Human and Murine IL-16 Conservation of Structure And

Conservation of Structure and Function Between Human and Murine IL-16 Joseph Keane, John Nicoll, Sue Kim, David M. H. Wu, William W. Cruikshank, William Brazer, Barbara Natke, Yujun Zhang, This information is current as David M. Center and Hardy Kornfeld of September 30, 2021. J Immunol 1998; 160:5945-5954; ; http://www.jimmunol.org/content/160/12/5945 Downloaded from References This article cites 42 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/160/12/5945.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Conservation of Structure and Function Between Human and Murine IL-161,2

Joseph Keane,3 John Nicoll,3 Sue Kim, David M. H. Wu, William W. Cruikshank,4 William Brazer, Barbara Natke, Yujun Zhang, David M. Center, and Hardy Kornfeld5

IL-16 is a proinﬂammatory cytokine that signals via CD4, inducing chemotactic and immunomodulatory responses of CD4؉ lymphocytes, monocytes, and eosinophils. Comparative analysis of murine and human IL-16 homologs could reveal conserved structures that would help to identify key functional regions of these cytokines. To that end, we cloned the murine IL-16 cDNA and found a high degree of amino acid similarity comparing the predicted murine and human IL-16 precursor proteins (pro- IL-16). The highest similarity (82.1%) was found in the C-terminal region, which is cleaved from pro-IL-16 to yield biologically active IL-16. Chemotaxis experiments with IL-16 of murine and human origin, using murine splenocytes or human T lymphocytes Downloaded from as targets, showed cross-species stimulation of motility. Synthetic oligopeptides and anti-peptide Ab were produced, based on the sequences of three predicted hydrophilic domains of IL-16 potentially presented in exposed positions. None of these peptides had intrinsic IL-16 bioactivity, but one (corresponding to a hydrophilic C-terminal domain of IL-16) partially displaced binding of OKT4 mAb to human lymphocytes. This peptide, and its cognate Ab, also inhibited IL-16 chemoattractant activity for human and murine cells. These studies demonstrate a high degree of structural and functional similarity between human and murine IL-16 and suggest that amino acids in the C terminus are critical for its chemoattractant function. The data suggest cross-species http://www.jimmunol.org/ conservation of IL-16 receptor structures as well. Inhibitory peptides may be useful in disease states where the proinﬂammatory functions of IL-16 are detrimental to the host. The Journal of Immunology, 1998, 160: 5945–5954.

n 1982, Cruikshank and Center first reported the functional identify any conserved features that might provide insight to its and biochemical characterization of lymphocyte chemoat- structure and function. Our results indicate a high degree of se- I tractant factor (1, 2), which has more recently been renamed quence similarity and cross-species biologic activity and provide IL-16 (3, 4). Numerous studies have provided evidence that CD4 evidence that the C terminus of IL-16 is required for CD4-medi- serves as a receptor for IL-16 and that IL-16-induced signal trans- ated functions and binding. ϩ duction through CD4 induces a variety of responses in CD4 cells, by guest on September 30, 2021 including G0 to G1a cell cycle shift; up-regulation of IL-2R and Materials and Methods HLA-DR expression; transient inhibition of TCR signal transduc- Cell preparations tion and mixed lymphocyte reactions; and repression of HIV-1 Mouse splenocytes were isolated from euthanized BALB/c mice and main- promoter activity (5). tained in RPMI 1640 medium (BioWhittaker, Walkersville, MD) supple- Analysis of human IL-16 cDNA clones indicates that the IL-16 mented with 10% FBS and 100 U/ml of both penicillin and streptomycin mRNA encodes a precursor protein (pro-IL-16), from which a C- (complete medium). Erythrocytes were lysed by suspension in one part complete medium to three parts Gey’s solution. This mixture was incu- terminal biologically active peptide is processed and secreted (6, bated on ice for 2 min. The reaction was stopped by the addition of 10 parts 7). Although the biologic activities of IL-16 have been studied in complete medium, and the cells were washed twice in complete medium some detail, little is known about possible functional domains before use in experiments. Human PBMC were isolated as described from within the secreted molecule. It is also unknown whether there are the blood of healthy normal volunteers by density centrifugation on Ficoll- important functional domains within the precursor protein. In the Paque (1, 2). Samples were enriched for T lymphocytes by nylon wool nonadherence (8). The nonadherent cells were Ͼ95% T lymphocytes by current studies, we isolated murine IL-16 cDNA from spleen and flow cytometry. lung and prepared recombinant and natural murine IL-16 protein to IL-16 cDNA clones IL-16 cDNA, 5Ј to the previously reported 2150 bp (6), was generated by 6 The Pulmonary Center, Boston University School of Medicine, Boston, MA 02118 rapid amplification of 5Ј ends (RACE), using human T lymphocyte total RNA as the starting material (9). RNA was reverse transcribed using 10 Received for publication August 28, 1997. Accepted for publication February pmol of IL-16-specific primers based on sequences within the 5Ј region of 12, 1998. the 2150 bp cDNA. The resulting cDNA was tailed with dATP in the The costs of publication of this article were defrayed in part by the payment of page presence of terminal transferase (Life Technologies, Gaithersburg, MD). charges. This article must therefore be hereby marked advertisement in accordance PCR was performed with this cDNA template, using anchored dT17 as the with 18 U.S.C. Section 1734 solely to indicate this fact. 5Ј primer and IL-16-specific sequences for 3Ј primers. The DNA sequence 1 Supported by National Institutes of Health Grant HL32802. of PCR products was analyzed on an ABI 373A Genetic Analyzer (Applied 2 The sequences reported in this paper have been deposited into the GenBank database Biosystems, Foster City, CA). Murine IL-16 cDNA clones were isolated (accession nos. AF006001 and M90391). from two cDNA libraries obtained from a commercial vendor (Clontech Laboratories, Palo Alto, CA). One library was generated from mRNA of 3 J.K. and J.N. made equal contributions to the work reported in this manuscript. Their names are listed in alphabetical order. NFS (The Jackson Laboratory, Bar Harbor, ME) mouse splenocytes that had been activated for 12 h with phorbol ester and calcium ionophore. The 4 W.W.C. is a Career Investigator of the American Lung Association. cDNA was produced by oligo(dT) priming, and the cloning vector was 5 Address correspondence and reprint requests to Dr. Hardy Kornfeld, Pulmonary Center, R-3, Boston University School of Medicine, 80 East Concord Street, Boston, MA 02118. 6 Abbreviation used in this paper: RACE, rapid amplification of cDNA ends.

