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The Pulmonary Center, Boston University School of Medicine, Boston, MA 02118
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Analysis of human IL-16 cDNA clones indicates that the IL-16 mRNA encodes a precursor protein (pro-IL-16), from which a C-terminal biologically active peptide is processed and secreted (6, 7). Although the biologic activities of IL-16 have been studied in some detail, little is known about possible functional domains within the secreted molecule. It is also unknown whether there are important functional domains within the precursor protein. In the current studies, we isolated murine IL-16 cDNA from spleen and lung and prepared recombinant and natural murine IL-16 protein to identify any conserved features that might provide insight to its structure and function. Our results indicate a high degree of sequence similarity and cross-species biologic activity and provide evidence that the C terminus of IL-16 is required for CD4-mediated functions and binding.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mouse splenocytes were isolated from euthanized BALB/c mice and maintained in RPMI 1640 medium (BioWhittaker, Walkersville, MD) supplemented with 10% FBS and 100 U/ml of both penicillin and streptomycin (complete medium). Erythrocytes were lysed by suspension in one part complete medium to three parts Gey’s solution. This mixture was incubated on ice for 2 min. The reaction was stopped by the addition of 10 parts complete medium, and the cells were washed twice in complete medium before use in experiments. Human PBMC were isolated as described from the blood of healthy normal volunteers by density centrifugation on Ficoll-Paque (1, 2). Samples were enriched for T lymphocytes by nylon wool nonadherence (8). The nonadherent cells were >95% T lymphocytes by flow cytometry.
IL-16 cDNA clones
IL-16 cDNA, 5' to the previously reported 2150 bp (6), was
generated by rapid amplification of 5' ends
(RACE),6 using human T
lymphocyte total RNA as the starting material (9). RNA was reverse
transcribed using 10 pmol of IL-16-specific primers based on sequences
within the 5' region of the 2150 bp cDNA. The resulting cDNA was tailed
with dATP in the presence of terminal transferase (Life Technologies,
Gaithersburg, MD). PCR was performed with this cDNA template, using
anchored dT17 as the 5' primer and IL-16-specific sequences
for 3' primers. The DNA sequence of PCR products was analyzed on an ABI
373A Genetic Analyzer (Applied Biosystems, Foster City, CA). Murine
IL-16 cDNA clones were isolated from two cDNA libraries obtained from a
commercial vendor (Clontech Laboratories, Palo Alto, CA). One library
was generated from mRNA of NFS (The Jackson Laboratory, Bar Harbor, ME)
mouse splenocytes that had been activated for 12 h with phorbol
ester and calcium ionophore. The cDNA was produced by oligo(dT)
priming, and the cloning vector was bacteriophage 
gt10. The other
cDNA library was prepared from BALB/c mouse lung using both oligo(dT)
and random priming, and the cloning vector was also bacteriophage
gt10. Both libraries were screened by plaque hybridization using
32P-labeled probes prepared by nick translation of the
2150-bp human IL-16 cDNA (Promega, Madison, WI). One clone was isolated
from 105 plaques of the spleen library but was found to be
incompletely extended 5'. A partially overlapping cDNA clone was
isolated from the lung library, providing additional 5' sequence
information. Human and murine IL-16 cDNA sequences were analyzed using
Lasergene software packages (DNASTAR, Madison, WI) for predicted amino
acid sequence similarity by the method of Lipman and Pearson (10),
hydrophilicity profiles by the method of Kyte and Doolittle (11), and
surface probability by the method of Emini (12). Potential coding
regions were assessed by Fickett’s testcode (13) in the Wisconsin
Package Version 9.0 (Genetics Computer Group, Madison, WI).
Recombinant human and murine IL-16
Recombinant human IL-16 was produced in Escherichia coli as a polyhistidine fusion protein containing 130 C-terminal residues encoded by the previously reported human cDNA (6), using the pET16b vector (Novagen, Madison, WI). It was purified by metal chelation chromatography, and the histidine tag was cleaved with factor Xa. Recombinant ß-galactosidase was produced and purified in an identical fashion for use as a negative control. Recombinant murine IL-16 was produced in E. coli as a polyhistidine fusion protein containing 118 C-terminal residues encoded by a murine cDNA, cloned into the pET30 vector (Novagen). It was purified by metal chelation chromatography, and the histidine tag was cleaved with enterokinase.
