expression. It has not been explored in vitro how these early findings impact on the more recent studies with dendritic cells noted above. A fifth in vitro activity noted is induction of RAG1 and RAG2 gene expression in mouse bone marrow progenitor B cells (Szabo et al., 1998). These cells express low levels of CD4 which is lost in further differentiation to pre-B cells. Last, IL-16 represses HIV-1 transcription (Baier et al., 1995; Maciaszek et al., 1997; Zhou et al., 1997). Unlike the chemokines, it does not inhibit viral entry or fusion. Rather, through a CD4-dependent process, IL-16 induces a repressor of HIV-1 transcription.

234 David M. Center, Hardy Kornfeld and William W. Cruikshank This effect was demonstrated using transfection of the CD4+ T cell line A3.01 with an HIV-1 long terminal repeat (LTR). Subsequent stimulation with Tat or PMA markedly upregulates transcription of this construct. Preincubation with IL-16 represses the PMAor Tat-induced transcription by up to 80%. The core enhancer region is required for the induction of the inhibitory effects as mutants that lack this region are not modulated by IL-16; and IL-16 apparently induces an activity that binds to sequences derived from the core enhancer, suggesting a repressor element. Similar experiments done in the CD4± derivation of A3.01, the A2.01 line, reveal no repression by IL-16. In addition, transfection of CD4+ Jurkat cells with IL-16 renders them relatively resistant to HIV-1 infection and replication (Zhou et al., 1997). Interestingly, in those experiments not all of the HIV-1 inhibitory activity could be attributed to autocrine secretion of IL-16 and signaling through CD4 as neutralizing antibodies did not abolish all of the IL-16 inhibitory effects. The nature of this intracellular inhibition has not been elucidated.

Regulatory molecules: Inhibitors and enhancers Four classes of inhibitory molecules for IL-16 function have been identified: those that correspond to essential functional regions of IL-16; those directed at regions of CD4 required for interaction with IL16; IL-16/CD4 signal inhibitors; and inhibitors of IL16 mRNA expression. First, there are two well-characterized monoclonal antibodies directed against the C-terminus of human recombinant IL-16 (Cruikshank et al., 1994). The epitope of these antibodies has recently been defined to include a tetrameric region near the C-terminus of the protein. As would be expected, soluble receptor molecules can act as competitive inhibitors in similar fashion. In this regard rsCD4, rsCD4 (D3D4 region) and peptides derived from a region of CD4 thought to be important in IL-16/CD4 interaction all inhibit IL-16 functions in vitro. The epitope mapping of the IL-16 inhibitory antibodies implies that peptides derived from this region of human IL-16 might also inhibit IL-16 functions. This is in fact the case, in vitro, where a C-terminal hexadecamer of human IL-16 has been demonstrated to inhibit either human or mouse rIL-16-induced chemotaxis for either mouse or human target CD4+ T cells (Keane et al., 1998). As noted above in Receptor utilization, in CD4+ T cells inhibitors of protein kinase C completely abolish

IL-16-induced chemotaxis (Parada et al., 1996). Their effects on other IL-16 functions are not known. Similarly, wortmannin inhibits IL-16-induced chemotaxis of T cells, while herbimycin A inhibits IL-16-induced chemotaxis of monocytes. The pathways responsible for the other functions of IL-16 noted above have not been sufficiently elucidated to yield other potential signal inhibitors. Last, the only existing information regarding regulation of IL-16 mRNA comes from an in vivo study in which corticosteroids were demonstrated to decrease the epithelial expression of IL-16 message in nasal mucosa (Laberge et al., 1997b). Prior local steroid treatment also inhibited the antigen-induced upregulation of IL-16 mRNA observed in untreated nasal mucosa.

Bioassays used Four major bioassays may be employed to identify IL-16 function. These are detailed in Protocols in Immunology (Center et al., 1997). Briefly, they are CD4+ (lymphocyte, monocyte, or eosinophil) chemotaxis in the presence of neutralizing IL-16 antibody or Fab of OKT4 monoclonal antibody. The modified microchemotaxis chamber assay with nitrocellulose filters is suitable for detecting IL-16 chemotactic activity. Prior activation of cells is not required, nor is coating of filters with connective tissue substrates. IL-16 at 10ÿ9 M should induce a 2-fold increase in migration. Induction of IL-2R on resting primary CD4+ T cells (also in the presence of neutralizing antibody to demonstrate specificity) occurs in 24±48 hours of incubation with IL-16. In addition to induction of IL-2R IL-16's effects on cell cycle changes can be determined by the induction of IL-2R , Acridine Orange staining demonstrating new RNA synthesis without DNA synthesis, and on human T cells or monocytes induction of MHC molecules (i.e. HLADR). The response will be limited to CD4+ T cells, 20±30% of which will express these markers under these conditions. Immunoregulatory assays include preincubation of T cells with IL-16 for one hour followed by stimulation with plate-bound anti-CD3. The effects of IL-16 can be monitored by subsequent assessment of proliferation by [3 H]thymidine incorporation at 5 days, IL-2 synthesis in 1±2 days, IL-2R expression in 1±2 days, or CD95 expression in 1±2 days. Preincubation with IL-16 will result in greater than a 50% decrease in proliferation, IL-2 synthesis or expression of IL2R or CD95.