bacteriophage ␭gt10. The other cDNA library was prepared from BALB/c Western blot analysis mouse lung using both oligo(dT) and random priming, and the cloning ␮ vector was also bacteriophage ␭gt10. Both libraries were screened by Affinity-purified native murine IL-16 (40 g) and recombinant IL-16 were plaque hybridization using 32P-labeled probes prepared by nick translation applied to SDS/15% polyacrylamide gels, then transferred to nitrocellulose by electroblotting (3500 Vh, 4°C). The nitrocellulose was incubated2hin of the 2150-bp human IL-16 cDNA (Promega, Madison, WI). One clone 125 was isolated from 105 plaques of the spleen library but was found to be PBS with 1% lactalbumin and 0.05% Tween 20, then probed with I- ␮ incompletely extended 5Ј. A partially overlapping cDNA clone was iso- conjugated polyclonal anti-human rIL-16 Ab added at 1 g/ml for 1 h. lated from the lung library, providing additional 5Ј sequence information. After washing in PBS, the dried blots were and visualized by autoradiog- Human and murine IL-16 cDNA sequences were analyzed using Lasergene raphy overnight at room temperature. software packages (DNASTAR, Madison, WI) for predicted amino acid Immunoprecipitation sequence similarity by the method of Lipman and Pearson (10), hydrophilicity profiles by the method of Kyte and Doolittle (11), and surface prob- Anti-IL-16 Abs were incubated with rIL-16 protein at 4°C for 4 h under ability by the method of Emini (12). Potential coding regions were assessed gentle rotation. Protein A-Sepharose beads were added, and the mixture by Fickett’s testcode (13) in the Wisconsin Package Version 9.0 (Genetics incubated for an additional 1 h. The beads were washed three times with Computer Group, Madison, WI). PBS, then resuspended in SDS-PAGE sample buffer and boiled. Released material was analyzed on a 15% SDS-polyacrylamide gel. After electro- Recombinant human and murine IL-16 phoresis, proteins were transferred to nitrocellulose membrane for immu- Recombinant human IL-16 was produced in Escherichia coli as a polyhis- noblotting analysis. tidine fusion protein containing 130 C-terminal residues encoded by the Chemotaxis assay previously reported human cDNA (6), using the pET16b vector (Novagen, Madison, WI). It was purified by metal chelation chromatography, and the Chemotaxis was performed using a modified Boyden chamber assay as histidine tag was cleaved with factor Xa. Recombinant ␤-galactosidase was described (1, 2). Cells were suspended at 5 ϫ 106/ml in complete medium. Downloaded from produced and purified in an identical fashion for use as a negative control. A12␮m nitrocelluose membrane separated cells in the upper wells from Recombinant murine IL-16 was produced in E. coli as a polyhistidine control buffer or experimental supernatants in the lower wells. Chambers fusion protein containing 118 C-terminal residues encoded by a murine were incubated at 37°C for 4 h, then the membranes were removed, stained cDNA, cloned into the pET30 vector (Novagen). It was purified by metal with hematoxylin, and dehydrated by sequential washes in ethanol, propa- chelation chromatography, and the histidine tag was cleaved with nol, and then xylene. Cell migration was quantitated by light microscopy, enterokinase. counting the number of cells migrating below a depth of 50 ␮m. Counts