Natural murine IL-16
Natural murine IL-16 was isolated by incubating 2 x 106 mouse splenocytes with Con A (2 µg/ml) for 48 h (37°C, 5% CO2) before harvesting supernatant and cell lysate. Supernatant was concentrated 10-fold using a Centricon-3 filter (Amicon, Beverly, MA). Splenocytes were lysed by incubation on ice for 3 min in lysis buffer (PBS containing 1% Nonidet P-40, 0.02% NaN3, 1 mM PMSF, and 10 µg apoprotinin). A polyclonal rabbit anti-human IL-16 Ab, raised against a 130 amino acid human rIL-16 produced in E. coli, was covalently bound to staphylococcal protein A-conjugated Sepharose beads (Pharmacia Biotech, Piscataway, NJ) for use in affinity chromatography. Stimulated murine splenocyte supernatant was applied to the column for 1 h, then washed. Ab-bound protein was specifically eluted into one tenth volume 1 M Tris buffer (pH 8.8) using glycine buffer (pH 4.0), then quantitated by BCA assay (Pierce, Rockford, IL).
Peptides and Abs
Synthetic oligopeptides corresponding to three hydrophilic domains identified within the human IL-16 sequence were synthesized. The sequence of peptide 1 (SLEGGKGSLHGD) corresponds to amino acids 546 to 557 of pro-IL-16. The sequence of peptide 2 (ASEQSETVQPGD) corresponds to amino acids 570 to 581. The sequence of peptide 3 (RRKSLQSKETTAAGDS) corresponds to amino acids 615 to 630. After conjugation to keyhole limpet hemocyanin, each peptide was used for rabbit immunizations. Anti-peptide IgG Ab were separated from immunized rabbit sera by column chromatography using staphylococcal protein A-conjugated Sepharose beads (Pharmacia), then specifically purified with CNBr-linked peptide Sepharose columns. Polyclonal rabbit anti-human IL-16 was raised against the 130-amino acid human rIL-16 and purified in a similar fashion on protein A and immobilized rIL-16 columns. Polyclonal rabbit anti-human IL-16 Ab and monoclonal anti-human IL-16 (14.1) were verified by Western blotting with rIL-16 and shown to be neutralizing, as demonstrated by inhibition of chemoattractant activity, IL-2R expression, and HIV-1 repression in IL-16-treated cells (6, 14).
Northern blot analysis
A commercial multiple tissue murine Northern blot (Mouse MTN; Clontech) consisting of 2 µg poly(A)+ RNA per lane from specific tissues transferred from a formaldehyde/1.2% agarose gel onto a positively 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 prehybridization solution (QuikHybe; Stratagene, La Jolla, CA) containing 10 mg/ml salmon sperm DNA, and subsequently hybridized with the cDNA probe for 1 h overnight at 68°C. After hybridization, the blot was washed twice at low stringency conditions of 2x SSC (300 mM NaCl, 30 mM sodium citrate, 0.5% sodium pyrophosphate, and 1% sodium lauryl sarkosine) at room temperature, followed by a wash at high stringency of 0.1x SSC at 60°C. Hybridization was visualized by autoradiography.
Western blot analysis
Affinity-purified native murine IL-16 (40 µg) and recombinant IL-16 were applied to SDS/15% polyacrylamide gels, then transferred to nitrocellulose by electroblotting (3500 Vh, 4°C). The nitrocellulose was incubated 2 h in PBS with 1% lactalbumin and 0.05% Tween 20, then probed with 125I-conjugated polyclonal anti-human rIL-16 Ab added at 1 µg/ml for 1 h. After washing in PBS, the dried blots were and visualized by autoradiography overnight at room temperature.
Immunoprecipitation
Anti-IL-16 Abs were incubated with rIL-16 protein at 4°C for 4 h under gentle rotation. Protein A-Sepharose beads were added, and the mixture incubated for an additional 1 h. The beads were washed three times with PBS, then resuspended in SDS-PAGE sample buffer and boiled. Released material was analyzed on a 15% SDS-polyacrylamide gel. After electrophoresis, proteins were transferred to nitrocellulose membrane for immunoblotting analysis.
