CCR5 and CXC chemokine receptor 4 (CXCR4) are coreceptors for CD4 as defined by HIV-1 glycoprotein (gp) 120 binding. Pretreatment of T cells with gp120 results in modulation of both CCR5 and CXCR4 responsiveness, which is dependent upon p56lck enzymatic activity. The recent findings that pretreatment of T cells with a natural CD4 ligand, IL-16, could alter cellular responsiveness to macrophage-inflammatory protein-1β (MIP-1β) stimulation, prompted us to investigate whether IL-16 could also alter CXCR4 signaling. These studies demonstrate that IL-16/CD4 signaling in T lymphocytes also results in loss of stromal derived factor-1α (SDF-1α)/CXCR4-induced chemotaxis; however, unlike MIP-1β/CCR5, the effects were not reciprocal. There was no effect on eotaxin/CCR3-induced chemotaxis. Desensitization of CXCR4 by IL-16 required at least 10–15 min pretreatment; no modulation of CXCR4 expression was observed, nor was SDF-1α binding altered. Using murine T cell hybridomas transfected to express native or mutated forms of CD4, it was determined that IL-16/CD4 induces a p56lck-dependent inhibitory signal for CXCR4, which is independent of its tyrosine catalytic activity. By contrast, IL-16/CD4 desensitization of MIP-1β/CCR5 responses requires p56lck enzymatic activity. IL-16/CD4 inhibition of SDF-1α/CXCR4 signals requires the presence of the Src homology 3 domain of p56lck and most likely involves activation of phosphatidylinositol-3 kinase. These studies indicate the mechanism of CXCR4 receptor desensitization induced by a natural ligand for CD4, IL-16, is distinct from the inhibitory effects induced by either gp120 or IL-16 on CCR5.

A coreceptor relationship has been established between CD4 and certain chemokine receptors such as CCR5 and CXC chemokine receptor 4 (CXCR4)4 (1, 2, 3, 4). Association between these receptors was first identified in the context of HIV-1 binding and internalization, which is mediated through its envelope glycoprotein, gp120 (5, 6, 7, 8). This coreceptor relationship was initially thought to exist only following the involvement of HIV binding. In fact, investigation of this relationship has led to the identification that signaling induced by gp120, thought to be facilitated solely by CD4 (6), may actually be transduced, in part, by the CD4-associated chemokine receptors, CCR5 (9) or CXCR4 (10). A direct physical association between CD4 and the chemokine receptors may not, however, require gp120 binding, as Xiao et al. (11) have recently reported that there is constitutive cell surface association between CD4 and CCR5 in unstimulated human T cells. Interestingly, this constitutive relationship does not appear to exist for CD4 and CXCR4, however, can be induced by the addition of gp120 (11). These data suggest that the relationship between CD4 and CCR5 is mechanistically different from that for CD4 and CXCR4, despite some functional similarities.

Functionally, binding of gp120 to CD4 results in subsequent unresponsiveness to either macrophage-inflammatory protein-1β (MIP-1β) (via CCR5) (12) or stromal derived factor-1α (SDF-1α) (via CXCR4) chemokine stimulation (13). The mechanism for this effect appears to be as a result of gp120-induced down-modulation of surface-expressed CCR5 or CXCR4 proteins. Loss of chemokine receptor expression and function following gp120 stimulation is dependent on phosphorylation of the CD4-associated tyrosine kinase, p56lck (13).

For many biological systems, the effects of gp120 stimulation mediated through CD4 (8, 14, 15, 16, 17) are similar to the effects induced following binding of a natural ligand for CD4, IL-16 (18, 19). A recent report has suggested that murine monocytes have an alternative receptor for IL-16 (20), and therefore, we have concentrated our current work on the interplay between IL-16/CD4 and chemokine receptors specifically in T cells. Along those lines, we have recently demonstrated that IL-16 has the selective ability to induce T cell unresponsiveness to MIP-1β/CCR5 stimulation; however, unlike gp120, does not affect signaling induced by monocyte chemoattractant proteins (MCP) 1 (via CCR2), 2 (via CCR1, 2, 3, and 5), and 4 (via CCR2 and 3) (21). This effect of IL-16 is not mediated by loss of CCR5 membrane expression, nor by steric inhibition of MIP-1β binding, but is mediated through the induction of intracellular signals that block CCR5 signaling. Similar to gp120, the inhibitory activity of IL-16/CD4 on CCR5 also requires enzymatic activity of p56lck (21). Interestingly, the inhibition is reciprocal, as pretreatment with MIP-1β results in loss of responsiveness to IL-16 stimulation (21).

In the present studies, we investigated whether IL-16 pretreatment could also induce unresponsiveness to the other major chemokine receptor utilized by HIV-1 binding, CXCR4 (22). Gp120 pretreatment, similar to its effects on CCR5, results in p56lck enzymatic activity-dependent modulation of CXCR4 associated with subsequent T cell unresponsiveness to SDF-1α stimulation (13). Our results indicate that IL-16 pretreatment does result in a transient unresponsiveness to CXCR4/SDF-1α stimulation; however, unlike CCR5, the effect is not reciprocal. In addition, we demonstrate that the presence and enzymatic activity of the SH1 domain of p56lck are not required, but that the inhibitory signal mediated through p56lck is transduced by the Src homology (SH) 3 domain and most likely involves activation of phosphatidylinositol-3 (PI3) kinase. These studies identify a natural functional relationship between CD4 and CXCR4, initiated by a natural ligand for CD4, which is mechanistically different from the CD4/CCR5 relationship.