were compared with control (unstimulated) migration, which was normal- http://www.jimmunol.org/ Natural murine IL-16 ized to 100%. All samples were tested in duplicate, and four high power Natural murine IL-16 was isolated by incubating 2 ϫ 106 mouse spleno- fields were examined in each duplicate. Results were analyzed using Stu- ␮ dent’s t test, and a P value Ͻ 0.05 was considered significant. cytes with Con A (2 g/ml) for 48 h (37°C, 5% CO2) before harvesting supernatant and cell lysate. Supernatant was concentrated 10-fold using a Flow cytometry Centricon-3 filter (Amicon, Beverly, MA). Splenocytes were lysed by in- cubation on ice for 3 min in lysis buffer (PBS containing 1% Nonidet P-40, To test the capacity of IL-16-derived peptides to inhibit anti-CD4 mAb ␮ ϫ 5 ␮ 0.02% NaN3, 1 mM PMSF, and 10 g apoprotinin). A polyclonal rabbit binding, 2 10 human T lymphocytes were incubated with 10 g of each anti-human IL-16 Ab, raised against a 130 amino acid human rIL-16 pro- peptide of the three IL-16-based peptides for2hatroom temperature, then duced in E. coli, was covalently bound to staphylococcal protein A-con- incubated with 10 ␮g of FITC-conjugated OKT4 or OKT4A mAb (Ortho jugated Sepharose beads (Pharmacia Biotech, Piscataway, NJ) for use in Diagnostics, Raritan, NJ) for 30 min. Cells were then washed twice in PBS by guest on September 30, 2021 affinity chromatography. Stimulated murine splenocyte supernatant was and resuspended at 1 ϫ 106 cells/ml, fixed with 10% formalin, and stored applied to the column for 1 h, then washed. Ab-bound protein was specif- in the dark at 4°C before analysis with a Becton Dickinson FACScan 440 ically eluted into one tenth volume 1 M Tris buffer (pH 8.8) using glycine (Becton Dickinson, Sunnyvale, CA) as described previously (15). Induc- buffer (pH 4.0), then quantitated by BCA assay (Pierce, Rockford, IL). tion of IL-2R on IL-16-stimulated murine splenocytes was detected by staining with FITC-conjugated anti-mouse IL-2R Ab (PharMingen, San Peptides and Abs Diego, CA). Synthetic oligopeptides corresponding to three hydrophilic domains identified within the human IL-16 sequence were synthesized. The sequence of Results peptide 1 (SLEGGKGSLHGD) corresponds to amino acids 546 to 557 of Murine IL-16 cDNA cloning and comparison of predicted pro-IL-16. The sequence of peptide 2 (ASEQSETVQPGD) corresponds to human and murine IL-16 proteins amino acids 570 to 581. The sequence of peptide 3 (RRKSLQSKETTA AGDS) corresponds to amino acids 615 to 630. After conjugation to key- Using fragments of human IL-16 cDNA as probes, we isolated hole limpet hemocyanin, each peptide was used for rabbit immunizations. murine IL-16 cDNA clones from bacteriophage ␭gt10 libraries of Anti-peptide IgG Ab were separated from immunized rabbit sera by col- mouse spleen and lung (GenBank accession no. AF006001). Fig- umn chromatography using staphylococcal protein A-conjugated Sepha- ure 1 shows the protein sequence predicted from the longest open rose beads (Pharmacia), then specifically purified with CNBr-linked peptide Sepharose columns. Polyclonal rabbit anti-human IL-16 was raised reading frame of murine IL-16. This 624-amino acid murine pu- against the 130-amino acid human rIL-16 and purified in a similar fashion tative pro-IL-16 is aligned with a human pro-IL-16 based on se- on protein A and immobilized rIL-16 columns. Polyclonal rabbit anti- quence data that we derived by a combination of RACE and human IL-16 Ab and monoclonal anti-human IL-16 (14.1) were verified by genomic DNA cloning (GenBank accession no. M90391), as well Western blotting with rIL-16 and shown to be neutralizing, as demonstrated by inhibition of chemoattractant activity, IL-2R expression, and as corrected DNA sequence analysis of our published human IL-16 HIV-1 repression in IL-16-treated cells (6, 14). cDNA clone (6). Our findings are in substantial agreement with the human pro-IL-16 sequence reported by Baier et al. (7), differing at Northern blot analysis only four residues (Glu104 to Asp, Arg225 to Gly, Thr233 to Phe, A commercial multiple tissue murine Northern blot (Mouse MTN; Clon- and Ala319 to Glu). The findings are also consistent with the dem- ϩ tech) consisting of 2 ␮g poly(A) RNA per lane from specific tissues onstration of ϳ80-kDa and ϳ67-kDa bands by Western blot anal- transferred from a formaldehyde/1.2% agarose gel onto a positively ysis of human PBMC lysate probed with an anti-human IL-16 Ab charged nylon membrane was probed with a 32P-labeled fragment of the murine IL-16 cDNA. The membrane was treated for 1 h with a prehybrid- (7, 16), suggesting the expression of a human pro-IL-16 in vivo. ization solution (QuikHybe; Stratagene, La Jolla, CA) containing 10 mg/ml Human IL-16 mRNA potentially encodes a 631-amino acid pre- salmon sperm DNA, and subsequently hybridized with the cDNA probe for cursor protein (Fig. 1). A pro-IL-16 of 605 amino acids would be 1 h overnight at 68°C. After hybridization, the blot was washed twice at produced if the downstream and in-frame Met28 were used as the low stringency conditions of 2ϫ SSC (300 mM NaCl, 30 mM sodium citrate, 0.5% sodium pyrophosphate, and 1% sodium lauryl sarkosine) at start site of translation. At both of these potential start sites, the room temperature, followed by a wash at high stringency of 0.1ϫ SSC at Met residues are weak initiator codons according to the scanning 60°C. Hybridization was visualized by autoradiography. model for translation, lacking key nucleotides normally present in The Journal of Immunology 5947

would yield a secreted murine IL-16 polypeptide of 118 amino acids. Comparing the predicted human and murine IL-16 protein sequences, we obtained a Lipman-Pearson similarity index of 82.1% for the putative cleaved and secreted C-terminal IL-16 regions, vs 75.1% for the remaining N-terminal precursor sequences. There are, however, several regions of higher homology within the residual precursors. Methionine residues at positions 258 and 502 of pro-IL-16 were previously postulated to be start sites for translation of human IL-16 (6, 19, 20). In the murine sequence, Met502 is replaced by Thr. Met502 is also not conserved in certain simian IL-16 homologs (20), indicating that it is very unlikely to serve as an initiator for human IL-16. In contrast, human Met258 is conserved (as Met254) in the predicted murine pro-IL-16 sequence. Whether this Met residue could serve as an alternative translational start site that might account for the reported appearance of a ϳ60- kDa natural human IL-16 band on immunoblot remains to be determined (7). Two GLGF sequence motifs present in human IL-16 are con- Downloaded from served in the murine homolog. One of these sequences lies within the precursor region (G422 to F425) and the other lies within the secreted C-terminal IL-16 (G542 to F545). This motif is contained within a domain pattern designated PDZ (previously called Discs- Large homology repeats) which are generally involved in intracellular protein-protein interactions (21, 22). Alignment of the upstream and downstream IL-16 PDZ domains (Fig. 2) indicates they http://www.jimmunol.org/ are more closely related to one another (Lipman-Pearson similarity index 33.3%) than to PDZ domains in other intracellular proteins, suggesting unique interactions. Any function for these PDZ domains, or for the other highly conserved regions within other pre- FIGURE 1. Alignment of the predicted human and murine pro-IL-16 cursor sequences of IL-16, is currently unknown. amino acid sequences. The sequence of the originally reported 2150-bp human IL-16 cDNA (6) was extended by 5Ј RACE, and cDNA clones of Northern blot of murine tissues murine IL-16 were isolated as described in Materials and Methods. Align- 32

