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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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In the current studies, we investigated the synthesis and release of IL-16 by CD4+ T lymphocytes after stimulation with specific Ag and anti-CD3 Ab and assessed the effect of costimulation of anti-CD3 with an activating anti-CD28 Ab. We observed that CD3 stimulation of peripheral blood CD4+ T cells results in the secretion of bioactive IL-16 without detectable changes in the level of expression of IL-16 mRNA. Although costimulation through CD28 results in a more rapid secretion, with increased levels of bioactive IL-16 in cell supernatants, we could not detect any change in IL-16 mRNA levels, rate of transcription, or stability. Furthermore, we could not detect any changes in the level of total pro-IL-16 protein. Rather, we found that, unlike CD8+ T cells, which contain active caspase-3 (1), CD4+ T cells require activation to induce the cleavage of pro-caspase-3 into its 20-kDa enzymatically active form. There is a temporal relationship between the appearance of active caspase-3 and bioactive IL-16 in anti-CD3-stimulated CD4+ T cells; in a similar fashion, CD28 costimulation augments the coincident appearance of both active caspase-3 and bioactive IL-16. These studies indicate that CD4+ and CD8+ T cells have distinct mechanisms and kinetics of caspase-dependent processing of IL-16. Whereas CD8+ T cells do not require antigenic activation for the release of IL-16, the cleavage of pro-IL-16 and the release of bioactive IL-16 by CD4+ T cells do require antigenic stimulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cycloheximide (CHX)3 was obtained from Sigma (St. Louis, MO). Actinomycin D was obtained from Life Technologies (Grand Island, NY). Caspase-1 and -3 inhibitors were obtained from Bachem (King of Prussia, PA).
Antibodies
An affinity-purified polyclonal rabbit anti-rIL-16 was prepared from rIL-16-immunized rabbit sera as described previously (3, 6). Mouse monoclonal anti-human CD4 Abs, mouse anti-human CD8 Abs, FITC-conjugated anti-CD4 or anti CD8 mAbs, and phycoerythrin-conjugated anti-CD3 were purchased from Bio-Source International (Camarillo, CA). Monoclonal anti-human CD3 Ab and mouse anti-human CD28 Ab were obtained from PharMingen (San Diego, CA).
Cell preparation
PBMCs were isolated by Ficoll-Paque (Pharmacia, Piscataway, NJ) density centrifugation of freshly drawn venous blood of healthy human volunteers. After centrifugation, cells were washed three times and resuspended at a concentration of 3.5 x 106/ml in culture medium 199 (Life Technologies) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 22 mM HEPES buffer, and 0.4% BSA. Nylon wool nonadherent human T cells (NWNA-Ts) were isolated by a modification (4) of the method of Julius et al. (13). For subset isolation, CD4- or CD8-depleted cell populations were prepared by magnetic bead negative selection (Biomag/Advanced Magnetics, Cambridge, MA) conjugated with goat anti-mouse IgG. NWNA-T lymphocytes were first labeled with mouse anti-human CD4 or mouse anti-human CD8 mAbs by incubating 1 x 106 cells with 5.0 µg of the appropriate Abs for 30 min at 4°C. Magnetic beads were washed with cold PBS (4°C) three times and mixed with the labeled lymphocytes for 30 min at 4°C in the dark. Nonadherent cells were collected in the supernatant. Immunostaining followed by FACScan analysis by flow cytometry (Becton Dickinson, FACS Division, Mountain View, CA) showed that CD4-depleted cell populations contained <1% of CD4+/CD3+ lymphocytes and that CD8-depleted cell populations contained <1% of CD8+/CD3+ cells.
