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Inhibition of CCR5 Expression by IL-12 Through Induction of ß-Chemokines in Human T Lymphocytes

Laboratory of Cell and Viral Regulation, Division of Therapeutic Proteins, Center for Biologics Evaluation and Research, Food and Drug Administration, National Institutes of Health, Bethesda, MD 20892

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
IL-12 induces initiation of the differentiation of naive CD4⁺ T lymphocytes into Th1 cells and is important for the control of cell-mediated immunity. ß-Chemokines serve to attract various types of blood leukocytes to sites of infection and inflammation. The specific receptor for the ß-chemokines (macrophage-inflammatory protein (MIP)-1, MIP-1ß, and RANTES), CCR5, also functions as the primary coreceptor for macrophage-tropic isolates of HIV-1. IL-12, but not IL-4, IL-10, or IL-13, now has been shown to down-modulate the surface expression of CCR5 induced by IL-2 on both CD4⁺ and CD8⁺ T lymphocytes. Decreased CCR5 surface expression was not secondary to transcriptional inhibition, given that CCR5 mRNA was enhanced in cells cultured in IL-12/IL-2 compared with those cultured in IL-2 only. The effect of IL-12 in down-modulation of CCR5 surface expression was shown to be mediated by soluble factors secreted from the T cells. Rapid and transient intracellular Ca²⁺ mobilization was induced in monocytes by IL-12-induced supernatants, which desensitized the response of monocytes to MIP-1, but not their response to stromal cell-derived factor-1. Neutralization with specific Abs identified these factors as MIP-1 and MIP-1ß from most donors. IL-4, IL-10, IFN-, and IL-18 primarily inhibited MIP-1ß secretion and also weakly suppressed MIP-1 secretion. HIV-1 replication was inhibited in IL-2/IL-12-containing cultures that correlated with chemokine and chemokine-receptor levels. These data suggest that the effects of IL-12 on ß-chemokine production and chemokine-receptor expression may contribute to the immunomodulatory activities of IL-12 and may have potential therapeutic relevance in controlling HIV-1 replication.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Interleukin-12 is a heterodimeric cytokine that consists of di-sulfide-linked p35 and p40 subunits. Bioactive IL-12 (p70) is produced predominantly by neutrophils and activated APC such as dendritic cells and macrophages (1). IL-12 induces the secretion of IFN- by T lymphocytes and NK cells and promotes the development of the Th1 phenotype of CD4⁺ cells (2, 3, 4). Th1 lymphocytes secrete IFN-, but not IL-4, and mediate cellular immunity, whereas Th2 lymphocytes secrete IL-4 and IL-5, but not IFN-, and promote humoral immunity. Studies of Th1 and Th2 clones reveal that expression of the ß-chemokine receptor CCR5 is characteristic of Th1 cells (5), whereas the CCR3 receptor is preferentially expressed by Th2 clones (6). However, another study showed that the expression of CCR5 did not differ between Th1 and Th2 subsets (7).

Various cytokines have been shown to modulate the expression of chemokine receptors on both monocytes and lymphocytes (8, 9, 10, 11). IL-2 induces expression of CCR5 in cultured lymphocytes, whereas IL-4 increases predominantly the expression of the chemokine receptor CXCR4 in these cells. In monocyte and macrophage cultures, M-CSF and GM-CSF up-regulate CCR5 expression, whereas IL-4 and IL-13 inhibit expression of this receptor (11). At high concentrations, GM-CSF also inhibits CCR5 expression (12). IFN- increases the expression of CCR1, CCR3, and CCR5 in U937 monocytic cells (8).

Cytokines also play a central role in modulating immune function and in the pathogenesis of HIV infection. Because of defects in the production of Th1 cytokines during the course of HIV infection, several cytokines, including IL-2 and IL-12, are being considered for use as immunotherapeutic agents in individuals with AIDS, both to increase the number of CD4⁺ cells and to enhance immune function. In vitro, IL-2 stimulates virus replication, whereas IL-12 can either enhance virus production when added with mitogens (13) or suppress replication in the presence of IL-2 (14). In clinical trials, IL-2 has been shown to increase CD4⁺ cell number in HIV-1-infected patients, but it also stimulates virus replication in the absence of antiviral drugs (15, 16). This latter effect is consistent with the activation of T cells by IL-2 and with the stimulatory effect of this cytokine on CCR5 expression in cultured lymphocytes, given that CCR5 functions as a coreceptor with CD4 for macrophage-tropic (M-tropic)² isolates of HIV-1. Deficiencies in the production of IL-12 have also been demonstrated in individuals infected with HIV-1. Because of the potential of IL-12 to reconstitute immune function in HIV-1-infected individuals, we have now investigated the effect of IL-12, as well as those of other cytokines, on CCR5 expression in human lymphocytes. Unlike the other cytokines examined (IL-4, IL-10, and IL-13), IL-12 down-regulated the expression of CCR5 on T cells through the induction of ß-chemokines.