Chemotaxis assay
Chemotaxis was performed using a modified Boyden chamber assay as described (1, 2). Cells were suspended at 5 x 106/ml in complete medium. A 12 µm nitrocelluose membrane separated cells in the upper wells from control buffer or experimental supernatants in the lower wells. Chambers were incubated at 37°C for 4 h, then the membranes were removed, stained with hematoxylin, and dehydrated by sequential washes in ethanol, propanol, and then xylene. Cell migration was quantitated by light microscopy, counting the number of cells migrating below a depth of 50 µm. Counts were compared with control (unstimulated) migration, which was normalized to 100%. All samples were tested in duplicate, and four high power fields were examined in each duplicate. Results were analyzed using Student’s t test, and a P value < 0.05 was considered significant.
Flow cytometry
To test the capacity of IL-16-derived peptides to inhibit anti-CD4 mAb binding, 2 x 105 human T lymphocytes were incubated with 10 µg of each peptide of the three IL-16-based peptides for 2 h at room temperature, then incubated with 10 µg of FITC-conjugated OKT4 or OKT4A mAb (Ortho Diagnostics, Raritan, NJ) for 30 min. Cells were then washed twice in PBS and resuspended at 1 x 106 cells/ml, fixed with 10% formalin, and stored in the dark at 4°C before analysis with a Becton Dickinson FACScan 440 (Becton Dickinson, Sunnyvale, CA) as described previously (15). Induction of IL-2R on IL-16-stimulated murine splenocytes was detected by staining with FITC-conjugated anti-mouse IL-2R Ab (PharMingen, San Diego, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using fragments of human IL-16 cDNA as probes, we isolated murine
IL-16 cDNA clones from bacteriophage gt10 libraries of mouse spleen
and lung (GenBank accession no. AF006001). Figure 1
shows the protein sequence predicted
from the longest open reading frame of murine IL-16. This 624-amino
acid murine putative pro-IL-16 is aligned with a human pro-IL-16 based
on sequence data that we derived by a combination of RACE and genomic
DNA cloning (GenBank accession no. M90391), as well as corrected DNA
sequence analysis of our published human IL-16 cDNA clone (6). Our
findings are in substantial agreement with the human pro-IL-16 sequence
reported by Baier et al. (7), differing at only four residues
(Glu104 to Asp, Arg225 to Gly,
Thr233 to Phe, and Ala319 to Glu). The findings
are also consistent with the demonstration of 
80-kDa and 67-kDa
bands by Western blot analysis of human PBMC lysate probed with an
anti-human IL-16 Ab (7, 16), suggesting the expression of a human
pro-IL-16 in vivo.
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). A pro-IL-16 of 605 amino acids would be produced if the
downstream and in-frame Met28 were used as the start site
of translation. At both of these potential start sites, the Met
residues are weak initiator codons according to the scanning model for
translation, lacking key nucleotides normally present in strong
initiator codons (17). Met1 lacks a purine at position -3,
while Met28 has an A rather than G at position +4.
Translation from the next downstream Met, at position 35, would yield a
298-amino acid protein. However, Met35 has neither a purine
at -3, nor a G at +4. Both Met1 and Met28 are conserved in the murine pro-IL-16, while Met35 is not. Potentially, either or both of the two upstream Met codons are translational start sites for the natural IL-16 precursor proteins of both species. Their conservation in the human and murine sequences supports this possibility, despite their associated weak initiator codons. Fickett’s testcode (13) was used to plot potential coding regions within the human IL-16 cDNA sequence, and both the Met1 and Met28 residues fall within the 95% confidence zone for coding. Lack of conservation of Met35 in the mouse provides additional evidence that it is unlikely to serve as a translational start site for human IL-16.
The mature, biologically active, secreted form of IL-16 is derived from
the C-terminal end of pro-IL-16. Baier et al. postulated that human
IL-16 is cleaved from pro-IL-16 after Asp510, yielding a
121-residue monomer. We have recently published evidence that cleavage
at this site is mediated by caspase-3 in human lymphocytes (18). The
caspase-3 cleavage site is conserved in murine IL-16 at
Asp506 and surrounding residues; cleavage at this site
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 60-kDa natural human IL-16 band on immunoblot remains to be
determined (7).