Cell cultures
Human PBMCs, CD4+ T cells, or CD8+ T
cells were cultured for varying time intervals at 37°C in U-bottom
microculture plates (Costar, Cambridge, MA) or P-60 culture plates in a
humidified atmosphere of 5% CO2 at concentrations of
3.0–4.0 x 106/ml in the presence or absence of
anti-CD3 (1–2.5 µg/ml). Costimulation studies involved the
addition of soluble anti-CD28 Ab (1 µg/ml). In certain
experiments, CD4- or CD8- subsets were
pretreated with a protein synthesis inhibitor (CHX, 20 µg/ml for
2 h) and washed twice before being added to plates containing
anti-CD3 Ab. After incubation, the cell-free supernatants were
harvested and assessed for IL-16 protein and bioactivity. In the
experiments using caspase inhibitors, the inhibitors were added to the
CD4+ T cells at the same time that the cells were added to
the culture wells containing immobilized anti-CD3 and anti-CD28
Abs. CD4+ T cells were incubated in the presence or absence
of 100 µM of the tetrapeptide inhibitors Ac-DEVD-CHO or Ac-YVAD-CHO
at 37°C in 5% CO2 for 
24 h. The culture medium was
collected at 6 and 24 h and assessed for IL-16 bioactivity by
chemotaxis.
Chemotaxis assay
The presence of IL-16 in cell-free supernatants was assessed by quantification of human NWNA-T lymphocyte migration using a modified Boyden chemotaxis chamber technique (4). A total of 52 µl of NWNA-T suspension (10 x 106 cells/ml) was placed in the upper compartments of the 48-well microchemotaxis chambers separated from 32 µl of test samples by 8-µm micropore nitrocellulose filters (Neuroprobe, Cabin John, MD) and was incubated at 37°C in a 5% CO2 atmosphere for 2 h. Test samples included cell-free supernatants diluted at various concentrations. The filters were fixed with hematoxylin, dehydrated, and mounted on glass slides. Cell migration was quantified by counting the total number of cells migrating beyond 50 µm into the filter by light microscopy, with baseline migration under control buffer conditions of 10–15 cells. Six high-power fields in duplicate for each sample and the means (±SD) were calculated and expressed as percent values of control migration (100%). For each set of experiments, four to five individual experiments were performed. A paired Student’s t test was used for statistical analysis to identify differences between experimental and control conditions using the mean values for lymphocyte migration. Significance was established at the p < 0.05 level of confidence.
To assess specificity for IL-16, neutralizing experiments were conducted by incubating test samples for 15 min with neutralizing concentrations of affinity-purified rabbit anti-human rIL-16 (rhuIL-16) antisera (5–10 µg/ml). At this concentration, the optimal chemotactic activity of 50 ng/ml rhuIl-16 is completely neutralized. There is no cross-neutralization by this Ab with the chemotactic activity induced by any of the lymphocyte chemoattractant chemokines tested thus far (14, 15, 16). Preimmune rabbit serum did not recognize IL-16 by Western blot analysis (9) and had no effect on IL-16-induced, chemokine-induced, or random migration of human T cells (data not shown).
RNA extraction and Northern hybridization
NWNA-Ts or PBMCs were stimulated with plate-bound monoclonal anti-CD3 (1–2.5 µg/ml) for 1, 2, 4, 6, or 24 h. Total cellular RNA was obtained from NWNA-T cell suspensions (20–30 x 106 cells) using the standard TriReagent (Molecular Research Center, Cincinnati, OH) extraction method according to the manufacturer’s recommendations. A total of 10–15 µg of total cellular RNA was fractionated by 1% agarose/17% formaldehyde gel electrophoresis and transferred to nylon membranes (Hybond N+, Amersham, Arlington Heights, IL) by capillary blotting. Short-wave UV light was used to fix the RNA on the membranes. The nylon membranes were treated for 20 min with prehybridization solution (Quick Hyb, Stratagene, La Jolla, CA) at 68°C and subsequently hybridized with 32P-labeled IL-16 cDNA probe with 10 mg/ml herring sperm DNA for 1 h at 68°C. After hybridization, blots were washed twice at low stringency conditions of 2x SSC (300 nM NaCl, 30 mM sodium citrate, 0.5% sodium pyrophosphate) and 0.1% SDS at 25°C followed by wash at high stringency conditions of 0.1x SSC/0.1% SDS at 60°C and were subjected to autoradiography. Equal loading of RNA was assessed by ethidium bromide staining and by rehybridizing blots with the ß-actin cDNA probe.