Recombinant human and murine SDF-1α, IL-16, and eotaxin were purchased from Biosource International (Camarillo, CA). Fluorescein-conjugated anti-CXCR4 Ab was purchased from PharMingen (San Diego, CA). Unconjugated anti-IL-16 Ab (clone 14.1) was isolated from hybridoma supernatants, purified using protein A affinity chromatography, and used at a concentration of 5 μg/ml, which is sufficient to neutralize 10−10 M rIL-16-induced migration of human T cells (23). HIV-1 gp120IIIB was purchased from Intracel (Issaquah, WA).

Normal human T lymphocytes were isolated from the blood of healthy volunteers using Hypaque-Ficoll separation of PBMCs, as previously described (19, 20). Preparations were enriched for T lymphocytes by nylon wool adherence. The nonadherent mononuclear cells were >95% T lymphocytes, as determined using flow cytometry to assess CD3+ cells. Enriched CD4+ T cells were generated as previously described (24). Briefly, the cells were then mixed with magnetic beads conjugated with anti-CD8 mAbs. Following incubation with the beads for 1 h at 37°C on a shaker, the CD4+ T cells were then isolated by negative selection using a strong magnet. The cells were cultured in medium 199 supplemented with 25 mM HEPES buffer, 100 U/ml penicillin, 100 μg/ml streptomycin, and containing 10% FBS for 18–24 h before use in the chemotaxis assay.

For the studies using herbimycin A, 2 × 106 cells/ml were incubated with herbimycin A (1 μM final concentration; Calbiochem, San Diego, CA) for 18 h before washing by centrifugation and resuspension with culture media. Following washing, rIL-16 (10−10 M) was added to the cells for 15 min, and the cells were washed again and resuspended at 8–10 × 106 cells/ml for use in the chemotaxis assay.

For the PI3 kinase inhibition experiments, cells were incubated with either wortmannin (1 or 10 nM) for 18–24 h, followed by a 1-h incubation period with IL-16, Ly294002 (10 or 40 μM; Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h, with IL-16 added for the last hour of pretreatment. After washing, the cells were then subjected to an SDF-1α dose response for induction of cell migration.

All CD4-expressing murine T cell hybridomas were generated as previously described (25, 26, 27) and were a generous gift from Steven J. Burakoff (Dana-Farber Cancer Institute, Boston, MA). To generate these cells, the L3T4-negative murine T cell hybridoma cell line By155.16 was infected with the mononuclear cell retroviral vector containing a neomycin resistance gene, a CMV promoter, and the gene for human CD4. Various constructs of CD4 were used to express either wild-type CD4 or point mutations corresponding to a Cys420 to Ser mutation, or a Cys430 to Ser mutation (25, 26). The mononuclear cell-CD4 transfectants were selected and assessed for CD4 surface expression. All cell lines were assessed for CD4 expression, as determined by anti-CD4 Ab binding and FACS analysis, and only those cell lines with comparable CD4 levels were used. In some studies, cells were infected to express a CD4/p56lck chimeric protein. Chimeric molecules containing the extracellular and transmembrane domains of CD4 directly ligated to different constructs of p56lck were expressed in the By155.16 cell line (27, 28) as either an intact p56lck molecule (full length); a truncated molecule containing only the N, SH2, and SH3 domains (N32); or a molecule comprised of only the N and SH3 domains (N3). Neomycin-resistant clones were screened for surface expression of CD4 by flow cytometric analysis using a FACScan (Becton Dickinson, Mountain View, CA), and only cells expressing comparable levels of CD4 were used. All cells were grown and maintained in RPMI 1640 medium (Sigma, St. Louis, MO) containing 200 U/ml penicillin, 200 μg/ml streptomycin, 2 mM glutamine, 20 mM HEPES (pH 7.4), and 10% FBS.

In vitro chemotaxis was performed as described previously (18, 29). Isolated CD4+ T cells were pretreated with rIL-16 (10−10 M) or control media for 0–1 h, depending on the experiment, at 37°C, then washed by centrifugation and resuspension in media twice before use in the chemotaxis assay. In the specificity experiments, the T cells were incubated under the same conditions with rIL-16 (10−10 M) and 10 μg/ml IL-16 mAb (14.1), an amount sufficient to neutralize 10−10 M rIL-16 activity (23). Migration was assessed using a 48-well microchemotaxis chamber with 8-μm nitrocellulose membrane filters used as the migration matrix (Neuroprobe, Cabin John, MD). Human CD4+ T lymphocytes (8–10 × 106/ml) or T cell hybridomas (5 × 106/ml), either with or without pretreatment with IL-16, were loaded into the upper well of the chamber, with 32 μl of various concentrations of chemoattractant or control buffer placed in the lower well. Chambers were incubated for 2 h for the human cells or 4 h for the T cell hybridomas (27), after which the filters were fixed in ethanol, stained with hematoxylin, and dehydrated by sequential washes in ethanol, propanol, and xylene. Cell migration was quantitated using light microscopy to visualize the number of cells that had migrated beyond a depth of 50 μm. All migration is expressed as percentage values of cell migration in control media (designated as 100%), and statistics were calculated using Student’s t test. As IL-16 is a chemoattractant factor, baseline migration was established as the migration of cells pretreated with IL-16, washed, and then stimulated with media alone. All samples were tested in duplicate, and four high-power fields were examined in each duplicate. Data are the mean value ± the SD of three or more experiments.