Two GLGF sequence motifs present in human IL-16 are conserved 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 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 precursor sequences of IL-16, is
currently unknown.
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Northern blot analysis of murine tissues, probed with a
32P-labeled fragment of murine IL-16 cDNA, revealed
strong signals from spleen and lung (Fig. 3). In a separate Northern blot
experiment, an identical strong signal was present in thymus (data not
shown). The relatively high constitutive expression of IL-16 mRNA in
mouse spleen and thymus is consistent with the primarily lymphoid
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 detectable. However, markedly
increased expression of IL-16 mRNA (and protein) in bronchial
epithelium of human asthmatics has been identified (23). It is unknown
if the cells expressing IL-16 mRNA in mouse lung are of lymphoid (e.g.,
bronchus-associated lymphoid tissue) or epithelial origin. Murine IL-16
mRNA expression also differs from that seen in human lymphoid tissues
in that it appears as two predominant forms of 2.5 kb and 3.5 kb,
whereas a single predominant band at 2.7 kb is found in human
tissues. The two mRNA species in the mouse may arise from usage of
different polyadenylation signals, but this remains to be
determined.
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Recombinant murine IL-16 corresponding to the 118 C-terminal
residues of the precursor protein was expressed in E. coli.
Nanomolar concentrations of murine rIL-16 stimulated a motile response
in both murine splenocytes and human T lymphocytes (Fig. 4A). This activity was
specifically inhibited by anti-human IL-16 Ab, and no
chemoattractant activity was found with recombinant ß-galactosidase
produced in E. coli and purified in an identical manner as
the murine rIL-16. Induction of IL-2R expression on resting
CD4+ T lymphocytes is another characteristic biologic
activity of IL-16 (24). Consistent with that function, murine rIL-16
induced IL-2R expression on a fraction of resting murine splenocytes
(Fig. 4B), and this activity was blocked when cells
were stimulated with murine rIL-16 in the presence of anti-IL16 Ab
(data not shown).
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60 kDa
(Fig. 4C). The HPLC column fractions were tested for
the presence of IL-16 by ELISA, and it was detected only in the
60-kDa range column fractions, which had also demonstrated IL-16
bioactivity. Together, these data are consistent with our earlier
findings and indicate that, like human IL-16, murine IL-16 monomers
undergo spontaneous noncovalent association to form tetramers that are
required for biologic activity. Identification and functional characterization of natural murine IL-16
Previous studies from our laboratory indicated that an
intracellular pool of biologically active IL-16 is stored in human
CD8+ lymphocytes and that IL-16 represents the
majority, if not all, of the chemoattractant activity that can be
recovered from lysate of unstimulated human lymphocytes (25). Based on
these observations, and the high level of sequence conservation between
murine and human IL-16, we predicted that testing unstimulated murine
leukocytes for the presence of preformed chemoattractant factors
capable of inducing motility of human target cells would provide a
strong bias for the identification of natural murine IL-16. As shown in
Figure 5A, crude lysate of
unstimulated BALB/c mouse splenocytes induced a motile response by
human PBMC (175% ± 14%; mean % control migration ± SEM,
p < 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-1ß, 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.
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17 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 molecular
mass of 13 kDa yet migrates as 17 kDa. These functional and
immunoblot results strongly suggest that the protein eluted from the
affinity column was the natural secreted murine IL-16 homolog. 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 10-10
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.
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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 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 rabbits. Of these three anti-peptide
Ab, anti-peptide 2 and anti-peptide 3 were capable of detecting
rIL-16 on Western blots, and anti-peptide 3 immunoprecipitated
rIL-16 (Fig. 7B).
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shows the
result of flow cytometry experiments where peptide 3, but not peptides
1 or 2, partially displaced the binding of OKT4. The binding of OKT4A
was not affected by any of the three oligopeptides. This result
suggests that peptide 3 might physically associate with CD4.
