IL-15 is a T cell growth factor that shares many biological activities with IL-2 and uses the same β/γ polypeptides of the IL-2R complex for signal transduction. Accumulating evidence implicates an important role for this cytokine in the inflammatory response of the host. Consistent with such a role, IL-15 has been shown to be a chemoattractant for T lymphocytes, NK cells, and neutrophils. Extending these observations, we now show that IL-15 is a potent inducer of CC-, CXC-, and C-type chemokines in T lymphocytes. In addition, we demonstrate that IL-15 induces CC chemokine receptors, but not CXC chemokine receptors, in a dose-dependent manner. Thus, our findings suggest that the proinflammatory effects of IL-15 at least in part may be due to the induction of chemokines and their receptors in T cells. Furthermore, we demonstrate that IL-15 promotes entry and replication of macrophage-tropic HIV in T lymphocytes and suggest a plausible mechanism by which IL-15, a cytokine that is elevated in HIV-infected individuals, may promote the transition of HIV displaying the M-tropic phenotype primarily associated with the initial transmission into the T cell-tropic phenotype that predominates as the disease progresses.

Interleukin-15 is a 15-kDa polypeptide that was discovered by its ability to promote the growth of T lymphocytes. It belongs to the four α-helix bundle family of cytokines 1, 2, 3 . IL-15 uses the β- and γ-components of the IL-2R (reviewed in 4 and, as anticipated from the receptor subunit sharing it, exhibits a spectrum of immune functions that largely overlaps with that of the T cell growth factor IL-2. However, IL-15 and IL-2 use different α-chain receptor components, i.e., IL-15Rα and IL-2Rα, respectively 5, 6 . IL-15Rα binds IL-15 with high affinity (Kd = ∼10−11 M) and is expressed in a wide variety of tissues, unlike its counterpart IL-2Rα, which binds IL-2 less avidly (Kd = ∼10−8 M) and is expressed largely in lymphocytes upon activation 7 .

Unlike IL-2, which is secreted by activated T lymphocytes, IL-15 mRNA is expressed by macrophages, dendritic cells, endothelial cells, keratinocytes, and other cell types as well in response to environmental/stress stimuli and infectious agents 3, 8, 9, 10, 11, 12, 13, 14 . There is increasing evidence to suggest that IL-15 may play an important role in protective immune responses, allograft rejection 15 , and the pathogenesis of autoimmune diseases 16, 17, 18, 19 where mononuclear cell infiltration is a hallmark feature. Recruitment of immune cells, especially lymphocytes, NK cells, and neutrophils, to sites of inflammation appears to be greatly influenced by IL-15 20, 21, 22 . More recently, it has been suggested that the effects of IL-15 on T cell motility are more similar to chemokinesis than chemotaxis due to its ability to stimulate motility in the absence of an established chemotactic gradient 23 . In parallel, chemokines such as RANTES, MIP-1α,2 and MIP-1β are not only potent inducers of lymphocyte chemotaxis at sites of immune and inflammatory reactions but also have the capacity to activate such cells reviewed in Refs. 24, 25, 26 . Thus, considering their effects on the mobility of immune cells as well as their abundance at sites of inflammation, IL-15 and chemokines may act in concert to sustain an inflammatory response. Alternatively, IL-15 could exert its effect at least in part by modulating the expression of chemokines and their receptors that are essential for migration of immune cells to such sites.

Addressing this issue, we demonstrate that IL-15 is a potent inducer of chemokines and their receptors in peripheral blood-derived T lymphocytes, thus establishing an important link between IL-15 expression and the induction of chemokines leading to a prolific inflammatory response. Furthermore, considering the importance of chemokines and their receptor expression in the pathogenesis of HIV infection (reviewed in Refs. 27–29), we suggest a plausible mechanism by which IL-15 may promote the spread into T lymphocytes of monocytotropic, nonsyncytial-inducing strains of HIV that are primarily responsible for the initial transmission 30, 31, 32 .

Peripheral blood-derived lymphocytes were obtained from normal volunteers by a two-step procedure: initiating with an automated leukopheresis and counterflow elutriation. Following elutriation, the lymphocytic fraction was collected, and any residual contaminating monocytes were further removed by incubation of the cells with carbonyl iron (100 mg/108 cells) to facilitate engulfment by monocytes and their subsequent removal by exposure to a magnetic field. NK cells and B lymphocytes were removed by using CD56 and CD19 microbeads with MACS separation columns from Miltenyi Biotech (Auburn, CA). The resultant T lymphocyte-enriched cell population was >96% CD3-positive as assessed by FACS analysis.