IL-16 235 Induction of HIV-1 replication can be inhibited by IL-16. A CD4+ T cell line (e.g. A3.01) must be chosen. Our best results were by transient transfection of the HIV-1 LTR followed by induction of transcription with PMA or Tat. IL-16 at 10ÿ9 M inhibits PMA- or Tat-induced CAT (chloramphenicol acyltransferase) activity by greater than 50% at one day. This assay does not require live virus and is one in which the chemokines have no inhibitory effects. Szabo et al. (1998) have suggested a fifth possible bioassay in their recent work with pre-B cells. They demonstrated that IL-16 induces mouse bone marrow-derived pre-B cell differentiation as measured by RAG1 activation. The assay requires harvesting bone marrow-derived pre-B cells and measuring RAG1 mRNA. Since other factors probably have similar activity, specificity would have to be defined by inhibition of this effect with neutralizing IL-16 monoclonal antibody.

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles IL-16 is present in over 95% of T cells, of both CD4+ and CD8+ types. The pro-IL-16 molecule is constitutively expressed in substantial quantities, while apparently a very small percentage of this is processed to mature IL-16 and secreted. Despite the abundance of mRNA and protein in lymphoid organs little is known about its normal function. Two studies point to some potential normal role. Aged mice and nude mice share a defect in pre-B cell differentiation, resulting in decreased numbers of B cells and antibody responses. Supernatants of T cells appear to be able to restore this defect, implying a role for T cells in B cell development in adult life. Of interest, the majority of the T cell activity can be derived from CD8+ T cells (the major in vitro lymphoid source of IL-16), leading those investigators to explore the possibility that IL-16 might provide this function (Szabo et al., 1998). In fact, neutralizing IL-16 antibodies inhibit 85±90% of the T cell activity; rIL-16 induces pre-B cell differentiation as determined by RAG1 and RAG2 expression. Most importantly, in vivo injection of rIL-16 restores RAG1+ RAG1+ B cell numbers in aged mice. Thus, IL-16 may play a role in normal B cell homeostasis. Any potential role in CD4+ T cell homeostasis would be speculative, however injection of rIL-16 into normal Balb/c mice results in increased

splenic and lymph node weight and increased numbers of both T cells and B cells. The role of IL-16 in airway hyperreactivity has been studied in a Balb/c mouse model of ovalbumin sensitization and airway aerosol challenge (Hessel et al., 1998) discussed in detail below.

Species differences There is very high sequence and functional homology among all species in IL-16 protein. Of note, the IL-16 of most simian species does not contain a cysteine, yet these molecules do oligomerize to 55 kDa aggregates wherein lies the bioactivity. IL-16's bioactivities cross species, including chemotaxis of CD4+ T cells, upregulation of IL-2R, induction of RAG1 in pre-B cells, and transient inhibition of T cell responses and inhibition of HIV-1 transcription. In fact, there are no reported qualitative differences in bioactivities of IL16 from different species, although there are quantitative differences in dose responses (e.g. simian IL-16 inhibits HIV-1 transcription at lower concentrations than human) (Baier et al., 1995; Lee et al., 1999).

Knockout mouse phenotypes No transgenic knockout animals have been studied to date.

Transgenic overexpression Only one model of transgenic overexpression of a human IL-16 is known (Chupp, personal communication). In those studies, a construct coding for the 55 kDa C- terminus of IL-16 directed by a CC-10 promoter was inserted into a Balb/c background mouse. There is airway epithelial cell (Clara cell)-specific IL-16 observed which is apparently processed to bioactive IL-16 and secreted into the airway lumen as determined by immunocytochemistry, ELISA, and chemotaxis. There is a minimal spontaneous phenotype (minimal mononuclear infiltration into the lungs), but apparently the animals are more sensitive to the inflammatory process induced by other agents. Neither the immunopathology nor subsequent sequelae of the inflammatory process in the presence of transgenic overexpression have been completely characterized.