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, A and
B). The human rIL-16-stimulated motility of 192% ±
18% (mean % control migration ± SEM) was reduced to 144% ±
14%, and the murine natural IL-16-stimulated motility of 185% ± 15%
was inhibited in the presence of peptide 3 to 130% ± 12%
(p < 0.05). In contrast, peptides 1 and 2
(derived from the N-terminal and central hydrophilic peaks of human
IL-16) demonstrated little or no blocking activity in these
experiments. Since peptide 3 appears to compete with the anti-CD4
mAb OKT4, inhibition of IL-16 biologic activity by this peptide is
postulated to be due to competition with IL-16 for CD4 binding. 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 whether peptide 3 could
neutralize IL-16 by disrupting autoaggregration was further assessed by
HPLC. Autoaggregation of rIL-16 monomers to the biologically active
tetrameric form was previously confirmed by the demonstration of
chemoattractant activity only in HPLC column fractions corresponding to
60 kDa (Fig. 4C). Preincubation of rIL-16 with
peptide 3 did not alter this peak of activity (data not shown),
suggesting that neutralization of biologic activity was not a result of
disaggregation of tetrameric IL-16.
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C). In these experiments, the rIL-16-stimulated
motility of 190% ± 22% was reduced to 111% ± 18% in the presence
of anti-peptide 3 (p < 0.05). Abs to
peptides 1 and 2 demonstrated no blocking effect. Finally, peptides 2
and 3, and their corresponding anti-peptide Ab, were tested on
murine splenocytes stimulated with murine rIL-16. Peptide 3 and
anti-peptide 3 inhibited murine rIL-16-stimulated splenocyte
migration, similar to their blocking effects against human IL-16 (Fig. 9D). None of these peptides or Ab, when used alone,
affected cell motility. Taken together, these data suggest that the
C-terminal hydrophilic domain of IL-16 might be involved in CD4 binding
and is critical for the expression of IL-16-stimulated lymphocyte
motility. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Analysis of crude lysate of unstimulated murine splenocytes revealed the presence of natural murine IL-16 in a preformed intracellular storage pool. Preformed bioactive IL-16 has previously been identified in unstimulated human CD8+ T cells (25). Anti-human rIL-16 Ab was used for immunoaffinity purification of the secreted form of natural murine IL-16 from supernatant of Con A-stimulated splenocytes. Affinity-purified murine IL-16 demonstrated chemoattractant activity similar to human IL-16; it was blocked by neutralizing anti-human rIL-16 Ab, and its mobility on SDS-PAGE was similar to human IL-16. Functional cross-reactivity between murine and human IL-16 was observed in experiments with natural murine IL-16 and human rIL-16, both of which stimulated motility in both human and murine mononuclear cells. The antigenic and functional cross-reactivity of these proteins is consistent with the 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 CD4+ 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 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 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. Presumably, 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. Consistent with the FACS data, peptide 3 demonstrated significant 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 displaced OKT4 binding to CD4, suggests that the C-terminal region of IL-16 may be involved in CD4 binding. In these experiments, peptide 3 is postulated to function as a competitive inhibitor for receptor binding, while anti-peptide 3 presumably blocks the receptor-binding domain on IL-16. The cross-species chemoattractant activity of murine and human IL-16, and the ability of peptide 3, which is based on the human IL-16 sequence, to block stimulated migration of murine target cells, also suggests interspecies conservation of the CD4 domain, which interacts with IL-16.
Sequence comparison between human and murine IL-16 provides additional insight to the structural and functional features of these cytokines. Although the mechanism of processing and secretion of murine IL-16 has not been characterized, conservation of sequences surrounding Asp506 (corresponding to Asp510 in human pro-IL-16) is consistent with our observation that cleavage of human pro-IL-16 is mediated caspase-3 (18). While a high degree of similarity in the secreted murine and human IL-16 sequences is readily understood in terms of their known cytokine properties, the functional significance of regions of high similarity within the residual N-terminal precursor domains is unclear. An intracellular function for pro-IL-16 was hypothesized, based on the presence of PDZ domains (19), although there is presently no functional data to support that concept. While the IL-16 precursor sequences could play a role in the regulation of processing and secretion, most of this region appears not to be absolutely required for IL-16 release. We originally isolated a truncated IL-16 cDNA encoding 374 residues of pro-IL-16 by expression cloning, yet the COS cells transfected with this construct secreted only the C-terminal portion (6). Similarly, Zhou et al. reported that transfection of Jurkat cells with a vector expressing only 130 C-terminal residues of IL-16 resulted in secretion of biologically active cytokine into the culture medium (28).