IL-16 mRNA stability was determined in PBMCs in the presence or absence of anti-CD3 (2.5 µg/ml) and anti-CD28 Ab (1 µg/ml) for costimulation studies. New transcription was inhibited by the addition of actinomycin D (10 µg/ml) at 24 h poststimulation. Relative steady-state mRNA levels for IL-16 and ß-actin were measured at 30 min and at 1, 2, and 3 h thereafter.
Western blot analysis
Purified CD4 or CD8 cells were prepared by negative selection as described above. Cells under control, anti-CD3, or CD28 costimulatory conditions were washed once with PBS, released by scraping, and harvested by centrifugation. Cells were immediately lysed by sonication in a buffer containing PBS (pH 7.5), 1 mM PMSF, 10 µg/ml aprotonin, and 10 µg/ml leupeptin and subjected to electrophoresis through a 15% SDS polyacrylamide gel. Proteins were electrophoretically transferred to a nitrocellulose membrane and probed with goat-polyclonal anti-caspase-3 against the P20 subunit (Santa Cruz Biotechnology, Santa Cruz, CA). The secondary Ab, which was an anti-goat Ig labeled with horseradish peroxidase (Santa Cruz Biotechnology), was used at a dilution of 1/5000, and the signal was detected by chemiluminescence (Pierce, Rockford, IL).
Staining for apoptosis
T cells harvested from the cultures were washed twice in PBS before staining with propidium iodide (1 µg/ml final concentration) for 5 min at room temperature. Apoptosis was determined using fluorescence microscopy and was quantitated by counting the percentage of stained nuclei.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The current studies were undertaken to determine under what
circumstances CD4+ T cells could process pro-IL-16 and
secrete bioactive IL-16. First, we investigated the effects of
immobilized anti-CD3 Ab and Ag stimulation on the secretion of
IL-16 in human T lymphocytes incubated as a mixed T cell population.
Monocytes represented 5% of the total cell numbers, which was
sufficient to facilitate T cell activation. The cells were stimulated
with either anti-CD3 Ab for 24 h. Cells isolated from normal
individuals were stimulated with anti-CD3 Ab (anti-
, 1
µg/ml). Because pro-IL-16 is recognized by current ELISAs but has no
chemoattractant activity (1, 9), we identified the presence of
bioactive IL-16 by chemotaxis of human T cells in the presence or
absence of IL-16 neutralizing Ab. As shown in Fig. 1
, anti-CD3 induced a significant
increase in the overall T cell chemoattractant activity detected in the
supernatants of mixed T cell cultures by 6 h, which increased
through 12, 18, and 24 h. The contribution of IL-16 to the overall
chemoattractant activity was determined by coincubating the
supernatants with neutralizing anti-IL-16 Ab for 30 min before
assessing cell motility. The IL-16 neutralizing Ab studies indicate
that IL-16 contributed the majority of the chemoattractant activity
that was present at the 6-h timepoint for anti-CD3 stimulation.
IL-16 contributed approximately one-half of the total chemoattractant
activity for anti-CD3 (Fig. 1). Although the total detectable
chemoattractant activity increased from 6 to 24 h, the greatest
contribution of IL-16, calculated as a percentage, was seen in the
earlier timepoints.