	Materials and Methods

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Cells and reagents

Human PBLs and monocytes were isolated from individual donors by countercurrent centrifugal elutriation and PBLs were cultured in RPMI 1640 medium supplemented with 10% FBS (HyClone, Logan, UT), 150 U/ml recombinant human IL-2 (rhIL-2) (Genzyme, Cambridge, MA), 100 U/ml penicillin, and 50 µg streptomycin. The following reagents were added to the medium, as indicated, at the beginning of cultures: 10 ng/ml rhIL-12 (kindly provided by Genetics Institute, Boston, MA); 100 ng/ml rhIFN-; 20 ng/ml TNF-, rhIL-4, rhIL-10, rhIL-13, and rhIL-18. Goat polyclonal Abs (20 ng/ml) to the human ß-chemokines RANTES, macrophage-inflammatory protein (MIP)-1, or MIP-1ß were from R&D Systems (Minneapolis, MN). MIP-1 and stromal cell-derived factor (SDF)-1 were from PeproTech (Rocky Hill, NJ).

Chemokine secretion

The amounts of RANTES (BioSource International, Camarillo, CA), MIP-1, and MIP-1ß (R&D Systems) secreted by PBLs into culture supernatants were determined with the use of ELISA kits.

Cell surface immunofluorescence staining

Cells were stained with the 5C7 (17) mAb to CCR5 and subjected to FCA as described previously (11). For analysis of CD4⁺ and CD8⁺ subsets expressing CCR5, two-color staining was performed with the 2D7 mAb to CCR5 (18), PE-conjugated F(ab')₂ fragments of goat polyclonal Abs to mouse IgG (Caltag, South San Francisco, CA), and then either with FITC-conjugated OKT4 mAb to CD4 (Ortho Diagnostics, Raritan, NJ) or with FITC-conjugated mAb to CD8 (Caltag).

RT-PCR

Total cellular RNA of human PBL was isolated by using Trizol reagent, treated with DNase I, and equal amounts of total RNA were subjected to first-strand DNA synthesis using RNase minus Superscript II reverse transcriptase (Life Technologies, Rockville, MD). Amplification of cDNA was performed for 30 cycles of PCR with human CCR5-specific sense primer 5'-CCT CCG CTC TAC TCA CTG GTG TTC (nt 100–123) and antisense primer 5'-GCA GGT GTA ATG AAG ACC TTC (nt 534–514), and human GAPDH sense and antisense-specific primers 5'-CAC CAC CAT GGA GAA GGC TGG (nt 306–326) and 5'-GCC ATG CCA GTG AGC TTC CCG (nt 695–675). PCR products were separated and stained with SYBR Green 1 (Molecular Probes, Eugene, OR).

Measurement of cytosolic-free Ca²⁺

Cells were labeled with the fluorochrome fura-2 acetoxymethyl ester (Molecular Probes, Eugene, OR). In brief, fura-2 acetoxymethyl ester (5 mM) and 10% Pluronic F-127 were premixed, added to cell suspension (1 x 10⁷/ml) in Ca²⁺ buffer (136 mM NaCl, 4.8 mM KCl, 1 mM CaCl₂, 5 mM glucose, and 20 mM HEPES, pH 7.4), and incubated at 37°C in the dark for 45 min. Cells were washed and resuspended at 2 x 10⁶/ml in Ca²⁺ buffer with 0.1% BSA. Cytosolic-free Ca²⁺ was measured using excitation at 340 and 380 nm with emission measured at 510 nm with a scan (Photon Technology International, Princeton, NJ), and data were recorded as the relative ratio of 340 and 380 nm measurements. The supernatants were concentrated (30x) and dialyzed against PBS buffer before testing.