The data presented here indicate a high degree of functional conservation between murine and human IL-16, consistent with their sequence homology. Both human and murine mononuclear cells respond to either human or murine IL-16 in the Boyden chamber chemotaxis assay. Furthermore, a requirement for autoaggregation to occur for murine IL-16 to exert biologic activities indicates that it functions in a similar fashion to human IL-16. A wealth of data indicates that multimeric human IL-16 activates cells by signaling through CD4, and it is hypothesized that receptor cross-linking occurs upon binding by these multimers (5). Our results suggest that murine IL-16 acts via an identical mechanism. It is unknown whether other cell surface molecules in addition to CD4 are required for IL-16 binding. However, transfection of L3T4- murine hybridoma cells with human CD4 alone is sufficient to enable IL-16-stimulated signal transduction and activation responses in these cells (6). The ability of human IL-16 to induce chemotaxis in murine splenocytes, and for murine IL-16 to act in a similar fashion on human T lymphocytes, also suggests that involvement of a second receptor molecule is unlikely.
IL-16 has been found to exert diverse activities via CD4 signaling in lymphocytes, monocytes, and eosinophils. Originally identified by its chemoattractant properties (1), IL-16 was subsequently found to induce signal transduction via CD4, to up-regulate IL-2R and HLA-DR expression, and to increase RNA content consistent with a G0 to G1a shift in resting CD4+T cells (6, 15, 24, 35). In addition to these activation and competence growth factor properties, IL-16 has been found to exert certain inhibitory effects, including inhibition of one way MLR, inhibition of Ag and anti-CD3 Ab-induced T lymphocyte proliferation, and repression of HIV-1 promoter activity (5, 14, 27). The circumstances in which each of these activities is physiologically significant in vivo remains to be determined. The capacity of IL-16 to signal through CD4 and to prime resting CD4+ T lymphocytes to respond to IL-2 raises the possibility of a role in T lymphocyte development, and the chemoattractant properties of IL-16 appear to be important in the pathophysiology of certain inflammatory diseases characterized by tissue accumulation of CD4+ lymphocytes, monocytes, and eosinophils. Conditions where IL-16 has been identified by ELISA and/or bioassay of body fluids, or by immunohistochemical and in situ hybridization techniques, include bronchial asthma (23, 26), inflammatory bowel disease (36), Graves’ disease (37), multiple sclerosis (38), and bullous pemphigoid (39). The ability to study IL-16 in murine model systems should facilitate understanding its roles in normal and pathologic immune function. We show here that a murine IL-16 homolog is expressed and that its structure and functions are very closely related to the known properties of human IL-16. It is therefore likely that murine model systems will be suitable for studies using hybridization and immunohistochemical techniques, as well as treatment with murine rIL-16 and IL-16 inhibitors (like peptide 3) to provide information relevant to IL-16 functions in humans.
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Footnotes |
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2 The sequences reported in this paper have been deposited into the GenBank database (accession nos. AF006001 and M90391).
3 J.K. and J.N. made equal contributions to the work reported in this manuscript. Their names are listed in alphabetical order.
4 W.W.C. is a Career Investigator of the American Lung Association.
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.