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, anti-CD3 stimulated the
release of chemoattractant activity at the 6-h timepoint from both cell
populations. Neutralizing anti-IL-16 Ab had no effect on total
chemoattraction from CD4+ cell supernatants, indicating a
lack of IL-16 contribution. Stimulating CD4+ cells from 6
to 24 h did result in increased chemoattractant activity, which
was partially (35%) inhibited by anti-IL-16 Ab (Fig. 2). Culturing
these cells from 24 to 48 h in fresh media resulted in a further
increase in overall activity, with the contribution of IL-16 increasing
to 
43%. In contrast, the stimulation of CD8+ T cells
for the initial 6 h resulted in supernatants with a
chemoattractant activity of 187% ± 8% of control cell migration,
with all but an insignificant 8% attributed to IL-16 (Fig. 2). When
the cells were washed and recultured from 6 to 24 h, the overall
chemoattractant activity remained approximately the same; however, the
contribution of IL-16 decreased to 40%. The same trend continued for
the 24–48 h cultures, with the contribution of IL-16 decreasing to
15% (Fig. 2). These kinetic studies indicate that CD8+
cells are the primary source of IL-16 immediately following
stimulation, whereas the CD4+ T cells become the primary
source at 24–48 h after stimulation. The different kinetics of IL-16
release by these two T cell subpopulations also argue against the
possibility that the IL-16 bioactivity measured in the
CD4+-enriched T cell supernatant was due to contaminating
CD8+ T cells.
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To determine whether the release of IL-16 was dependent upon the
generation of de novo protein, T cell subsets were treated with the
protein synthesis inhibitor CHX (20 µg/ml; sufficient to inhibit all
[35S]methionine incorporation (data not shown)) for
2 h before stimulation with anti-CD3. At 6 h after
stimulation, there was no significant chemoattractant activity in the
supernatants of CD4+ T cells treated with CHX, indicating
that either the synthesis or secretion of all lymphocyte
chemoattractant activities required new protein synthesis (Fig. 3). Similarly, at 24 h, all of the
anti-CD3 induced chemoattractant activity was inhibited by CHX
treatment. These findings are consistent with our previous reports of a
lack of preformed chemoattractant activity contained in
CD4+ T cell lysates.
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). The addition of anti-IL-16 Ab to
the chemotaxis assay resulted in the loss of all chemoattractant
activity. All of the chemoattractant activity associated with either
unstimulated CD8+ cells or cells stimulated with
anti-CD3 for 6 h is attributable to IL-16, which is consistent
with the presence of preformed, bioactive, IL-16. CHX
pretreatment did reduce the chemoattractant activity released by
stimulated CD8+ T cells at 24 h by 50%; however,
all of the CHX-insensitive chemoattractant activity was inhibited by
anti-IL-16 Abs. The stimulation of CD8+ T cells induces
the release of preformed IL-16 by 6 and 24 h, whereas the
additional chemoattractant activity produced by CD8+ T
cells over 24 h is not IL-16 and requires new protein synthesis.
Chemokines are a likely source (14, 15) for the residual
chemoattractant activity. In that regard, we identified RANTES and
macrophage inflammatory protein (MIP)-1ß as the other two
chemoattractant factors by neutralizing Ab studies. No MIP-1
bioactivity was detected. A combination of anti-RANTES,
anti-IL-16, and anti-MIP-1ß Abs completely neutralized all
chemoattractant activity (data not shown). Costimulation with CD28 increases IL-16 protein production
To assess the effect of CD28 costimulation (16, 17, 18) on IL-16
expression, we assayed supernatants from CD4+ and
CD8+ subsets stimulated with anti-CD3, anti-CD28,
or the combination of anti-CD3 and anti-CD28 for 6 and 24
h. The major difference noted with CD28 costimulation was the
appearance of chemoattractant activity attributable to IL-16 in
CD4+ T cell supernatants at 6 h (Fig. 4A). The majority of the
chemoattractant activity seen at this timepoint was blocked with CHX
treatment (Fig. 4A). There was only a slight increase in the
proportion of IL-16 bioactivity induced by costimulation at the 24
h timepoint (Fig. 4B). As noted above for anti-CD3
stimulation, CHX treatment blocked the synthesis and/or release of
almost all chemoattractant activity in costimulated cells at both
timepoints. The same experiment was performed with purified
CD8+ T cells. In these studies, we did not find any
significant increase in overall chemoattractant activity or in the
proportion of IL-16 chemoattractant activity at 6 h after CD28
costimulation when compared with anti-CD3 alone (Fig. 4C). Again, treatment with CHX had no effect on the
appearance of chemoattractant activity secreted over 6 h. However,
CHX did reduce the overall chemoattractant activity at 24 h for
both anti-CD3 alone and costimulated supernatants (Fig. 4D). All of the CHX-insensitive chemoattractant activity
appeared to be attributable to IL-16, as the addition of anti-IL-16
Ab blocked all residual activity.