Virus infection

After culture in medium containing IL-2, or IL-12 and IL-2 for 7 days, PBLs were infected with M-tropic HIV-1 strain Ba-L (equivalent to 50 ng/ml of p24) overnight. After extensive washing with PBS, PBLs were refed with medium containing IL-2 (150 U/ml) and were cultured for 7 days. Supernatant was collected and assayed for p24 by ELISA (Coulter, Hialeah, FL).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of IL-12 on CCR5 expression by PBLs

To investigate the effects of IL-12 and other cytokines on CCR5 expression by T lymphocytes, we used a simple culture system in which PBLs were incubated in the absence of polyclonal mitogens. Initial experiments revealed that incubation of the cells with IL-12 alone did not induce the expression of CCR5 in short-term culture and that this cytokine was not able to sustain lymphocyte viability. In contrast, IL-2 induced CCR5 expression and stimulated cell replication, consistent with previous observations (9, 19). Cells cultured after 8 days with IL-2 comprised predominantly CD8⁺ (70%) and CD4⁺ (30%) lymphocytes.

We next investigated the effect of IL-12 on cells stimulated to express CCR5 by culture with IL-2. After 8 days of culture, CCR5 expression was examined by immunofluorescence staining with the mAb 5C7, which reacts with the amino terminus of CCR5, and by flow cytometric analysis (FCA). The presence of IL-12 (10 ng/ml) in the culture medium markedly inhibited the expression of CCR5 induced by IL-2 (Fig. 1A). In contrast, the Th2 cytokines IL-4, IL-10, and IL-13 each slightly increased the CCR5 fluorescence intensity of IL-2-treated cells. The inhibitory effect of IL-12 on CCR5 expression was dose-dependent, being less apparent at a concentration of 1 ng/ml. CCR5 expression (mean fluorescence intensity (MFI)) after IL-2 or IL-2 plus IL-12 treatment from six donors is shown in Fig. 1B. IL-12 inhibited MFI of CCR5 50–90%, although the level of expression of CCR5 varied from donor to donor. Similar results were obtained by immunofluorescence staining with the mAb 2D7, which recognizes the second extracellular loop of CCR5. However, 2D7 staining was more intense than the 5C7 staining. Cells treated with IL-12 (10 ng/ml) showed virtually no 5C7 staining, but showed a low level of 2D7 staining (data not shown).

View larger version (16K): [in this window] [in a new window]   FIGURE 1. Effects of cytokines on CCR5 expression by PBLs. Cells were cultured for 8 days in medium supplemented with IL-2 either alone (Med) or in the additional presence 20 ng/ml of IL-4, IL-10, IL-13, or IL-12, as indicated. They were stained with the 5C7 mAb to CCR5 (filled trace) or with an isotype control (dotted line), and then were subjected to FCA (A). Similar staining patterns of IL-12-treated cells were obtained with six different PBL donors. CCR5 expression (MFI) for these donors is shown in B.

Both CD4⁺ and CD8⁺ T cells previously have been shown to express CCR5 after sequential stimulation with Abs to CD3 and with IL-2 (17). The effects of IL-12 on the expression of CCR5 by CD4⁺ and CD8⁺ T cells were assessed by two-color immunofluorescence staining (with mAb 2D7 to detect CCR5) after culture of PBLs in medium supplemented with IL-2 (Fig. 2). CCR5 was expressed on the surface of both CD4⁺ (67% CCR5 positive; MFI, 225) and CD8⁺ (76% CCR5 positive; MFI, 203) T cells incubated with IL-2 alone (Med in Fig. 2). IL-12 inhibited the expression of CCR5 on both CD4⁺ T cells and CD8⁺ T cells, reducing the percentage of CCR5 positive cells to 29% and 6% and the corresponding MFI to 57 and 85, respectively.

View larger version (45K): [in this window] [in a new window]   FIGURE 2. Effects of IL-12 on CCR5 expression by CD4+ and CD8+ T lymphocytes. PBLs were cultured for 8 days in medium containing IL-2 in the absence (center panels) or presence (lower panels) of IL-12 (10 ng/ml). The cells were stained with mAb 2D7 to CCR5 and mAbs either to CD4 (left panels) or to CD8 (right panels), and then were analyzed by FCA. Quadrants were set according to the staining pattern obtained with the isotype control mouse IgG2a (top panels). The pairs of numbers in the upper right and upper left quadrants represent the percentage of CD4+ or CD8+ T cells positive for CCR5 (left) and the corresponding MFI (right). Similar staining patterns were obtained with three different PBL donors.