Human recombinant IL-15 and IL-2 were purchased from PeproTech (Rocky Hill, NJ). Rabbit polyclonal Abs to human IL-1β and human TNF-α were purchased from Genzyme (Cambridge, MA). The mAb to IL-2Rβ (Mikβ-1) was a gift from Matsuru Tsudo (Kyoto, Japan). MIP-1α, MIP-1β, and RANTES ELISA kits were purchased from R&D Systems (Minneapolis, MN) and were used to measure the levels of these chemokines in the cell culture supernatants.

Total cellular RNA was isolated from cytokine-treated T lymphocytes using TRIzol (Life Science Technologies, Gaithersburg, MD) according to the manufacturer’s instructions. The expression of various chemokines and their receptors was measured by multiprobe RPAs 33 . Template sets for the multiprobe RPA were purchased from PharMingen (San Diego, CA), and the assays were performed according to the manufacturer’s instructions. Briefly, 50 ng of DNA from each multiprobe set was used to generate 32P-labeled riboprobes of defined length with T7 RNA polymerase in the presence of 150 μCi of [32P]UTP. Template DNA was then eliminated by digestion with DNase free of RNase, followed by precipitation of labeled RNA. Fifteen micrograms of total cellular RNA was then mixed with 6 × 105 cpm of 32P-labeled riboprobe mixture in a hybridization buffer consisting of 40 mM PIPES, 1 mM EDTA, and 0.4 M NaCl in 80% formamide and incubated at 90°C for 5 min followed by 56°C for 12 h. The hybridized RNA duplexes were then treated with an RNase mixture consisting of RNase A and RNase T1 followed by proteinase K digestion. RNase-resistant duplex RNA was extracted with phenol once and precipitated by the addition of an equal volume of 4 M ammonium acetate and 2 vol of ethyl alcohol. The RNA pellet was then solubilized and resolved on a 6% sequencing gel, dried, and subjected to autoradiography or phosphorimage analysis.

To evaluate the effect of IL-15 on chemokine receptor expression in peripheral blood-derived T lymphocytes, cells were cultured in the presence of recombinant human IL-15 (10 ng/ml). In parallel, T lymphocytes were treated with IL-2 (10 ng/ml)-supplemented medium for comparison of these functionally related cytokines. Cells were harvested 12 h later, and total cellular RNA was extracted and subjected to a RPA to evaluate the modulation of chemokine receptor expression. As shown in Fig. 1 A, the mRNAs for CXCR1 and CXCR2 receptors that bind IL-8 and for the CXCR3 receptor that binds IP10/Mig were not detectable in resting T cells or in T cells cultured in the presence of IL-15 or IL-2. However, there was abundant expression of the CXCR4 receptor that binds stromal-derived growth factor-1 (SDF-1) and functions as a coreceptor for syncytial-inducing, T cell-tropic HIV in resting T lymphocytes 34, 35, 36 . The levels of CXCR4 expression remained unaltered regardless of whether the cells were cultured in the presence of IL-2 or IL-15. In addition, there was abundant expression of BLR-2 (CCR7) receptor 37 but not BLR-1 (CXCR5) in resting T lymphocytes, although no modulation of its expression was evident in the presence of either of these cytokines. We also noted a detectable signal for the orphan receptor V28 38 , which appears to function as an entry molecule for certain strains of HIV-1 and HIV-2 39 , but again no modulation of its expression was evident in the presence of IL-15 or IL-2. Thus, resting T lymphocytes express a subset of CXC chemokine receptors, and the expressions of these chemokine receptors remain unchanged by the presence of T cell growth factors IL-15 or IL-2.

FIGURE 1.

Effects of IL-2 and IL-15 on chemokine receptor expression. Purified T lymphocytes (2 × 107) obtained from a healthy donor were cultured in the presence of recombinant IL-2 or IL-15 at a concentration of 10 ng/ml for 12 h. Total cellular RNA was extracted, and RPAs were performed as described in Materials and Methods. A illustrates the expression profile of CXC chemokine receptors evaluated by RPA. B illustrates the expression profile of CC chemokine receptors. The expression levels of ribosomal L32 and cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serve as internal controls. In lanes 1, RPA was performed with yeast transfer RNA; in lanes 2, RPA was performed with RNA derived from cells cultured in medium alone; in lanes 3, RPA was performed with RNA derived from cells cultured in medium supplemented with IL-2; in lanes 4, RPA was performed with RNA derived from cells cultured in medium supplemented with IL-15. Similar results were obtained from cells derived from two other donors.