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To directly investigate whether anti-CD3 stimulation could
modulate IL-16 mRNA, human CD4+ T cells were
stimulated with immobilized anti-CD3 for 6 or 24 h before
isolation of total RNA and Northern blot analysis. IL-16 mRNA was
constitutively expressed and did not appear to increase after 6 or
24 h of anti-CD3 stimulation (Fig. 5). Furthermore, no change in mRNA
following anti-CD3 activation could be identified in
CD8+ subsets (data not shown). We subsequently examined the
effect of costimulation on IL-16 mRNA expression in CD4+ T
cells. CD3/CD28 costimulation had no significant effect on the levels
of IL-16 mRNA at any timepoint up to 6 h of stimulation (Fig. 6). Finally, there was no detectable
effect of CD3/CD28 costimulation on the stability of IL-16 mRNA (Fig. 7). In these experiments,
CD4+ T cells were incubated in the presence or absence of
anti-CD3/anti-CD28 for 24 h, followed by the addition of
actinomycin D (10 µg/ml). In both conditions, IL-16 mRNA was
detectable at 1 h, decreased at 2 h, and was not detectable
by 3 h, indicating no change in mRNA stability. Taken together,
these experiments suggest that IL-16 secretion in CD4 cells must be
regulated at some critical step after transcription and translation. We
hypothesized that this critical step might lie with the
pro-IL-16-processing enzyme, caspase-3.
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We first investigated whether stimulated CD4+ T
lymphocytes contain active caspase-3 and then determined the time
course of processing of pro-caspase-3 following anti-CD3 or
anti-CD3/anti-CD28 costimulation. In these experiments,
purified CD4+ lymphocytes were stimulated for 6 and 24
h with anti-CD3 or anti-CD3/CD28 costimulation. Cell lysates at
these two timepoints were subjected to SDS-PAGE followed by caspase-3
Western blot analysis. As seen in Fig. 8,
no active caspase-3 is observed in unstimulated CD4+ T
cells. At 4 h, a timepoint when bioactive IL-16 is not
observed in supernatants from anti-CD3-activated CD4+ T
cells, there is only a faint (20-kDa) detectable band correlating to
active caspase-3. However, at this timepoint, a 20-kDa band of
10-fold the intensity (lane 3) compared with
anti-CD3 stimulated alone (lane 2) is observed
with anti-CD3/CD28 costimulation. By 24 h, cells stimulated
with anti-CD3 alone or costimulated with CD3/CD28 all express
similar levels of active caspase-3 (Fig. 8). These data suggest that
caspase-3 is activated after the stimulation of CD4+ T
cells with anti-CD3 or anti-CD3/CD28 in a time course
compatible with the appearance of the bioactive IL-16 detected in the
cell supernatants.
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). Similar effects were observed for
stimulation by anti-CD3 alone (data not shown). The involvement of
caspase-3 appears to be specific, as the inhibition of caspase-1 by the
peptide Ac-YVAD-aldehyde did not alter the magnitude or the time course
required for expression of bioactive IL-16. It is interesting to
note that treatment with the caspase-3 inhibitor did not appear to
affect non-IL-16-induced migration for either CD3 alone or for
CD3/CD28-costimulated cells, suggesting that IL-16 is the only
detectable chemoattractant processed by caspase-3. To confirm the
bioactivity of the caspase-1 and -3 inhibitors, their effect on
CD3-stimulated apoptosis was assessed at the 24-h timepoint by
propidium iodide staining. Based on apoptotic staining, cells treated
with the inhibitors demonstrated 23% and 18% apoptotic staining for
caspase-1 and -3 inhibitors, respectively, compared with 57% apoptotic
staining for untreated cells. This represents a 60.7% and 69.5%
decrease in apoptotic cells for the caspase-1 and -3 inhibitors,
respectively, compared with untreated cells. Therefore, the data
indicate that the caspase-1 inhibitor was functionally active in
reducing apoptosis but had no effect on IL-16 release. These findings
suggest that the release of bioactive IL-16 is coincident with and
dependent upon activation of the enzyme that facilitates its cleavage,
caspase-3, and not on transcriptional regulation of the IL-16 mRNA.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Of interest, in contrast to CD8+ T cells, IL-16 does not
represent a majority of the lymphocyte chemoattractant activity
identified in CD4+ T cell supernatants following CD3 or
CD3/CD28 stimulation. However, the contribution of IL-16 increases
proportionally to 40% of the overall chemoattractant activity
during the first 48 h poststimulation. All other chemoattractant
activities did not appear following CHX treatment, suggesting new
protein synthesis was required. In that regard, members of the CC
chemokine family are likely candidates for this activity.