Interactions with cytokine network Two cytokine interactions have been identified based upon the observations that IL-16 induces IL-2R and

236 David M. Center, Hardy Kornfeld and William W. Cruikshank IL-2R expression in resting CD4+ T cells (Parada et al., 1998). In that regard, sequential addition of IL2 or IL-15, but not IL-4 results in selective CD4+ T cell upregulation of CD25 with ultimate increases in T cell proliferation. Several weeks of IL-16/ IL-2 stimulation results in marked increases of CD4+CD25+ CD45ROCD95+ T cells. They do not appear to be polarized to a specific TH subset. IL-16 stimulation of CD4+ T cells results in direct synthesis and secretion of GM-CSF, IL-3, TNF, and IL-6 (Parada et al., 1998; Amiel et al., 1999) and IFN mRNA is preferentially induced however, no IFN protein can be detected unless a second stimulus is supplied.

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Normal levels and effects Amiel et al. (1999) have compiled the most extensive information on circulating levels of IL-16 in normal individuals. In their study the average normal levels were about 200 pg/mL with a range from undetectable to 800 pg/mL. There are no studies that have followed IL-16 levels sequentially over time in normals, nor determined the normal range at various ages. In HIV-1-infected individuals, the IL-16 levels vary depending upon disease stage. The levels in stages A and B are similar to normals. With advancement to stage C there is a significant decrease in circulating levels of IL-16 to an average of 100 pg/mL. IL-16 levels have been reported to rise 1000-fold following indinavir treatment (Bisset et al., 1997). The clinical significance of this observation is not known, but it correlates with the increased release of IL-16 from cloned PBMCs obtained from HIV-1-infected long-term survivors in vitro (Scala et al., 1997; Amiel et al., 1999).

Role in experiments of nature and disease states IL-16 in inflammation IL-16 has the ability to increase CD4+ cell numbers at sites of inflammation by three different mechanisms: to induce cell migration (Center and Cruikshank, 1982); prime T cells for proliferation

(Parada et al., 1998); and potentially protect T cells from activation-induced cell death (AICD) (Idziorek et al., 1998). As such, IL-16 can be classified as a proinflammatory cytokine. Work directed at identifying a role for IL-16 in inflammation has focused on diseases characterized by CD4+ cellular infiltrates, specifically asthma and granulomatous diseases. Asthma was the first disease to be directly associated with IL-16 production (Bellini et al., 1993). IL-16 bioactivity was identified in cultures of primary epithelial cells obtained from asthmatics but not from normals stimulated with histamine. This association was confirmed by studies which identified IL-16 in the bronchoalveolar lavage (BAL) fluid obtained 6 hours following antigen challenge of asthmatic subjects (Cruikshank et al., 1995). IL-16 was not detected in the BAL fluid obtained from either normal or atopic nonasthmatic individuals. At this 4 hour time point IL-16 represented the major chemoattractant activity, approximately 80% of total activity, with the balance of the activity attributable to macrophage inflammatory protein 1 (MIP-1 ) bioactivity. These findings were confirmed in studies in which direct subsegmental histamine challenge of asthmatic subjects resulted in the elaboration of IL-16 protein detected in the BAL fluid (Vallen-Mashikian et al., 1998). In contrast to antigen challenge, histamine challenge resulted in release of only IL-16 into the BAL fluid. Histamine challenge of normals or atopic nonasthmatic individuals did not result in IL16 production. Detection of IL-16 following airway challenge with either histamine or antigen from asthmatics but not from normals or atopic nonasthmatics suggested the existence of a phenotypic difference between asthmatics and nonasthmatics. A phenotypic difference between asthmatics and nonasthmatics was identified by immunohistochemical staining and in situ hybridization for IL-16 protein and message. Analysis of biopsies from asthmatics revealed readily detectable and uniformly distributed IL-16 protein and message in their airway epithelium (and infiltrating CD4+ cells) (Laberge et al., 1996). There was a high correlation between the amount of detectable IL16 protein and mRNA in the airway epithelium with the number of infiltrating CD4+ mononuclear cells. In contrast, nonasthmatics had little detectable IL-16 protein and message. Of interest, the first identification of IL-16 in a human disease was in the blister fluid of patients with bullous pemphigoid (Center et al., 1979, 1983b). It was noted as one of four lymphocyte chemotactic factors whose appearance correlated with a mononuclear cell infiltrate. Subsequently, Laberge et al. (1998) has found IL-16 in the keratinocytes of skin biopsies of patients with atopic dermatitis. The level of