FIGURE 1.

Effects of IL-2 and IL-15 on chemokine receptor expression. Purified T lymphocytes (2 × 107) obtained from a healthy donor were cultured in the presence of recombinant IL-2 or IL-15 at a concentration of 10 ng/ml for 12 h. Total cellular RNA was extracted, and RPAs were performed as described in Materials and Methods. A illustrates the expression profile of CXC chemokine receptors evaluated by RPA. B illustrates the expression profile of CC chemokine receptors. The expression levels of ribosomal L32 and cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serve as internal controls. In lanes 1, RPA was performed with yeast transfer RNA; in lanes 2, RPA was performed with RNA derived from cells cultured in medium alone; in lanes 3, RPA was performed with RNA derived from cells cultured in medium supplemented with IL-2; in lanes 4, RPA was performed with RNA derived from cells cultured in medium supplemented with IL-15. Similar results were obtained from cells derived from two other donors.

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In contrast to the CXC chemokine receptors that were evaluated previously and remained unmodulated in the presence of IL-15 or IL-2, the receptors for C-C chemokines examined in Fig. 1 B displayed differential responses to IL-15 and IL-2. The receptors CCR1, CCR4, CCR5, CCR2a, and CCR2b were expressed, although in low abundance, in resting T lymphocytes and were detected by the exceedingly sensitive RPA. Transcripts for CCR3 and TER-1 were, however, not detected. More importantly, all the transcripts that were constitutively expressed in resting T cells were induced when cultured in the presence of IL-15, and the magnitude of the induction was fivefold or more (compare lane 2 vs lane 4), whereas in the presence of IL-2, no discernible induction was apparent (compare lane 2 vs lane 3).

Although both IL-15 and IL-2 signaling occurs through the engagement of IL-2/15Rβ and IL-2/15Rγ (γc) components of the receptor complex, IL-2 requires the presence of IL-2Rα for high affinity interactions and subsequent signal transduction (reviewed in Refs. 4 and 7). However, IL-2Rα, which is induced by the engagement of the TCR/CD3 complex, is minimally expressed in resting T lymphocytes, unlike the IL-2/15Rβ and γc polypeptides 4, 7 . Thus, it is conceivable that in resting T cells, IL-15 efficiently engages IL-2/15Rβ and IL-2/15Rγ in the presence of IL-15Rα for signal transduction, leading to significant up-regulation of receptors for C-C chemokines relatively rapidly, while IL-2 is unable to do so. Nonetheless, it is important to note that the inability of IL-2 to up-regulate these receptors in T cells does not appear to be absolute. Recently, Loetscher et al. 40 reported that when T lymphocytes were cultured in the presence of IL-2, in addition to displaying chemotaxis to both MCP-1 (monocyte chemoattractant protein-1, and RANTES, their cognate receptor expression was also up-regulated coincidentally with that of the IL-2Rα expression. However, this induction required prolonged culture of cells (>4–10 days) in the presence of IL-2.

The expression of chemokines in response to IL-15 and IL-2 was next examined. When resting T lymphocytes were cultured in the presence of either recombinant human IL-15 or IL-2 there was significant up-regulation of steady state mRNA levels for a number of chemokines (compare lanes 3 and 4 of Fig. 2 with lane 2). The ability of IL-2 to induce chemokines but not their receptors (see above) was somewhat perplexing and may reflect threshold differences in the induction pathways. Nonetheless, unlike the induction of chemokine receptor expression that was limited to C-C-type chemokine receptors, up-regulation of chemokines themselves was not restricted to a particular subclass. For example, IP10, which is a CXC-type chemokine, was induced over sixfold as were MIP-1α, MIP-1β, and RANTES, which belong to the C-C type of chemokines. In addition, mRNA levels of lymphotactin, which is the sole member of C-type chemokine subclass (reviewed in Refs. 25 and 26), were up-regulated in the presence of both IL-15 and IL-2, although prolonged exposure of the gel was necessary to visualize this induction. In addition, an increase in the levels of MCP-1 mRNA and IL-8 mRNA was detected in T lymphocytes similar to their reported responsiveness to IL-15 in human monocytes 41 .