Northern blot analysis of murine tissues, probed with a P-labeled by guest on September 30, 2021 ment of the predicted amino acid sequences for the longest open reading frames in both the human (hu) and murine (mu) IL-16 cDNAs are shown. fragment of murine IL-16 cDNA, revealed strong signals from Between each residue of the two sequences a vertical bar represents a spleen and lung (Fig. 3). In a separate Northern blot experiment, an perfect match, a colon represents a favorable mismatch (weight table identical strong signal was present in thymus (data not shown). value Ͼ 0), a period represents a neutral mismatch (weight table value ϭ The relatively high constitutive expression of IL-16 mRNA in 0), and a blank space represents an unfavorable mismatch (weight table mouse spleen and thymus is consistent with the primarily lym- value Ͻ 0). phoid tissue distribution and constitutive expression of human IL-16 mRNA. The strong IL-16 signal in mRNA from mouse lung contrasts with normal human lung where IL-16 mRNA is barely strong initiator codons (17). Met1 lacks a purine at position Ϫ3, detectable. However, markedly increased expression of IL-16 while Met28 has an A rather than G at position ϩ4. Translation mRNA (and protein) in bronchial epithelium of human asthmatics from the next downstream Met, at position 35, would yield a 298- has been identified (23). It is unknown if the cells expressing IL-16 amino acid protein. However, Met35 has neither a purine at Ϫ3, mRNA in mouse lung are of lymphoid (e.g., bronchus-associated noraGatϩ4. lymphoid tissue) or epithelial origin. Murine IL-16 mRNA expres- Both Met1 and Met28 are conserved in the murine pro-IL-16, sion also differs from that seen in human lymphoid tissues in that while Met35 is not. Potentially, either or both of the two upstream it appears as two predominant forms of ϳ2.5 kb and ϳ3.5 kb, Met codons are translational start sites for the natural IL-16 pre- whereas a single predominant band at ϳ2.7 kb is found in human cursor proteins of both species. Their conservation in the human tissues. The two mRNA species in the mouse may arise from usage and murine sequences supports this possibility, despite their asso- of different polyadenylation signals, but this remains to be ciated weak initiator codons. Fickett’s testcode (13) was used to determined. plot potential coding regions within the human IL-16 cDNA sequence, and both the Met1 and Met28 residues fall within the 95% Biologic activity of recombinant murine IL-16 confidence zone for coding. Lack of conservation of Met35 in the Recombinant murine IL-16 corresponding to the 118 C-terminal mouse provides additional evidence that it is unlikely to serve as a residues of the precursor protein was expressed in E. coli. Nano- translational start site for human IL-16. molar concentrations of murine rIL-16 stimulated a motile re- The mature, biologically active, secreted form of IL-16 is de- sponse in both murine splenocytes and human T lymphocytes (Fig. rived from the C-terminal end of pro-IL-16. Baier et al. postulated 4A). This activity was specifically inhibited by anti-human IL-16 that human IL-16 is cleaved from pro-IL-16 after Asp510, yielding Ab, and no chemoattractant activity was found with recombinant a 121-residue monomer. We have recently published evidence that ␤-galactosidase produced in E. coli and purified in an identical cleavage at this site is mediated by caspase-3 in human lympho- manner as the murine rIL-16. Induction of IL-2R expression on cytes (18). The caspase-3 cleavage site is conserved in murine resting CD4ϩ T lymphocytes is another characteristic biologic ac- IL-16 at Asp506 and surrounding residues; cleavage at this site tivity of IL-16 (24). Consistent with that function, murine rIL-16 5948 MURINE IL-16

FIGURE 2. Alignment of PDZ domains of IL-16 with PDZ domains of selected other proteins. The predicted IL-16 protein sequences examined for this alignment are I414 to A484 for the upstream domain and A529 to R617 for the downstream domain. Boxed residues match the IL-16 upstream PDZ domain exactly. PDZ-1, 2, and 3 represent domains within postsynaptic protein PSD-95 (40). DLG, Discs Large repeat 3 (41). ZO-1 and ZO-2, tight junction proteins (42, 43). nNOS, neuronal nitric oxide synthase (44). Downloaded from

induced IL-2R expression on a fraction of resting murine spleno- spontaneous noncovalent association to form tetramers that are cytes (Fig. 4B), and this activity was blocked when cells were required for biologic activity. stimulated with murine rIL-16 in the presence of anti-IL16 Ab http://www.jimmunol.org/ (data not shown). Previous experiments analyzing natural and recombinant human Identiﬁcation and functional characterization of natural murine IL-16 by Sephadex G-100 molecular sieve chromatography and IL-16 HPLC indicated that the monomeric IL-16 peptides must form Previous studies from our laboratory indicated that an intracellular aggregates, presumably noncovalently associated homotetramers, pool of biologically active IL-16 is stored in human CD8ϩ lym- to exhibit bioactivity (2;6). We have postulated that tetrameric phocytes and that IL-16 represents the majority, if not all, of the IL-16 induces CD4 signaling by cross-linking CD4 receptors (5). chemoattractant activity that can be recovered from lysate of un- Recombinant murine IL-16 (predicted molecular mass 12,234 Da)