The requirement of caspase activation for protein processing and
secretion has been reported previously for both IL-1ß (20) and IL-18
(21). Both of these ILs require activation of caspase-1. The efficiency
for IL-1 and -1ß processing and release is increased with induced
apoptosis but not with necrosis (22). The mechanism for the release of
IL-16 has not been determined; however, because IL-16 also lacks a
signal peptide, there may be some similarities with IL-1 and -1ß.
Thus far, our data suggest that apoptosis is not required for the
processing and release of IL-16 as the prevention of apoptosis with a
caspase-1 inhibitor also did not effect the magnitude or the time
course for the release of IL-16. However, we have noted that induction
of apoptosis with staurosporin does result in an enhanced release of
bioactive IL-16 (Y.Z., unpublished observations). These findings
indicate that the processing and release of IL-16 by CD4+ T
cells is not contingent upon subsequent cell apoptosis, and further
supports the concept that, although activation of caspase-3 enzymatic
activity is essential for an apoptotic signal (23, 24), activation of
caspase-3 alone is not sufficient for the induction of apoptosis (25).
Based on the kinetics data, one could hypothesize that CD8 cells would represent a rapid release mechanism initiated by the mediators of early inflammation, such as vasoactive amines, and would contribute to the recruitment of CD4+ T cells by the secretion of preformed IL-16. The ability of CD4+ T cells to synthesize and secrete IL-16 may serve to function as a positive feedback mechanism for further cell recruitment and activation. A similar situation may occur with another proinflammatory cell, the eosinophil, which has been shown to generate and release IL-16 protein after activation (11).
In summary, we have shown that CD4+ T cells are a source of IL-16 following TCR activation or CD28 costimulatory conditions. The active form of caspase-3, an important mechanism for further processing of pro-IL-16 and the secretion of bioactive IL-16, can be identified in activated CD4+ T cells in a time course that is similar to that seen for IL-16 release. However, an apparent separation does exist between the release of IL-16 following caspase-3 activation, pro-IL-16 processing, and the induction of apoptosis. In addition, our data demonstrate that the mechanisms and requirements for the synthesis and release of IL-16 from CD4+ and CD8+ T cells are entirely different. Taken together, these studies suggest that Ag-activated CD4+ T cells could be a source of IL-16, which might amplify the accumulation of unsensitized CD4+ T cells at sites of Ag-induced inflammation.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. William Cruikshank, Pulmonary Center, Housman R-304, Boston University School of Medicine, Boston, MA 02118. E-mail address:
3 Abbreviations used in this paper: CHX, cycloheximide; NWNA-T, nylon wool nonadherent human T cell; MIP, macrophage inflammatory protein.
Received for publication July 16, 1998. Accepted for publication October 9, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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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D. Sciaky, W. Brazer, D. M. Center, W. W. Cruikshank, and T. J. Smith Cultured Human Fibroblasts Express Constitutive IL-16 mRNA: Cytokine Induction of Active IL-16 Protein Synthesis Through a Caspase-3-Dependent Mechanism J. Immunol., April 1, 2000; 164(7): 3806 - 3814. [Abstract] [Full Text] [PDF]
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