Surface expression of chemokine receptor was accomplished using a previously reported method (21). Briefly, CXCR4 expression was analyzed using fluorescein-conjugated anti-CXCR4 Abs (clone C-20; Santa Cruz Biotechnology). Human T cells (1 × 106 cells/ml) were either left untreated or pretreated with IL-16 (10−10 M) or with gp120IIIB (5 μg/ml) for 1, 4, 12, or 24 h, washed, and resuspended in staining buffer (PBS with 0.4% BSA and 0.1% sodium azide), and incubated with 0.25 μg of labeled Ab for 30 min at 4°C. Cells were then washed three times in cold PBS, resuspended and fixed with 10% Formalin, and analyzed with a FACScan (Becton Dickinson).

Binding assays were conducted, as previously described (13), by stimulating 2 × 106 human T cells in 200 μl of culture media with either rIL-16 (10–1000 pg) or cold SDF-1α (10–1000 pg) for 30 min at room temperature before the addition of labeled SDF-1α. Radiolabeled 125I-SDF-1α (0.12 nM, 185 kBq; New England Nuclear, Boston, MA) was added to each sample for 120 min at room temperature. Following a 120-min incubation, the samples were quickly aspirated through GF/C microfiber filters (Whatman, Maidstone, U.K.) using a vacuum harvester. The filters were air dried and counted in a gamma counter. Nonspecific radioactivity present with 10–1000-fold excess of cold SDF-1α was subtracted from total bound counts for each dose and used to calculate specifically bound counts. Percent inhibition of SDF-1α binding induced by IL-16 or cold SDF-1α was calculated by subtracting counts from IL-16- or SDF-1α-treated cells from specific counts, divided by specific binding counts, and multiplied by 100%.

Recent studies have identified that IL-16 pretreatment results in selective desensitization of MIP-1β/CCR5-induced chemotaxis (21). We continued these observations by investigating whether IL-16 stimulation could also desensitize another CD4-related chemokine receptor, CXCR4. Human CD4+ T cells were isolated and then incubated with IL-16 (10−10 M) for 1 h before washing and stimulation by the CXCR4-specific ligand SDF-1α. As shown in Fig. 1,A, SDF-1α induced a migratory response in the T cells in a dose-dependent fashion. Cells prestimulated with IL-16, however, did not respond to any concentration of SDF-1α (Fig. 1,A). This effect was selective, as previous studies have identified no IL-16 effect on MCP-1-, MCP-2-, and MCP-4 (21)-induced migration, and for these studies we also demonstrate that migration to eotaxin, mediated through CCR3 was not altered (Fig. 1 A). Coincubation of IL-16 with neutralizing concentrations of anti-IL-16 before pretreatment of the T cells completely eliminated the inhibitory effect of IL-16 on SDF-1α stimulation (data not shown).

FIGURE 1.

Effect of IL-16 pretreatment on chemokine-induced migration. A, Isolated human CD4+ T cells were incubated with IL-16 (10−10 M) for 1 h before induction of chemotaxis by various concentrations of SDF-1α or eotaxin. A baseline migratory response was established by cells pretreated with IL-16 with no further stimulation by any chemokine. The data are expressed as percentage of migration beyond baseline migration, which has been normalized to 100%. Migration by cells exposed to IL-16 is shown in the closed squares, while migration by cells not exposed to IL-16 is shown in the open diamonds. B, The migratory response of human CD4+ T cells stimulated with 100 ng/ml SDF-1α for 1 h before washing and assessment for IL-16-induced migration. The data represent the averages of four separate experiments. The asterisk denotes significantly different migration in the treated cells as compared with migration seen in the untreated cells, p < 0.05.

FIGURE 1.

Effect of IL-16 pretreatment on chemokine-induced migration. A, Isolated human CD4+ T cells were incubated with IL-16 (10−10 M) for 1 h before induction of chemotaxis by various concentrations of SDF-1α or eotaxin. A baseline migratory response was established by cells pretreated with IL-16 with no further stimulation by any chemokine. The data are expressed as percentage of migration beyond baseline migration, which has been normalized to 100%. Migration by cells exposed to IL-16 is shown in the closed squares, while migration by cells not exposed to IL-16 is shown in the open diamonds. B, The migratory response of human CD4+ T cells stimulated with 100 ng/ml SDF-1α for 1 h before washing and assessment for IL-16-induced migration. The data represent the averages of four separate experiments. The asterisk denotes significantly different migration in the treated cells as compared with migration seen in the untreated cells, p < 0.05.

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To determine whether the receptor cross-desensitization was reciprocal, as has been demonstrated for CCR5 signaling (21), cells were pretreated with SDF-1α for 1 h before assessing for IL-16-induced chemotaxis. Concentrations of SDF-1α used for pretreatment ranged from 1 to 100 ng/ml; however, none of the concentrations used were found to have any effect on IL-16 stimulation (Fig. 1 B, only the 100 ng/ml concentration of SDF-1α is shown).