stimulated human lymphocytes (25). Based on these observations, by guest on September 30, 2021 was subjected to HPLC, and column fractions were tested for che- and the high level of sequence conservation between murine and moattractant activity. Peak activity was eluted in the column frac- human IL-16, we predicted that testing unstimulated murine leu- tion corresponding to a molecular size of ϳ60 kDa (Fig. 4C). The kocytes for the presence of preformed chemoattractant factors ca- HPLC column fractions were tested for the presence of IL-16 by pable of inducing motility of human target cells would provide a ELISA, and it was detected only in the ϳ60-kDa range column fractions, which had also demonstrated IL-16 bioactivity. To- strong bias for the identification of natural murine IL-16. As shown gether, these data are consistent with our earlier findings and in- in Figure 5A, crude lysate of unstimulated BALB/c mouse spleno- Ϯ dicate that, like human IL-16, murine IL-16 monomers undergo cytes induced a motile response by human PBMC (175% 14%; mean % control migration Ϯ SEM, p Ͻ 0.05). This activity was strongly inhibited by an Ab raised against a 130-amino acid human rIL-16. The specificity of this anti-IL-16 Ab for neutralizing IL-16 chemoattractant activity was previously confirmed in experiments where it was found not to inhibit motility induced by MIP-1␣, MIP-1␤, or RANTES (26). Similar to our previous findings with human lymphocytes, there appears to be a preformed pool of biologically active murine IL-16 in resting splenocytes. A column for affinity chromatography was constructed by linking anti-human rIL-16 Ab to staphylococcus protein A-conjugated Sepharose beads, and this was used for partial purification of natural murine IL-16. Affinity-purified supernatant of Con-A-stimulated mouse splenocytes was subjected to Western blot analysis using 125I-conjugated anti-human rIL-16 Ab as probe, and a band of ϳ17 kDa was identified (Fig. 5B). This is similar to the SDS- PAGE mobility of murine rIL-16 (not shown) as well as recombinant and natural human IL-16 (6). The migration of IL-16 on SDS-PAGE appears to be somewhat aberrant, since rIL-16 produced in E. coli, and therefore unmodified, has a predicted molec- FIGURE 3. Constitutive expression of IL-16 mRNA in murine tissues. ϳ ϳ A Northern blot of poly(A)ϩ RNA from the murine tissues indicated above ular mass of 13 kDa yet migrates as 17 kDa. These functional each lane was probed with a 32P-labeled fragment of murine IL-16 cDNA. and immunoblot results strongly suggest that the protein eluted Horizontal bars on the left of the figure represent size markers of 9.5 kb, 7.5 from the affinity column was the natural secreted murine IL-16 kb, 4.4 kb, 2.4 kb, and 1.35 kb, from top to bottom. homolog. The Journal of Immunology 5949

Cross-species bioactivity of IL-16 To further test the hypothesis that the murine and human IL-16 homologs would demonstrate cross-species functionality, human rIL-16 and affinity-purified natural murine IL-16 were assayed for the induction of motility using either murine splenocytes or human T lymphocytes as responding cells. The results presented in Figure 6, A and B, show that both the murine and the human cytokines are chemoattractant for either murine or human target cells in a dose- dependent manner. Peak motility was induced at IL-16 concentrations in the 10Ϫ10 M range, and high dose inhibition (a characteristic of chemoattractant cytokines) was observed with both preparations. While murine IL-16 appeared to be marginally more potent for murine cells, and human IL-16 appeared to be more potent for human cells, the differences in motility in these experiments were not significant.

Identiﬁcation of structural requirements for IL-16 biologic activity Downloaded from Kyte-Doolittle hydrophilicity (11) and Emini surface probability plots (12) were generated, based on the predicted amino acid sequence of secreted human IL-16 (Fig. 7A). This analysis indicated the presence of three hydrophilic domains (labeled 1, 2, and 3, respectively from the N terminus to the C terminus). The surface

probability prediction was highest in the region corresponding to http://www.jimmunol.org/ the hydrophilic domain 3 located at the C-terminal end of the protein, although it was also positive in the central hydrophilic domain 2. A very similar pattern was observed in plots based on murine IL-16. We postulated that one or more of these domains would be exposed on the surface of naturally folded IL-16, and thus likely to be involved in CD4 binding. Synthetic oligopeptides corresponding to each of the three hydrophilic domains were produced, and Ab against these three synthetic oligopeptides were raised in rab- bits. Of these three anti-peptide Ab, anti-peptide 2 and anti-peptide by guest on September 30, 2021 3 were capable of detecting rIL-16 on Western blots, and anti- peptide 3 immunoprecipitated rIL-16 (Fig. 7B). Previous work from our laboratory indicated that the anti-CD4 mAb OKT4 binds to an epitope on CD4 near the domain involved in IL-16 recognition. We therefore tested the effect of the three IL-16-derived oligopeptides on the binding of FITC-conjugated OKT4 and OKT4A mAb to human T lymphocytes. Figure 8 shows