IL-16 237 expression correlated with the CD4+ T cell numbers in the dermis and the stage of the disease. IL-16 is also present as one of the lymphocyte chemotactic factors derived from T cells stimulated in vitro with myelin basic protein of patients with multiple sclerosis (Biddison et al., 1997a, 1997b, 1998). Biddison et al. (1998b) has also identified IL-16 as a T cell chemotactic factor from T cell clones derived from patients infected with HTLV-I. The clinical significance of these findings in multiple sclerosis and HTLV-I is not known, but the collection of studies suggests that IL-16 is probably a common lymphocyte chemotactic factor in immune-mediated diseases. In a mouse model of allergic inflammation in the lung, ovalbumin-sensitized Balb/c mice express IL-16 protein in airway epithelium while unsensitized mice do not. Neutralizing anti-IL-16 antibodies administered prior to ovalbumin challenge significantly reduced the airway hyperreactivity and IgE antibody production observed in animals treated with control antibodies (Hessel et al., 1998). Similar findings were obtained using IL-16-neutralizing peptides derived from the putative bioactive site of IL-16 (D. Center, unpublished observation). These findings suggest that IL-16 may contribute to the accumulation of CD4+ T cells and overall pathophysiology seen in asthmatic inflammation. The potential role of IL-16 has also been examined in the inflammation associated with inflammatory bowel disease. Analysis of colonic tissue sections from patients with Crohn's disease demonstrate increases in both IL-16 message and protein when compared with either uninvolved colonic tissue from the same patient or with tissue from normal individuals. In addition, using a mouse model of inflammatory bowel disease, animals treated with neutralizing anti-IL-16 antibodies demonstrated significantly less weight loss, mucosal ulcerations and myeloperoxidase activity as compared with animals receiving control antibodies (A. Keates, unpublished observation). Taken together, these studies indicate that IL-16 is present at sites of inflammation and neutralization of IL-16 bioactivity may significantly alter the inflammatory process. IL-16 in HIV-1 infection As noted above, a number of investigators have explored the potential role of IL-16 in progression of HIV-1 infection to AIDS. In that regard, IL-16 serum levels drop with progression to stage C disease, while asymptomatic individuals have IL-16 levels that are comparable to normals (Amiel et al., 1999). Moreover, there was a correlation between IL-16

levels and long-term nonprogressors. IL-16 levels appear to rise following treatment with indinavir, corresponding to a rise in CD4 counts and fall in viral load (Bisset et al., 1997). It is not clear whether in these circumstances IL-16 is playing an active role in viral repression and preservation of CD4 cell numbers or if it represents a marker of CD4+ T cell `health'. In the former circumstance, it would be logical to include IL-16 therapy along with IL-2 in HIV-1 infected individuals. In either circumstance, following sequential IL-16 levels along with viral load and CD4 cell counts may provide additional predictive value for prognosis and progression of disease (Amiel et al., 1999).

IN THERAPY

Preclinical ± How does it affect disease models in animals? IL-16 has been used in two animal models. Aged and nude mice suffer from poor B cell development characterized by a lack of RAG1 and RAG2 mRNA expression in bone marrow pre-B cells. IL-16 injected i.p. restores pro-B cell development into pre-B cells in both animals, characterized by RAG1 expression in their bone marrow pre-B cells. Limited information exists utilizing IL-16 for immune reconstitution in vivo and control of HIV-1 replication exists. Normal mice injected i.p. experience increases in splenic and lymphoid T cells and B cells. Animal trials are under way in huPBL-SCID and huPBL-SCID-HIV-1 animals to determine the effect on CD4+ T cell reconstitution and in SIV-1infected monkeys, although no data are yet available.

Effects of therapy: Cytokine, antibody to cytokine inhibitors, etc. There are no reports of human trials.

Toxicity No formal toxicity studies on IL-16 or inhibitors have been performed. No toxic effects have been reported by investigators who have injected IL-16 into the peritoneum. Mice injected intraperitoneally with neutralizing antibodies or exposed to aerosols of IL-16derived peptides have not experienced local or systemic effects.

238 David M. Center, Hardy Kornfeld and William W. Cruikshank

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240 David M. Center, Hardy Kornfeld and William W. Cruikshank Zhou, P., Goldstein, S., Devadas, K., Tewari, D., and Notkins, A. L. (1997). Human CD4+ cells transfected with IL-16 cDNA are resistant to HIV-1 infection: inhibition of mRNA expression. Nature Med. 3, 659±664.

LICENSED PRODUCTS Recombinant IL-16 is available from PharMingen and R&D Systems.

IL-16 ELISA kits are available from PharMingen, R&D Systems, Genzyme, and Endogen. Neutralizing IL-16 antibodies are available from PharMingen and R&D Systems.