A time course was then conducted to establish the minimal amount of time required for IL-16/CD4 stimulation to cross-desensitize CXCR4-induced migration. Human CD4+ T cells were incubated with IL-16 (10−10 M) for a variety of time points up to 1 h. The cells were then washed and assessed for SDF-1α-induced migration. As shown in Fig. 2,A, receptor cross-desensitization induced by IL-16 required between 10 and 15 min of IL-16 stimulation, and was maximal by 60 min. To determine whether the inhibitory effect was reversible, cells were incubated with IL-16 for 1 h, washed three times in media, and then incubated in culture media alone for varying lengths of time up to 48 h before subjected to SDF-1α-induced chemotaxis. As shown in Fig. 2 B, the cells required 18–24 h of incubation before they demonstrated a normal dose response to SDF-1α stimulation, indicating a transient effect by IL-16.

FIGURE 2.

A time course for the IL-16-inhibitory effect. A, The migratory response of human CD4+ T cells incubated with IL-16 (10−10 M) for the times noted before washing and induced migration by SDF-1α (10 ng/ml). B, The migratory response of T cells incubated with IL-16 for 1 h, washed, and then incubated for various time points before SDF-1α-induced migration. These experiments were conducted four separate times, and the data represent the averages for those experiments. The asterisk denotes migration that was statistically different in cells that were stimulated by IL-16 as compared with cells not stimulated with IL-16, p < 0.05.

FIGURE 2.

A time course for the IL-16-inhibitory effect. A, The migratory response of human CD4+ T cells incubated with IL-16 (10−10 M) for the times noted before washing and induced migration by SDF-1α (10 ng/ml). B, The migratory response of T cells incubated with IL-16 for 1 h, washed, and then incubated for various time points before SDF-1α-induced migration. These experiments were conducted four separate times, and the data represent the averages for those experiments. The asterisk denotes migration that was statistically different in cells that were stimulated by IL-16 as compared with cells not stimulated with IL-16, p < 0.05.

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Gp120-induced desensitization of CXCR4 signaling results from the loss of CXCR4 from the cell surface (13), most likely due to the induction of phosphorylation. To determine whether the effects of IL-16 were mechanistically similar to gp120, surface expression of CXCR4 was monitored following IL-16 stimulation. Human CD4+ T cells were incubated with IL-16 (10−10 M) for 1 h before staining with FITC-conjugated anti-CXCR4 Abs and FACS analysis. As shown in Fig. 3,A, CXCR4 expression was not altered by IL-16 stimulation. CXCR4 expression was monitored for up to 24 h following IL-16 stimulation, with no observable changes (data not shown). Similarly, there was no detectable induction of CXCR4 phosphorylation at either 1 or 24 h following IL-16 stimulation (data not shown). The cells were responsive to gp120 stimulation, however, as CXCR4 expression was significantly reduced following exposure to 5 μg/ml gp120 for 1 h (Fig. 3 A).

FIGURE 3.

Effect of IL-16 stimulation on CXCR4 expression and SDF-1α binding. A total of 1 × 106 human CD4+ T cells was stimulated with IL-16 (10−10 M) for 24 h. A, The surface expression of CXCR4, as determined by anti-CXCR4 Ab binding and FACS analysis, of unstimulated cells (upper panel), cells stimulated with IL-16 (middle panel), or cells stimulated with gp120 (lower panel). These FACS analyses are representative of four separate experiments with similar findings. B, The effect of IL-16 stimulation on SDF-1α binding. A total of 3 × 106 human CD4+ T cells was incubated with either cold SDF-1α (□) or IL-16 (⋄) for 30 min before the addition of radiolabeled SDF-1α. The doses of cold SDF-1α and IL-16 effected up to a 1000-fold excess as compared with the amount of labeled SDF-1α. Cells were incubated for 120 min before filtration through microfiber filters and assessment of binding using a gamma counter. Percent inhibition was calculated by subtracting counts from IL-16-treated cells from specifically bound counts, divided by specifically bound counts, and multiplied by 100%. This experiment was conducted three separate times, each with similar findings.

FIGURE 3.

Effect of IL-16 stimulation on CXCR4 expression and SDF-1α binding. A total of 1 × 106 human CD4+ T cells was stimulated with IL-16 (10−10 M) for 24 h. A, The surface expression of CXCR4, as determined by anti-CXCR4 Ab binding and FACS analysis, of unstimulated cells (upper panel), cells stimulated with IL-16 (middle panel), or cells stimulated with gp120 (lower panel). These FACS analyses are representative of four separate experiments with similar findings. B, The effect of IL-16 stimulation on SDF-1α binding. A total of 3 × 106 human CD4+ T cells was incubated with either cold SDF-1α (□) or IL-16 (⋄) for 30 min before the addition of radiolabeled SDF-1α. The doses of cold SDF-1α and IL-16 effected up to a 1000-fold excess as compared with the amount of labeled SDF-1α. Cells were incubated for 120 min before filtration through microfiber filters and assessment of binding using a gamma counter. Percent inhibition was calculated by subtracting counts from IL-16-treated cells from specifically bound counts, divided by specifically bound counts, and multiplied by 100%. This experiment was conducted three separate times, each with similar findings.