were counted, and the mean was obtained for each sample. Results are expressed as the mean % control migration Ϯ SEM for three experiments. Comparisons between control and experimental conditions were analyzed by Student’s t test; the asterisk indicates statistical significance ( p Ͻ 0.05) for a difference compared with control migration. B, Induction of IL-2R on murine splenocytes. Splenocytes of BALB/c mice were incubated in control buffer (upper panel) or stimulated with 1.4 ng/ml murine rIL-16 (lower panel), then stained with FITC-conjugated anti-mouse IL-2R mAb for FACS analysis as described in Materials and Methods. The unstimulated splenocytes were 12% IL-2Rϩ and this increased to 20% following IL-16 stimulation. The effect was ablated in the presence of anti-IL-16. C, Au- toaggregation of murine rIL-16. Molecular sieve HPLC was performed with murine rIL-16 run in PBS, pH 8.0. Fractions were collected and assayed for lymphocyte chemoattractant activity (bars), as indicated on the left ordinate. Results are expressed as mean % control migration Ϯ SD for one experiment where four high power fields were counted in duplicates of FIGURE 4. Biologic activity of recombinant murine IL-16. A, Chemoat- each condition. Peak chemoattractant activity was observed in the fraction tractant activity of murine rIL-16 was tested in Boyden chamber chemotaxis corresponding to ϳ60 kDa. Blue dextran eluted in fraction 7, BSA in assays using murine or human T lymphocytes as target cells. Migration in fraction 9, OVA in fraction 11, and chymotrypsinogen in fraction 14. The buffer control was compared with 10 ng/ml of recombinant ␤-galactosidase column bed volume corresponded to fractions 6 through 15; there was no (b-gal), 1.4 ng/ml murine rIL-16, or murine rIL-16 preincubated with 10 ␮g/ml chemoattractant activity in the void volume fractions. Column fractions anti-human IL-16 Ab. Cell counts in the experimental wells are compared with were also tested for IL-16 by ELISA (solid line and boxes) as indicated on unstimulated (control) migration, represented by 100%. Ten high power fields the right ordinate. 5950 MURINE IL-16 Downloaded from http://www.jimmunol.org/

FIGURE 6. Cross-species bioactivity of human and murine IL-16. A, by guest on September 30, 2021 Chemoattractant activity of natural murine IL-16 was tested, using human T lymphocytes or murine splenocytes as the target cells. The response to FIGURE 5. Identification of natural murine IL-16. A, Chemoattractant serial dilutions of natural murine IL-16 from 140 ng/ml to 0.14 ng/ml was activity of unstimulated murine splenocyte lysate is inhibited by anti-IL-16 compared with buffer control. B, Chemoattractant activity of human rIL-16 Ab. Boyden chamber chemotaxis assays were performed using human T was tested in serial dilutions, using human T lymphocytes or murine lymphocytes as target cells. Conditions included buffer control, 1.4 ng/ml splenocytes as targets. Results are expressed as the mean % control mi- of human rIL-16, 1:100 dilution of murine splenocyte lysate, and murine gration Ϯ SEM for three experiments. lysate preincubated with 10 ␮g/ml anti-human IL-16 Ab. The asterisk indicates statistical significance ( p Ͻ 0.05) for a difference in migration in response to murine lysate vs lysate plus anti-IL-16 Ab. The anti-IL-16 Ab alone did not affect the motility of unstimulated lymphocytes, or lympho- motility of 185% Ϯ 15% was inhibited in the presence of peptide cytes stimulated with chemokines (data not shown). B, Identification of 3 to 130% Ϯ 12% ( p Ͻ 0.05). In contrast, peptides 1 and 2 (de- natural murine IL-16 by Western blot. Western blotting of affinity-purified rived from the N-terminal and central hydrophilic peaks of human natural murine IL-16 and human rIL-16 was performed as described in IL-16) demonstrated little or no blocking activity in these exper- Materials and Methods and the blot was probed with 125I-labeled anti- iments. Since peptide 3 appears to compete with the anti-CD4 human IL-16 Ab. Lane 1, standards (kDa). Lane 2, human rIL-16. Lane 3, mAb OKT4, inhibition of IL-16 biologic activity by this peptide is IL-16 immunoaffinity-purified supernatant of Con A-stimulated murine postulated to be due to competition with IL-16 for CD4 binding. splenocytes. This is an important distinction since inhibition of IL-16 bioactivity would also be predicted to occur if multimer formation was disrupted, thus preventing receptor cross-linking. The question the result of flow cytometry experiments where peptide 3, but not whether peptide 3 could neutralize IL-16 by disrupting autoaggre- peptides 1 or 2, partially displaced the binding of OKT4. The bind- gration was further assessed by HPLC. Autoaggregation of rIL-16 ing of OKT4A was not affected by any of the three oligopeptides. monomers to the biologically active tetrameric form was previ- This result suggests that peptide 3 might physically associate ously confirmed by the demonstration of chemoattractant activity with CD4. only in HPLC column fractions corresponding to ϳ60 kDa (Fig. We next tested the effect of the synthetic IL-16 peptide frag- 4C). Preincubation of rIL-16 with peptide 3 did not alter this peak ments on rIL-16-stimulated lymphocyte motility. In these experi- of activity (data not shown), suggesting that neutralization of biologic ments, peptide 3 partially inhibited the chemoattractant activity of activity was not a result of disaggregation of tetrameric IL-16. both human rIL-16 and affinity-purified murine natural IL-16 for The effect of rabbit anti-peptide Ab on human IL-16-stimulated human target cells (Fig. 9, A and B). The human rIL-16-stimulated motility of human T lymphocytes was also tested (Fig. 9C). In motility of 192% Ϯ 18% (mean % control migration Ϯ SEM) was these experiments, the rIL-16-stimulated motility of 190% Ϯ 22% reduced to 144% Ϯ 14%, and the murine natural IL-16-stimulated was reduced to 111% Ϯ 18% in the presence of anti-peptide 3 The Journal of Immunology 5951

FIGURE 7. A, Hydrophilicity and surface probability plots of IL-16. Kyte-Doolittle hydrophilicity (11) and Emini surface probability plots (12) were generated based on the predicted amino acid sequence of the 121 C-terminal residues of human IL-16 using Lasergene software (DNASTAR). Synthetic oligopeptides were produced based on the sequence of the three major hydrophilic peaks as indicated at the top of the ﬁgure. B, Immunoprecipitation of IL-16. Recombinant human IL-16 was immunoprecipitated with anti- rIL-16 mAb 14.1 (lane 1), anti- peptide 3 Ab (lane 2), or mouse IgG (lane 3), as described in Materials and Methods. Downloaded from