FIGURE 2.

Induction of chemokine gene expression by IL-2 and IL-15. Purified T lymphocytes (2 × 107) obtained from a healthy donor were cultured in the presence of recombinant IL-2 or IL-15 at a concentration of 10 ng/ml for 12 h. Total cellular RNA was extracted, and ribonuclease protection assays were performed. The expression levels of ribosomal L32 and cellular GAPDH serve as internal controls. In lane 1, RPA was performed with yeast transfer RNA; in lane 2, RPA was performed with RNA derived from cells cultured in medium alone; in lane 3, RPA was performed with RNA derived from cells cultured in medium supplemented with IL-2; in lane 4, RPA was performed with RNA derived from cells cultured in medium supplemented with IL-15. Similar results were obtained from cells derived from two other donors.

FIGURE 2.

Induction of chemokine gene expression by IL-2 and IL-15. Purified T lymphocytes (2 × 107) obtained from a healthy donor were cultured in the presence of recombinant IL-2 or IL-15 at a concentration of 10 ng/ml for 12 h. Total cellular RNA was extracted, and ribonuclease protection assays were performed. The expression levels of ribosomal L32 and cellular GAPDH serve as internal controls. In lane 1, RPA was performed with yeast transfer RNA; in lane 2, RPA was performed with RNA derived from cells cultured in medium alone; in lane 3, RPA was performed with RNA derived from cells cultured in medium supplemented with IL-2; in lane 4, RPA was performed with RNA derived from cells cultured in medium supplemented with IL-15. Similar results were obtained from cells derived from two other donors.

Close modal

Having demonstrated that the expression of both chemokines and their receptors are induced in response to IL-15 treatment in T cells, we next examined the kinetics of this induction by culturing cells in the presence of IL-15 at a concentration of 10 ng/ml and evaluating the mRNA profiles of chemokines and their receptors at various post-treatment time points up to 24 h by RPA. As shown in Fig. 3, by 12 h following IL-15 treatment an increase in the mRNA levels of all responsive chemokines was apparent, although highest induction levels were seen in the samples harvested 18 h after IL-15 treatment. It should be noted that the gradual decline in the mRNA levels seen in samples collected after 24 h suggests that the induction in response to IL-15 is probably transitory. Moreover, when cells were exposed to increasing amounts of IL-15, there was a concordant increase in the magnitude of the induction of mRNA expression for all the responsive chemokines examined (Fig. 3).

FIGURE 3.

Kinetics and dose dependence of IL-15-mediated induction of chemokines. To examine the kinetics of chemokine expression in response to IL-15, purified T lymphocytes were cultured in medium supplemented with 10 ng/ml rIL-15, and cells were harvested at the indicated times (3 h (lane 3), 6 h (lane 4), 12 h (lane 5), 18 h (lane 6), and 24 h (lane 7)), and total cellular RNA was extracted. To examine the dose-response relationship, cells were cultured in medium supplemented with increasing amounts of IL-15, as indicated, for 12 h, and cellular RNA was extracted (lane 8, 10 ng/ml IL-15; lane 9, 100 ng/ml IL-15; lane 10, 500 ng/ml IL-15; lane 11, 1 μg/ml IL-15). RPAs were performed as described in Materials and Methods. As controls in lane 1, RPA was performed with yeast transfer RNA, and in lane 2, RPA was performed with RNA extracted from cells cultured in medium without IL-15.

FIGURE 3.

Kinetics and dose dependence of IL-15-mediated induction of chemokines. To examine the kinetics of chemokine expression in response to IL-15, purified T lymphocytes were cultured in medium supplemented with 10 ng/ml rIL-15, and cells were harvested at the indicated times (3 h (lane 3), 6 h (lane 4), 12 h (lane 5), 18 h (lane 6), and 24 h (lane 7)), and total cellular RNA was extracted. To examine the dose-response relationship, cells were cultured in medium supplemented with increasing amounts of IL-15, as indicated, for 12 h, and cellular RNA was extracted (lane 8, 10 ng/ml IL-15; lane 9, 100 ng/ml IL-15; lane 10, 500 ng/ml IL-15; lane 11, 1 μg/ml IL-15). RPAs were performed as described in Materials and Methods. As controls in lane 1, RPA was performed with yeast transfer RNA, and in lane 2, RPA was performed with RNA extracted from cells cultured in medium without IL-15.