Received for publication August 28, 1997. Accepted for publication February 12, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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  F. Venet, C.-S. Chung, X. Huang, J. Lomas-Neira, Y. Chen, and A. Ayala Lymphocytes in the Development of Lung Inflammation: A Role for Regulatory CD4+ T Cells in Indirect Pulmonary Lung Injury J. Immunol., September 1, 2009; 183(5): 3472 - 3480. [Abstract] [Full Text] [PDF]
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Y. Zhang, M. Tuzova, Z.-X. J. Xiao, W. W. Cruikshank, and D. M. Center Pro-IL-16 Recruits Histone Deacetylase 3 to the Skp2 Core Promoter through Interaction with Transcription Factor GABP J. Immunol., January 1, 2008; 180(1): 402 - 408. [Abstract] [Full Text] [PDF]
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M. D. Mannie and D. J. Abbott A Fusion Protein Consisting of IL-16 and the Encephalitogenic Peptide of Myelin Basic Protein Constitutes an Antigen-Specific Tolerogenic Vaccine That Inhibits Experimental Autoimmune Encephalomyelitis J. Immunol., August 1, 2007; 179(3): 1458 - 1465. [Abstract] [Full Text] [PDF]
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F. Ren, X. Zhan, G. Martens, J. Lee, D. Center, S. K. Hanson, and H. Kornfeld Pro-IL-16 Regulation in Activated Murine CD4+ Lymphocytes J. Immunol., March 1, 2005; 174(5): 2738 - 2745. [Abstract] [Full Text] [PDF]
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D. M. Center, W. W. Cruikshank, and Y. Zhang Nuclear Pro-IL-16 Regulation of T Cell Proliferation: p27KIP1-Dependent G0/G1 Arrest Mediated by Inhibition of Skp2 Transcription J. Immunol., February 1, 2004; 172(3): 1654 - 1660. [Abstract] [Full Text] [PDF]
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 N. M. Archin, L. van den Boom, L. Perelygina, J. M. Hilliard, and S. S. Atherton Delayed Spread and Reduction in Virus Titer after Anterior Chamber Inoculation of a Recombinant of HSV-1 Expressing IL-16 Invest. Ophthalmol. Vis. Sci., July 1, 2003; 44(7): 3066 - 3076. [Abstract] [Full Text] [PDF]
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 F. F. Little, E. Lynch, G. Fine, D. M. Center, and W. W. Cruikshank Tumor Necrosis Factor-{alpha}-Induced Synthesis of Interleukin-16 in Airway Epithelial Cells: Priming for Serotonin Stimulation Am. J. Respir. Cell Mol. Biol., March 1, 2003; 28(3): 354 - 362. [Abstract] [Full Text] [PDF]
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 C. M. Leutenegger, F. S. Boretti, C. N. Mislin, J. N. Flynn, M. Schroff, A. Habel, C. Junghans, S. A. Koenig-Merediz, B. Sigrist, A. Aubert, et al. Immunization of Cats against Feline Immunodeficiency Virus (FIV) Infection by Using Minimalistic Immunogenic Defined Gene Expression Vector Vaccines Expressing FIV gp140 Alone or with Feline Interleukin-12 (IL-12), IL-16, or a CpG Motif J. Virol., November 15, 2000; 74(22): 10447 - 10457. [Abstract] [Full Text]
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 T. Yoshimoto, C.-R. Wang, T. Yoneto, A. Matsuzawa, W. W. Cruikshank, and H. Nariuchi Role of IL-16 in delayed-type hypersensitivity reaction Blood, May 1, 2000; 95(9): 2869 - 2874. [Abstract] [Full Text] [PDF]
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 C. Kurschner and M. Yuzaki Neuronal Interleukin-16 (NIL-16): A Dual Function PDZ Domain Protein J. Neurosci., September 15, 1999; 19(18): 7770 - 7780. [Abstract] [Full Text] [PDF]
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M. V. Mashikian, T. C. Ryan, A. Seman, W. Brazer, D. M. Center, and W. W. Cruikshank Reciprocal Desensitization of CCR5 and CD4 Is Mediated by IL-16 and Macrophage-Inflammatory Protein-1{beta}, Respectively J. Immunol., September 15, 1999; 163(6): 3123 - 3130. [Abstract] [Full Text] [PDF]
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J. Nicoll, W. W. Cruikshank, W. Brazer, Y. Liu, D. M. Center, and H. Kornfeld Identification of Domains in IL-16 Critical for Biological Activity J. Immunol., August 15, 1999; 163(4): 1827 - 1832. [Abstract] [Full Text] [PDF]
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 Y. Liu, W. W. Cruikshank, T. O'Loughlin, P. O'Reilly, D. M. Center, and H. Kornfeld Identification of a CD4 Domain Required for Interleukin-16 Binding and Lymphocyte Activation J. Biol. Chem., August 13, 1999; 274(33): 23387 - 23395. [Abstract] [Full Text] [PDF]
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Y. Zhang, H. Kornfeld, W. W. Cruikshank, S. Kim, C. C. Reardon, and D. M. Center Nuclear Translocation of the N-terminal Prodomain of Interleukin-16 J. Biol. Chem., January 5, 2001; 276(2): 1299 - 1303. [Abstract] [Full Text] [PDF]
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