( p Ͻ 0.05). Abs to peptides 1 and 2 demonstrated no blocking in the C-terminal region that is postulated to be cleaved and se- 506

effect. Finally, peptides 2 and 3, and their corresponding anti- creted. Recombinant murine IL-16, based on the Asp cleavage http://www.jimmunol.org/ peptide Ab, were tested on murine splenocytes stimulated with site and expressed in E. coli, demonstrated chemoattractant activity murine rIL-16. Peptide 3 and anti-peptide 3 inhibited murine rIL- and IL-2R induction similar to human IL-16, and these activities were 16-stimulated splenocyte migration, similar to their blocking ef- neutralized by anti-human IL-16 Ab. Furthermore, murine rIL-16 was fects against human IL-16 (Fig. 9D). None of these peptides or Ab, shown to undergo autoaggregation, forming homotetramers required when used alone, affected cell motility. Taken together, these data for biologic activity, a unique property of IL-16. suggest that the C-terminal hydrophilic domain of IL-16 might be Analysis of crude lysate of unstimulated murine splenocytes involved in CD4 binding and is critical for the expression of IL- revealed the presence of natural murine IL-16 in a preformed 16-stimulated lymphocyte motility. intracellular storage pool. Preformed bioactive IL-16 has previously been identified in unstimulated human CD8ϩ T cells by guest on September 30, 2021 Discussion (25). Anti-human rIL-16 Ab was used for immunoaffinity pu- We isolated cDNA clones of the murine IL-16 homolog and com- rification of the secreted form of natural murine IL-16 from pared them with a full-length human IL-16 cDNA sequence de- supernatant of Con A-stimulated splenocytes. Affinity-purified rived by 5Ј RACE and genomic cloning. Similar to the 631-amino murine IL-16 demonstrated chemoattractant activity similar to acid putative human pro-IL-16, murine IL-16 appears to be pro- human IL-16; it was blocked by neutralizing anti-human rIL-16 duced as a precursor protein of up to 624 amino acids. The bio- Ab, and its mobility on SDS-PAGE was similar to human IL- logically active and secreted mature form of IL-16 is derived by 16. Functional cross-reactivity between murine and human caspase-3 cleavage of the C-terminal region from pro-IL-16 (18). IL-16 was observed in experiments with natural murine IL-16 Comparing the predicted amino acid sequences of murine and hu- and human rIL-16, both of which stimulated motility in both man IL-16, there is a high degree of homology throughout the human and murine mononuclear cells. The antigenic and func- putative pro-IL-16 molecule, but the highest conservation is found tional cross-reactivity of these proteins is consistent with the

FIGURE 8. Partial displacement of OKT4 by peptide 3. Human T lymphocytes were incubated with 10 ␮g of each of the three IL-16-based peptides for 30 min at room temperature, then incubated with 10 ␮g of FITC-conjugated OKT4 mAb for 30 min. Cells were then ﬁxed in formalin and analyzed by FACS. In similar experiments, binding of OKT4A mAB was not affected by any of the three peptides (data not shown). 5952 MURINE IL-16

high degree of structural similarity revealed by molecular cloning of the murine IL-16 cDNA. Previous experiments suggested that the anti-CD4 mAb OKT4 binds to an epitope that is proximate to, but probably not identical with, the domain of CD4 that interacts with IL-16. Fab fragments of OKT4 have been shown to inhibit IL-16 in a variety of functional assays including chemotaxis, IL-2R induction, and signal transduction (intracellular calcium flux and inositol tris phosphate generation; (6, 15)). Theodore et al. reported that coincubation of T lymphocytes with IL-16 resulted in partial displacement of OKT4, but not OKT4A mAb assessed by flow cytometry (14). Similarly, Maciaszek et al. found that OKT4 mAb partially inhibited IL-16-mediated repression of HIV-1 promoter activity in CD4ϩ lymphoid cells (27). In the present studies (based on the hydrophilicity and surface probability plots of human and murine IL-16) we identified two regions within the secreted cytokine likely to be exposed on the surface and thus possibly involved in receptor binding. A third N-terminal hydrophilic domain did not Downloaded from have a corresponding high surface probability prediction, making it a less likely candidate for receptor binding. Synthetic oligopeptides representing the amino acid sequences of these three regions in the predicted human IL-16 protein sequence were tested for their ability to inhibit OKT4 and OKT4A mAb binding to lymphocytes. Only the peptide that corresponded to the C-terminal hydrophilic domain of IL-16 (peptide 3) was found to partially http://www.jimmunol.org/ displace OKT4, while none of the peptides displaced OKT4A. Consistent with the studies of IL-16 and OKT4 binding cited above, the inhibitory activity of peptide 3 was incomplete. Pre- sumably, this sixteen-residue peptide would provide less steric interference with OKT4 binding than the 130-amino acid rIL-16. The ability of the three IL-16 peptides (and anti-peptide Ab) to neutralize IL-16 bioactivity was tested by chemotaxis assay. Con-

sistent with the FACS data, peptide 3 demonstrated signiﬁcant by guest on September 30, 2021 IL-16 blocking ability in experiments with human rIL-16 and natural as well as recombinant murine IL-16 stimulating human or murine target cells. Anti-peptide 3 Ab was also a potent inhibitor. Since autoaggregation is required for IL-16 to exert biologic effects, a peptide or Ab that disrupted this association could also function as an IL-16 inhibitor. However, chemotaxis assays of HPLC column fractions of rIL-16 preincubated with peptide 3 provided no evidence of interference with IL-16 tetramer formation. This ﬁnding, together with the observation that peptide 3 partially