Close modal

The inductive kinetics of the C-C chemokine receptor expression paralleled those of chemokines themselves as shown in Fig. 4, with peak expression occurring 18 h post-treatment. In addition, as demonstrated above for the expression of chemokine gene expression, exposure of cells to increasing amounts of IL-15 resulted in an enhancement of receptor gene expression in a dose-dependent manner up to 500 ng/ml, although at 1 μg/ml some diminution in the level of induction was apparent.

FIGURE 4.

Kinetics and dose-dependence of IL-15-mediated induction of C-C chemokine receptors. To determine the kinetics and dose-response relationship of IL-15-mediated activation of CC chemokine receptor expression, RPA was performed as described in Fig. 3. The inset shows a shorter exposure of the area of the gel containing the housekeeping genes ribosomal L32 and GAPDH.

FIGURE 4.

Kinetics and dose-dependence of IL-15-mediated induction of C-C chemokine receptors. To determine the kinetics and dose-response relationship of IL-15-mediated activation of CC chemokine receptor expression, RPA was performed as described in Fig. 3. The inset shows a shorter exposure of the area of the gel containing the housekeeping genes ribosomal L32 and GAPDH.

Close modal

To determine whether the increase in the steady state mRNA levels of chemokines seen above in the presence of IL-2 or IL-15 actually results in augmented secretion of these chemokines, we measured the chemokine levels in the culture supernatants of treated cells using an ELISA. The basal levels of chemokines assessed varied from donor to donor, but as shown in Fig. 5, a dose-dependent increase in the secreted levels of MIP-1α, MIP-1β, and RANTES was observed. This enhanced secretion was not affected by the presence of Abs to TNF-α or IL-1β, thus excluding the possibility that the chemokine secretion observed was secondary to induction of other proinflammatory cytokines, such as IL-1β or TNF-α, by IL-15 (data not shown). More importantly, the observed dose dependency extending to high concentrations of IL-15, exemplified by cultures treated with 500 or 1000 ng/ml, was somewhat surprising considering the fact that T lymphocytes express only about 2 × 103 IL-2/15Rβ and γc receptors on the cell surface, and saturation of these receptors should have occurred at the lower amounts of IL-15 used 42 . Since IL-15 binds to the IL-2/15Rβ,γc heterodimer in the absence of IL-15Rα with low (10−8–10−9 M) affinity, one possible explanation for this could be that peripheral blood-derived T lymphocytes use a receptor system that does not involve IL-15Rα. Alternatively, IL-15 might use a receptor that does not involve any of the IL-2/15R elements in freshly isolated peripheral blood T lymphocytes. To explore this possibility, a mAb, Mikβ-1, that binds to IL2Rβ and prevents IL-15 signaling via the IL-2R complex subunits 43 was added (20 μg/ml) to cells 15 min before the addition of various amounts of IL-15. As shown in Fig. 6, Mikβ-1, which has an IC50 of 5 nM for the induction of IL-15-mediated proliferation of the Kit225/K6 T lymphocytic cell line via the IL-2/15R complex 44 , inhibited 75% of the MIP-1α secretion induced by low concentrations of IL-15 in peripheral blood-derived T lymphocytes, in accord with the use of IL-2/15Rβ,γc with or without IL-15Rα. More importantly, when T cells were treated with higher doses of IL-15, the Ab was less effective (<25% inhibition) in blocking IL-15-mediated MIP-1α secretion. From the results presented in Fig. 6, it is conceivable that the response elicited with lower amounts of IL-15, which is inhibitable by Mikβ-1, represents a more efficient signaling pathway, whereas when IL-15 is present in excess, an alternate receptor system other than the IL-2/15R with attenuated ligand affinity becomes operational. In this regard it is noteworthy that an alternate receptor system for IL-15 has been described in mast cells, although the actual components of this system have yet to be identified 45 . However, if the receptor densities of IL-2/15Rβ and γc are markedly different in peripheral blood-derived T lymphocytes compared with those in the well-studied T lymphocytic cell line Kit225/k6, then the failure of Mikβ-1 Ab to block high dose IL-15 effects may simply be due to a limitation of the Ab.

FIGURE 5.