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The data to date do not exclude the possibility that IL-16 bound to CD4 might sterically inhibit SDF-1α binding, and/or that IL-16/CD4 signaling results in a conformational change in CXCR4, which then reduces the avidity for SDF-1α binding. To address these possibilities, binding studies were conducted with radiolabeled SDF-1α in the presence of cold SDF-1α or IL-16. Human CD4+ T cells were incubated with an increasing concentration of either SDF-1α or IL-16 for 1 h. Without washing, radiolabeled SDF-1α (0.1 nM) was then added for an additional hour, and bound counts were analyzed. As shown in Fig. 3 B, increasing concentrations of cold SDF-1α inhibited bound counts by up to 80%, while IL-16, for all concentrations used, was unable to alter SDF-1α binding. Taken together, these data indicate that IL-16 stimulation results in loss of CXCR4 responsiveness, but that neither CXCR4 expression nor the ability of SDF-1α to bind to CXCR4 is altered by the IL-16/CD4 interaction. In addition, these studies indicate that while both gp120 and IL-16 bind to CD4 and are capable of inhibiting SDF-1α/CXCR4 signaling, the mechanism for inhibition is different for the two CD4 ligands.

The findings that IL-16 stimulation does not result in loss of surface-expressed CXCR4 protein nor alters SDF-1α binding suggest that IL-16/CD4 association generates a signal that results in the inhibition of CXCR4-induced chemotaxis. To further define the mechanism by which IL-16 inhibits CXCR4 signaling, murine T cell hybridomas were retrovirally infected to express point-mutated human CD4 molecules. As previously reported (25, 26), L3T4-negative T cells were infected to express either wild-type CD4; a Cys420 to Ser (CS420); or Cys430 to Ser (CS430) point-mutated CD4 molecules. Cysteine at position 420 is essential for a CD4/p56lck association, and, therefore, mutation of Cys420 to Ser420 results in the disruption of this association (25, 26). Cysteine at position 430 is outside of the binding region, and therefore, the CS430 point mutation does not alter this association. We have previously used these cells to demonstrate that a CD4/p56lck association is required to confer IL-16-induced cell migration and second messenger signaling (27). To determine the effects of IL-16 on CXCR4-induced migration, it was first established that murine SDF-1α could induce a migratory response in these cell lines similar to what was observed for human CD4+ T cells. As shown in Fig. 4, SDF-1α induced a migratory response in a dose-dependent fashion. Cells expressing wild-type CD4, CS420, or CS430 were then exposed to IL-16 for 1 h and then stimulated with SDF-1α. IL-16, as anticipated, blocked SDF-1α-induced migration in cells expressing the wild-type or CS430 CD4 molecules; however, there was no IL-16 effect in the CS420-expressing cells (Fig. 4). This finding was consistent with the hypothesis that an IL-16/CD4-generated signal was inducing the observed inhibition, and, furthermore, that a CD4/p56lck association was required to transmit the inhibitory signal.

FIGURE 4.

Effects of IL-16 pretreatment on murine T cell hybridomas. Murine T cell hybridomas expressing either wild-type human CD4 (wild type), a cysteine to serine point mutation at position 430 (CS430), or a similar point mutation at position 420 (CS420) were subjected to murine SDF-1α-induced migration either with (⋄) or without (▪) a 1-h pretreatment with IL-16 (10−10 M). The data are expressed as percent control migration, in which control migration is determined by the migration of unstimulated cells for each CD4 construct. The asterisk denotes significantly different migration in the treated cells as compared with migration seen in the untreated cells, p < 0.05. The data represent the averages of five separate experiments.

FIGURE 4.

Effects of IL-16 pretreatment on murine T cell hybridomas. Murine T cell hybridomas expressing either wild-type human CD4 (wild type), a cysteine to serine point mutation at position 430 (CS430), or a similar point mutation at position 420 (CS420) were subjected to murine SDF-1α-induced migration either with (⋄) or without (▪) a 1-h pretreatment with IL-16 (10−10 M). The data are expressed as percent control migration, in which control migration is determined by the migration of unstimulated cells for each CD4 construct. The asterisk denotes significantly different migration in the treated cells as compared with migration seen in the untreated cells, p < 0.05. The data represent the averages of five separate experiments.

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We have previously reported that IL-16-initiated inhibition of CCR5 signaling requires enzymatic activity of p56lck (21). To determine whether the inhibitory effects of IL-16 on CXCR4 signaling were also dependent on p56lck enzymatic activity, we incubated isolated human T cells with the specific src tyrosine kinase inhibitor herbimycin A. These studies are feasible because cell migration induced directly by SDF-1α/CXCR4 is not herbimycin A sensitive (data not shown). Human CD4+ T cells were treated with herbimycin A (1 μM) for 1 h before the addition of IL-16 for an additional hour. The cells were then assessed for SDF-1α-induced cell migration. Unlike CCR5 inhibition, herbimycin A treatment did not reverse the desensitization effect of IL-16 (Fig. 5,A). To demonstrate that herbimycin A treatment was sufficient to eliminate enzymatic activity of p56lck, herbimycin A-pretreated cells were also pretreated with gp120 before SDF-1α stimulation. Under these conditions, herbimycin A completely ablated the gp120-induced inhibitory effect compared with cells treated with herbimycin alone (Fig. 5,B). We next assessed whether herbimycin A treatment had an effect on gp120-induced modulation of CXCR4 expression. Flow cytometric analysis of cell surface expression of CXCR4 indicated that herbimycin A treatment did prevent gp120-induced loss of CXCR4 (Fig. 5 C). Therefore, unlike the effects of gp120, p56lck enzymatic activity does not appear to be essential for transmission of the IL-16/CD4-mediated inhibitory signal.