tion Ϯ SEM for three experiments. The asterisk indicates a significant difference in migration ( p Ͻ .05) of cells treated with peptide 3 plus human rIL-16, compared with cells stimulated by human rIL-16 alone. B, Human T lymphocytes were stimulated with 1.4 ng/ml affinity-purified natural murine IL-16 alone, or in the presence of 10 ␮g each of the three IL-16 oligopeptides. The asterisk indicates a significant difference in migration ( p Ͻ .05) comparing cells treated with peptide 3 plus natural murine IL-16, compared with cells stimulated by murine IL-16 alone. C, Inhibition of IL-16 by anti-peptide 3 Ab. Human T lymphocytes were stimulated with 1.4 ng/ml human rIL-16 alone, or in the presence of 10 ␮g each of three Ab raised against the IL-16-based synthetic oligopeptides. Results are expressed as the mean % control migration Ϯ SEM for three experiments. The asterisk indicates a significant difference in migration ( p Ͻ .05) of cells treated with anti-peptide 3 Ab plus human IL-16, compared with cell stimulated by IL-16 alone. D, Inhibition of murine rIL-16-induced murine splenocyte migration by IL-16 peptides and anti-peptide Ab. Murine FIGURE 9. Inhibition of IL-16 biologic activity by a synthetic oli- splenocytes were stimulated with 1.4 ng/ml murine rIL-16 alone, or in the gopeptide and anti-peptide Ab. A, Human T lymphocytes were stimulated presence of 10 ␮g each of peptide 2, peptide 3, anti-peptide 2 Ab, or with 1.4 ng/ml human rIL-16 alone, or in the presence of 10 ␮g each of anti-peptide 3 Ab. Results of one experiment are expressed as mean % three IL-16-based oligopeptides described in Materials and Methods and control Ϯ SD where four high power fields were counted in duplicates of indicated in Fig. 6. Results are expressed as the mean % control migra- each condition. The Journal of Immunology 5953 displaced OKT4 binding to CD4, suggests that the C-terminal re- prime resting CD4ϩ T lymphocytes to respond to IL-2 raises the gion of IL-16 may be involved in CD4 binding. In these experi- possibility of a role in T lymphocyte development, and the che- ments, peptide 3 is postulated to function as a competitive inhibitor moattractant properties of IL-16 appear to be important in the for receptor binding, while anti-peptide 3 presumably blocks the pathophysiology of certain inflammatory diseases characterized by receptor-binding domain on IL-16. The cross-species chemoattrac- tissue accumulation of CD4ϩ lymphocytes, monocytes, and eosin- tant activity of murine and human IL-16, and the ability of peptide ophils. Conditions where IL-16 has been identified by ELISA 3, which is based on the human IL-16 sequence, to block stimu- and/or bioassay of body fluids, or by immunohistochemical and in lated migration of murine target cells, also suggests interspecies situ hybridization techniques, include bronchial asthma (23, 26), conservation of the CD4 domain, which interacts with IL-16. inflammatory bowel disease (36), Graves’ disease (37), multiple Sequence comparison between human and murine IL-16 pro- sclerosis (38), and bullous pemphigoid (39). The ability to study vides additional insight to the structural and functional features of IL-16 in murine model systems should facilitate understanding its these cytokines. Although the mechanism of processing and se- roles in normal and pathologic immune function. We show here cretion of murine IL-16 has not been characterized, conservation that a murine IL-16 homolog is expressed and that its structure and of sequences surrounding Asp506 (corresponding to Asp510 in hu- functions are very closely related to the known properties of human pro-IL-16) is consistent with our observation that cleavage of man IL-16. It is therefore likely that murine model systems will be human pro-IL-16 is mediated caspase-3 (18). While a high degree suitable for studies using hybridization and immunohistochemical of similarity in the secreted murine and human IL-16 sequences is techniques, as well as treatment with murine rIL-16 and IL-16 readily understood in terms of their known cytokine properties, the inhibitors (like peptide 3) to provide information relevant to IL-16 Downloaded from functional significance of regions of high similarity within the re- functions in humans. sidual N-terminal precursor domains is unclear. An intracellular function for pro-IL-16 was hypothesized, based on the presence of References PDZ domains (19), although there is presently no functional data 1. Center, D. M., and W. W. Cruikshank. 1982. Modulation of lymphocyte migra- to support that concept. While the IL-16 precursor sequences could tion by human lymphokines. I. Identification of characterization of chemoattrac- play a role in the regulation of processing and secretion, most of tant activity for lymphoyctes from mitogen-stimulated mononuclear cells. J. Im- this region appears not to be absolutely required for IL-16 release. munol. 128:2563. http://www.jimmunol.org/ 2. Cruikshank, W. W., and D. M. Center. 1982. Modulation of lymphocyte migra- We originally isolated a truncated IL-16 cDNA encoding 374 res- tion by human lymphokines. II. Purification of a lymphotactic factor (LCF). idues of pro-IL-16 by expression cloning, yet the COS cells trans- J. Immunol. 128:2569. fected with this construct secreted only the C-terminal portion (6). 3. Anonymous. 1996. Interleukin-16 (IL-16). A terminology note. IUIS/WHO Standing Committee on Interleukin Designation. J. Immunol. Methods 196:103. Similarly, Zhou et al. reported that transfection of Jurkat cells with 4. Anonymous. 1996. Interleukin 16 (IL-16). IUIS/WHO Standing Committee on a vector expressing only 130 C-terminal residues of IL-16 resulted Interleukin Designation. Bull. W. H. O. 74:451. in secretion of biologically active cytokine into the culture 5. Center, D. M., H. Kornfeld, and W. W. Cruikshank. 1996. Interleukin 16 and its function as a CD4 ligand. Immunol. Today 17:476. medium (28). 6. Cruikshank, W. W., D. M. Center, N. Nisar, M. Wu, B. Natke, A. C. Theodore, and H. Kornfeld. 1994. Molecular and functional analysis of a lymphocyte che-

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