IL-15-mediated chemokine secretion by T lymphocytes. Purified T lymphocytes (2 × 107) were cultured in medium supplemented with increasing amounts of rIL-15 for 24 h, and the culture supernatants were tested for the presence of MIP-1α (A), MIP-1β (B), and RANTES (C) by an ELISA. The mean chemokine concentration in the supernatants from triplicate wells is shown (±SEM). Similar results were obtained in two additional experiments.

FIGURE 5.

IL-15-mediated chemokine secretion by T lymphocytes. Purified T lymphocytes (2 × 107) were cultured in medium supplemented with increasing amounts of rIL-15 for 24 h, and the culture supernatants were tested for the presence of MIP-1α (A), MIP-1β (B), and RANTES (C) by an ELISA. The mean chemokine concentration in the supernatants from triplicate wells is shown (±SEM). Similar results were obtained in two additional experiments.

Close modal
FIGURE 6.

Ab against IL-2/15Rβ (Mikβ-1) differentially inhibits the IL-15-mediated secretion of chemokines. Purified T lymphocytes (2 × 107) were cultured in medium supplemented with increasing amounts of IL-15. In a parallel set of experiments, mAb Mikβ-1, which binds to the IL-2/15Rβ polypeptide, was added (20 μg/ml) to the culture medium 15 min before the addition of IL-15. MIP-1α levels in the culture supernatants were measured after 24 h by an ELISA, and the reduction in MIP-1α levels in the presence of Mikβ-1 Ab is expressed as a percentage and represents the mean ± SEM of samples performed in triplicate.

FIGURE 6.

Ab against IL-2/15Rβ (Mikβ-1) differentially inhibits the IL-15-mediated secretion of chemokines. Purified T lymphocytes (2 × 107) were cultured in medium supplemented with increasing amounts of IL-15. In a parallel set of experiments, mAb Mikβ-1, which binds to the IL-2/15Rβ polypeptide, was added (20 μg/ml) to the culture medium 15 min before the addition of IL-15. MIP-1α levels in the culture supernatants were measured after 24 h by an ELISA, and the reduction in MIP-1α levels in the presence of Mikβ-1 Ab is expressed as a percentage and represents the mean ± SEM of samples performed in triplicate.

Close modal

The importance of chemokine receptors for HIV entry and AIDS pathogenesis has recently become increasingly apparent 27, 28, 29 . The C-C chemokine receptor CCR5 and, to a lesser extent, CCR3 and CCR2b mediate entry of M-tropic, nonsyncytial-inducing strains of HIV that are primarily responsible for the transmission of HIV 30, 31, 32 . In quiescent T cells these C-C chemokine receptors are minimally expressed 46 ; as a result, T cells are refractory to HIV infection. Our observation of an enhanced expression of these receptors in the presence of IL-15 in T lymphocytes taken together with the fact that unlike IL-2 production, which declines as the disease progresses, the elevated levels of serum IL-15 often persist in AIDS patients 47 suggest a potential role for this cytokine in the pathogenesis of HIV infection. It is possible that IL-15 could facilitate the spread into the T cell population of M-tropic virus that is usually confined to monocytes/macrophages. IL-15 is ideally poised for this function because of its inherent ability to induce the expression of CCR5 receptor as well as CD3-independent proliferation of T lymphocytes, two prerequisites for efficient replication of HIV. To examine this possibility, in the presence of IL-15, freshly isolated, quiescent T lymphocytes were infected with a primary M-tropic, nonsyncytial-inducing HIV isolate (HIVUS-1) that had previously been shown to use the CCR-5 receptor exclusively for entry 48 . Viral replication was monitored by measuring the secreted p24 levels in infected culture supernatants. It is important to note that unlike previous studies 49, 50, 51 that used either cell lines or artificially activated T cells with mitogens or CD3 cross-linking, our studies were performed using unactivated cells and thus are more likely to represent the natural state of T cells that would encounter the virus in the body. As can be seen in Fig. 7, there was efficient replication of HIV in T cells as assessed by the detection of p24 as early as day 3 postinfection in cultures treated with IL-15 but not in untreated control cultures. The addition of IL-15 and virus simultaneously to cells or pretreatment of cells with IL-15 before the addition of virus did not significantly affect the virus replication profile (data not shown). It is established that CCR-5-mediated entry can be competitively inhibited by the presence of its ligands, MIP-1α, MIP-1β, and RANTES, in T lymphocytes 52 . The fact that IL-15 induced these chemokines efficiently with kinetics similar to those of the CCR-5 gene and yet was able to promote HIV entry suggests that the receptor induction is more pronounced than that of its ligands, thus quantitatively favoring virus entry. The levels of IL-15-induced MIP-1α, MIP-1β, and RANTES in peripheral blood-derived T lymphocytes (<6 ng/ml) may not sufficiently saturate the induced cell surface CCR-5 coreceptors. This is in contrast to what is seen in NK cells, in which, either in combination with IL-12 or following CD16 cross-linking, IL-15 leads to copious secretion of these chemokines 53, 54 and, in fact, is able to efficiently suppress M-tropic HIV entry and replication in T lymphocytes 54 . Thus, the overall impact of IL-15 on HIV replication and pathogenesis may be dependent upon the balance of the HIV-suppressive and HIV-inductive activities of this cytokine.