FIGURE 5.

Effect of herbimycin A on IL-16 and gp120 pretreatment of human T cells. A, Human CD4+ T cells stimulated for a migratory response with SDF-1α alone (first group of bars), or following pretreatment with IL-16 (10−10 M for 1 h) (second set of bars), or pretreated with a combination of IL-16 (1 h) and herbimycin A (2 μM for 18 h) (last set of bars). B, The same experimental protocol as shown in A; however, gp120 (5 μM for 1 h) was used for pretreatment. These experiments were conducted four separate times, and the data represent the averages of those experiments. The asterisk denotes significantly different migration in the treated cells as compared with migration seen in the cells stimulated with SDF-1α alone, p < 0.05. C, The surface expression of CXCR4 as determined by anti-CXCR4 Ab binding and FACS analysis of unstimulated cells (upper panel); cells stimulated with gp120 (5 μM for 4 h) (middle panel); or cells pretreated with herbimycin A for 18 h before gp120 stimulation for 4 h (lower panel). This is a representative analysis of three separate experiments.

FIGURE 5.

Effect of herbimycin A on IL-16 and gp120 pretreatment of human T cells. A, Human CD4+ T cells stimulated for a migratory response with SDF-1α alone (first group of bars), or following pretreatment with IL-16 (10−10 M for 1 h) (second set of bars), or pretreated with a combination of IL-16 (1 h) and herbimycin A (2 μM for 18 h) (last set of bars). B, The same experimental protocol as shown in A; however, gp120 (5 μM for 1 h) was used for pretreatment. These experiments were conducted four separate times, and the data represent the averages of those experiments. The asterisk denotes significantly different migration in the treated cells as compared with migration seen in the cells stimulated with SDF-1α alone, p < 0.05. C, The surface expression of CXCR4 as determined by anti-CXCR4 Ab binding and FACS analysis of unstimulated cells (upper panel); cells stimulated with gp120 (5 μM for 4 h) (middle panel); or cells pretreated with herbimycin A for 18 h before gp120 stimulation for 4 h (lower panel). This is a representative analysis of three separate experiments.

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In an attempt to confirm these findings and further delineate the mechanism for transmission of the inhibitory signal, murine hybridoma T cells were infected with several different CD4/p56lck chimeric constructs. These cells, as previously described (27, 28), express chimeric constructs of wild-type CD4 with either wild-type p56lck or deletional mutations of p56lck synthesized on the C-terminal end of CD4. The constructs include CD4 chimerized to wild-type p56lck (full length); CD4 chimerized to p56lck containing the N, SH3, and SH2 domains, however, lacking the enzymatic SH1 domain (N32); CD4 chimerized to p56lck containing only the N and SH3 domains (N3); and cells expressing only the N construct (N). We have previously used all of these cell lines to identify p56lck components essential to transduce an IL-16/CD4 migratory signal (T. Ryan, unpublished observations). For these studies, an initial dose-response curve demonstrated that each of these cell types were responsive to either murine SDF-1α (Fig. 6) or eotaxin (data not shown) stimulation. The same dose response was repeated following a 1-h incubation in the presence of IL-16 (10−10 M). Similar to our findings with primary T cells, hybridoma cells expressing the wild-type p56lck construct (full length), exposed to IL-16, were subsequently unresponsive to SDF-1α stimulation (Fig. 6). Also similar to the data generated with primary T cells, cellular response to eotaxin stimulation was unaffected by IL-16 pretreatment (data not shown). Cells expressing the SH1 deletional mutation (N32) also demonstrated an IL-16-induced inhibitory effect. This finding is consistent with the data generated on human T cells following incubation with herbimycin A (Fig. 5). Interestingly, cells expressing only the SH3 domain (N3) also demonstrated inhibitory activity following IL-16 pretreatment (Fig. 6). This indicates that the SH3 domain is at least in part capable of transmitting the inhibitory signal. These data do not exclude the potential contribution by other domains, such as SH2. The N domain is not involved in transmitting the inhibitory effect of IL-16, as cells expressing only the N domain are unresponsive to IL-16 pretreatment (Fig. 6).

FIGURE 6.

Effects of IL-16 pretreatment on the migratory response of CD4/p56lck chimeric T cell hybridomas. L3T4 murine T cell hybridoma cells expressing human CD4 chimerized either to a full-length p56lck chimeric molecule (full length); to a p56lck molecule lacking the SH1 domain (N32); to a p56lck molecule lacking the SH1 and SH2 domains (N3); or to a p56lck molecule containing only the N domain (N) were used in the migration assay. These cell lines were stimulated in a dose-dependent fashion with murine SDF-1α either directly (▪) or following a 1-h pretreatment with IL-16 (10−10 M) (⋄). The data are expressed as the averages of four separate experiments. The asterisk denotes significantly different migration in the cells pretreated with IL-16 as compared with untreated cells, p < 0.05.

FIGURE 6.

Effects of IL-16 pretreatment on the migratory response of CD4/p56lck chimeric T cell hybridomas. L3T4 murine T cell hybridoma cells expressing human CD4 chimerized either to a full-length p56lck chimeric molecule (full length); to a p56lck molecule lacking the SH1 domain (N32); to a p56lck molecule lacking the SH1 and SH2 domains (N3); or to a p56lck molecule containing only the N domain (N) were used in the migration assay. These cell lines were stimulated in a dose-dependent fashion with murine SDF-1α either directly (▪) or following a 1-h pretreatment with IL-16 (10−10 M) (⋄). The data are expressed as the averages of four separate experiments. The asterisk denotes significantly different migration in the cells pretreated with IL-16 as compared with untreated cells, p < 0.05.

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The ability of the SH3 domain to transmit the inhibitory signal is similar to preliminary data identifying that the SH3 domain is involved in transmission of an IL-16-induced migratory signal (T. Ryan, unpublished observations). Those studies also suggest that the migratory signal transduced by the SH3 domain is dependent on PI3 kinase activity. To determine whether PI3 kinase is also involved in transmission of the CXCR4-inhibitory signal, human T cells were incubated with the PI3 kinase-specific inhibitors, wortmannin or Ly294002 (29). In these studies, T cells were incubated with either wortmannin (1 nM, for 18–24 h) or Ly294002 (40 μM, for 1 h), followed by IL-16 pretreatment for 1 h before conducting a SDF-1α dose response. As shown in Fig. 7, both wortmannin- and Ly294002-treated cells demonstrated a partial reduction in the dose response as compared with no inhibitor treatment, and support previous studies indicating that SDF-1α signaling is in part PI3 kinase dependent (30, 31). When cells were pretreated with either inhibitor and IL-16, response to SDF-1α-induced chemotaxis was identical to the dose response of cells pretreated with inhibitor alone, indicating that the inhibitory effect of IL-16 was blocked (Fig. 7). These data suggest that the IL-16/CD4/p56lck-transmitted inhibitory signal of CXCR4 involves PI3 kinase activity. Cells pretreated with higher concentrations of wortmannin (10 nM) or Ly294002 (80 μM) were completely unresponsive to SDF-1α stimulation (data not shown).

FIGURE 7.

The effects of wortmannin and Ly294002 on IL-16 pretreatment in human CD4+ T cells. Human CD4+ T cells were incubated with either wortmannin (1 nM) for 18 h or Ly294002 (40 μM for 1 h) before washing and a 1-h incubation with IL-16 (10−10 M). The cells were then subjected to SDF-1α-induced migration. The figure represents the migratory response of T cells either without any PI3 kinase inhibitor or IL-16 pretreatment (□); with inhibitor pretreatment only (▪); with both inhibitor and IL-16 pretreatment (▨); or with IL-16 pretreatment only (|og). This experiment was conducted five separate times, and the data are expressed as the averages for those experiments. The asterisk represents migration that was statistically different from the migration observed for untreated cells for the same concentration of SDF-1α, p < 0.05.

FIGURE 7.

The effects of wortmannin and Ly294002 on IL-16 pretreatment in human CD4+ T cells. Human CD4+ T cells were incubated with either wortmannin (1 nM) for 18 h or Ly294002 (40 μM for 1 h) before washing and a 1-h incubation with IL-16 (10−10 M). The cells were then subjected to SDF-1α-induced migration. The figure represents the migratory response of T cells either without any PI3 kinase inhibitor or IL-16 pretreatment (□); with inhibitor pretreatment only (▪); with both inhibitor and IL-16 pretreatment (▨); or with IL-16 pretreatment only (|og). This experiment was conducted five separate times, and the data are expressed as the averages for those experiments. The asterisk represents migration that was statistically different from the migration observed for untreated cells for the same concentration of SDF-1α, p < 0.05.

Close modal

A coreceptor relationship between CD4 and certain of the chemokine receptors has been clearly established following interaction with the CD4 ligand HIV-1 gp120 (1, 2, 3, 4, 5, 6, 7, 11). Gp120 binding to CD4 results in the loss of signaling by a variety of chemokine receptors, including CCR5 and CXCR4 for both lymphocytes and monocytes (12, 13). Interestingly, monocytes also lose their responsiveness to endotoxin stimulation, suggesting that gp120 stimulation results in a nonspecific modulation of monocyte responsiveness. The mechanism for gp120-induced loss of MIP-1β/CCR5 or SDF-1α/CXCR4 signaling has been well defined. Following binding by gp120, CCR5, CXCR4, as well as CD4 are modulated from the cell surface, resulting in cellular unresponsiveness to chemokine stimulation. This effect requires enzymatic activity of the src kinase family member, p56lck, mediated through the SH1 domain of p56lck (13). The ability to cross-desensitize chemokine signaling is not restricted to the CD4 ligand gp120, nor is it restricted mechanistically to the modulation of chemokine receptors from the cell surface. We have previously reported that another CD4 ligand, IL-16, could also desensitize CCR5 signaling (21). Although IL-16, similar to gp120, requires enzymatic activity of p56lck, unlike gp120, IL-16 does not induce modulation of CCR5 from the cell surface. The CD4-CCR5 desensitization relationship is also reciprocal, whereby MIP-1β pretreatment results in cellular unresponsiveness to IL-16/CD4 stimulation (21). This indicates that the relationship between CD4 and CCR5, first identified by gp120, exists also for the natural ligands for CD4 and CCR5. The present studies were conducted to investigate whether a similar relationship existed for CD4 and CXCR4.