FIGURE 7.

T cells were purified from elutriated lymphocytes obtained from a healthy, HIV-seronegative donor as described in Materials and Methods. From this cell population, CD8+ cells were removed by using CD8 microbeads with MACS separation columns (Miltenyi Biotech). CD4-enriched T cells (7 × 106) were added to medium containing IL-15 (20 ng/ml) and infected with 1 × 105 TCID50 of the M-tropic isolate HIVUS-1. Two hours postinfection, cells were washed three times and resuspended in medium containing IL-15 (20 ng/ml). Parallel control experiments were performed in medium without any IL-15 supplementation. Aliquots of culture supernatants were collected daily, and virus production was measured by an ELISA (New England Nuclear, Boston, MA) for HIV-1 p24 Ag. Similar results were obtained in two additional experiments.

FIGURE 7.

T cells were purified from elutriated lymphocytes obtained from a healthy, HIV-seronegative donor as described in Materials and Methods. From this cell population, CD8+ cells were removed by using CD8 microbeads with MACS separation columns (Miltenyi Biotech). CD4-enriched T cells (7 × 106) were added to medium containing IL-15 (20 ng/ml) and infected with 1 × 105 TCID50 of the M-tropic isolate HIVUS-1. Two hours postinfection, cells were washed three times and resuspended in medium containing IL-15 (20 ng/ml). Parallel control experiments were performed in medium without any IL-15 supplementation. Aliquots of culture supernatants were collected daily, and virus production was measured by an ELISA (New England Nuclear, Boston, MA) for HIV-1 p24 Ag. Similar results were obtained in two additional experiments.

Close modal

Additionally, the association of IL-15 with aberrant inflammatory responses such as rheumatoid arthritis 55, 56 , ulcerative colitis 57, 58 , pulmonary sarcoidosis 19 , and multiple sclerosis 59 strongly suggests an etiologic role for this cytokine in the perpetuation of inflammation. Although physiological inflammation is pivotal in host defense, a breakdown in this finely tuned response may result in an exaggerated response that is detrimental to the host, leading to chronic inflammatory, autoimmune, or allergic diseases. Parallel evidence from animal models of such deranged inflammatory processes have clearly defined a critical role for chemokines 60 . For example, lung reperfusion injury, and urate-crystal-induced arthritis in rabbits showed regression after treatment with an anti-IL-8 Ab 61, 62 . Adjuvant-induced arthritis in Lewis rats, which mimics rheumatoid arthritis, responded to anti-RANTES therapy 63 . Similarly, administration of Abs to MIP-2 and monocyte chemoattractant protein-1 led to rapid resolution of glomerulonephritis and cutaneous delayed hypersensitivity in rats, respectively 64, 65 . In addition, Abs against MIP-1α and RANTES reversed allergic airway inflammation in mice 66 . In this context, the ability of IL-15 to directly and rapidly augment the synthesis of both C-C as well as CXC chemokines by T lymphocytes as shown in this study suggests that IL-15 acts at the earliest phase of immune reactivity to induce chemokine-mediated recruitment and activation of mononuclear cells into the evolving nidus of immune reaction or inflammatory lesion. In summary, the increase in IL-15 observed in HIV infection and inflammatory autoimmune disease may in part exert its pathogenic action through the induction of inflammatory chemokines and their receptors.

We thank Drs. Keizo Furuke, Hiroyuki Moriuchi, Masako Moriuchi, Joe Mosca, and Pin-Yu Perera for their assistance.

2

Abbreviations used in this paper: MIP, macrophage inflammatory protein; RPA, ribonuclease protection assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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