Our studies indicate that, like CCR5, a functional relationship does exist for CD4 and CXCR4, which is facilitated by IL-16. Although there are similarities between the effects of IL-16 on CXCR4 and CCR5 (inhibition of induced migration, time course required for inhibition, and lack of an effect on chemokine receptor expression or chemokine binding), there are some major differences. The effects of IL-16 on SDF-1α/CXCR4 signaling are not reciprocal. Pretreatment of primary T cells or T cell hybridomas with SDF-1α had no effect on IL-16-induced migration. This lack of reciprocity with CXCR4 may relate to the findings that CD4 and CXCR4 are not constitutively associated, whereas reciprocity is present for CCR5 and CD4, in which constitutive association has been reported (11). Although gp120 is capable of inducing CD4-CXCR4 receptor complex formation, it has not as yet been determined whether IL-16 stimulation results in the induction of a similar complex.

A second major difference between the interaction of CD4 with CXCR4 as compared with that for CCR5 is that p56lck enzymatic activity is not required to inhibit SDF-1α/CXCR4 signaling. Rather, based on the studies using p56lck deletional mutant cell lines and on the PI3 kinase inhibitor experiments, the inhibitory signal is transmitted, at least in part, through the interaction of the SH3 domain and appears to require the involvement of PI3 kinase. Interestingly, preliminary studies have indicated that an IL-16/CD4-mediated migratory response in T cells is also transmitted via the SH3 domain of p56lck, which results in activation of PI3 kinase (T. Ryan, unpublished observations). The preliminary observation that IL-16-induced migration also involves PI3 kinase activation suggests common signaling pathways for these two chemoattractant cytokines, despite SDF-1α using a seven-membrane, G protein-associated receptor (CXCR4) and IL-16 using a type I Ig family receptor (CD4). Without direct evidence for IL-16-induced CD4-CXCR4 receptor complex formation, common use of intracellular signaling pathways, such as depletion or occupancy of intracellular pools of active kinases, is a potential mechanism for receptor cross-desensitization.

The inhibitory effects of IL-16 are selective. We have previously demonstrated that IL-16 stimulation lacks an effect on MCP-1, MCP-2, or MCP-4 stimulation. In these studies, we identify that pretreatment of primary T cells by IL-16 did not effect the migratory response induced by eotaxin stimulation. Eotaxin binds exclusively to the chemokine receptor CCR3 and, similar to CCR5 and CXCR4, has been shown to facilitate HIV entry into CD4+ T cells (32). The lack of an effect on eotaxin/CCR3 stimulation by IL-16/CD4 indicates that the specificity of IL-16-inhibitory activity is not completely coincident with the subset of CD4-chemokine coreceptors, as defined by HIV-1 gp120 binding and internalization. Further studies are required to fully elucidate the spectrum of chemokine receptors that are affected by IL-16 signaling.

At present, we can only speculate as to the biological importance of these findings. Clearly, the process of leukocyte migration from circulation to sites of inflammation is complex and involves a variety of stimuli acting either concomitantly or sequentially on selectively recruited cells. As a result of exposure to multiple microenvironments, proper interpretation of sequential stimuli is essential for directed and efficient cellular recruitment. Previous reports have identified both positive and inhibitory effects of one chemoattractant on subsequent stimulation by a second chemoattractant (33, 34, 35, 36). Foxman et al. (36) have reported on the ability of neutrophils to maintain their ability to respond to IL-8-induced chemotaxis even after preexposure to another neutrophil chemoattractant leukotriene B4. There was also no inhibitory effect when the chemoattractants were reversed. In contrast, sequential stimulation of neutrophils by fMLP and IL-8 identifies a different regulatory process (34). Pretreatment of neutrophils with IL-8 has no effect on subsequent stimulation by fMLP; however, fMLP stimulation totally abrogated the adhesive and chemoattractant responses to subsequent IL-8 stimulation. Our data indicate that this same paradigm exists for IL-16/CD4 and CXCR4, whereby CD4+ T cells would be transiently unable to respond to SDF-1α as a secondary stimulus following IL-16 stimulation.

In summary, our findings suggest that there is a functional relationship between two chemoattractant cytokines of completely different classes, IL-16 and SDF-1α. A regulatory component exists whereby pretreatment with IL-16 results in cross-desensitization of SDF-1α. The mechanism for the IL-16 effect on CXCR4 appears to be quite distinct from the mechanism used by HIV-1 gp120, as inhibition occurs as a result of a p56lck-independent inhibitory signal, which is also in sharp contrast to the mechanism for IL-16-induced inhibition of CCR5. As both of these cytokines have been detected coincidently in association with inflammation, it is feasible to hypothesize that this represents an adaptive process to restrict and regulate recruitment of immune cells to sites of inflammation.

1

This work was supported in part by Grants AI35680, HL32802, and AI41994 from the National Institutes of Health. W.W.C. is a recipient of a Career Investigator Award from the American Lung Association.

4

Abbreviations used in this paper: CXCR, CXC chemokine receptor; gp, glycoprotein; MCP, monocyte chemoattractant protein; MIP, macrophage-inflammatory protein; PI3, phosphatidylinositol 3; SDF, stromal-derived factor; SH, Src homology.

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