Cytokines have been reported to regulate the expression of CCR5 both in macrophages and in T cells (8, 9). IL-2 directly increases the expression of CCR5 on activated T cells (9), and T cells polarized with IL-12 to differentiate toward a Th1 phenotype express CCR5 (5). To determine whether the reduction in cell surface CCR5 expression in response to IL-12 in our system was secondary to effects on mRNA synthesis, cytokine-treated cells were analyzed for CCR5 mRNA by RT-PCR. Consistent with previous reports, PBLs cultured in medium containing IL-2 expressed CCR5 mRNA, as shown as an expected CCR5-specific 435-bp band in Fig. 3. With the addition of IL-12, increased levels of CCR5 mRNA were detected in the treated cells. No CCR5 band was detected without adding reverse transcriptase, indicating that the CCR5 band detected is specific for CCR5 mRNA and is not from CCR5 genome DNA (data not shown). These data indicate that IL-12 does not directly inhibit, but rather enhances, CCR5 mRNA in cultured T cells, and demonstrates that inhibition of CCR5 surface expression is not a consequence of CCR5 transcriptional regulation.

View larger version (44K): [in this window] [in a new window]   FIGURE 3. Transcriptional regulation of CCR5 gene by IL-12. PBLs were cultured in medium containing IL-2 (Med), or IL-12 and IL-2 (IL-12) for 8 days; total RNA was isolated and subjected to RT-PCR (described in Materials and Methods). Representative results from two donors are shown.

We next investigated whether the inhibitory effect of IL-12 on CCR5 surface expression by T cells is mediated by IL-12-induced secretion of a soluble factor (or factors) that induces down-regulation of CCR5. PBLs were cultured in IL-2-containing medium for 8 days to induce CCR5 expression; then they were washed and cultured overnight in medium conditioned by incubation of PBLs either in the absence or presence of IL-12. PBLs incubated overnight with the culture supernatant of IL-12-treated cells from the same donor (Fig. 4A) or from an unrelated donor (Fig. 4B) showed a marked decrease in the extent of CCR5 expression. In contrast, the extent of CCR5 expression by PBLs similarly incubated with medium conditioned by culture of cells in the absence of IL-12 did not differ substantially from that CCR5 expression of cells incubated overnight with control medium. These results indicate that IL-12 induced the secretion of a soluble factor that is capable of directly down-modulating the surface expression of CCR5 in homologous or heterologous PBLs.

View larger version (20K): [in this window] [in a new window]   FIGURE 4. Effects of IL-12-treated PBLs medium on CCR5 expression of autologous and heterologous cells. PBLs from each of two donors (A and B) were cultured for 8 days in medium containing IL-2; they were then washed and incubated overnight with medium containing IL-2 (Med) or with the culture supernatant of autologous (A, from donor 3) or heterologous (B, from donor 5) PBLs that had been cultured for 8 days in the absence or presence of IL-12. Cells were stained with the 5C7 mAb to CCR5 or with an isotype control (IgG), as indicated, and then subjected to FCA.

Given that chemokines have been shown to induce down-regulation of their receptors through endocytosis (20), we next examined whether IL-12 stimulates the secretion of chemokines that interact specifically with CCR5 (21, 22). Therefore, we measured the amounts of the ß-chemokines MIP-1, MIP-1ß, and RANTES in the culture supernatants of PBLs incubated for 8 days in medium containing IL-2, either in the absence or presence of IL-12. Only small amounts of these three chemokines were produced by IL-2-treated PBLs from eight unrelated donors (Fig. 5). However, culture of the PBLs in the presence of IL-12 induced a marked increase in the amounts of MIP-1, MIP-1ß, and RANTES in the culture supernatants; the maximal concentrations detected were 40, 90, and 10 ng/ml, respectively, and there was some individual variability in the total amount of chemokines produced. The concentrations of MIP-1 and MIP-1ß in the culture supernatants were higher than that of RANTES for PBLs from most donors; however, cells from one donor (donor 5) secreted substantial amounts of RANTES but little MIP-1 or MIP-1ß. These data suggested that the inhibition of CCR5 expression by IL-12 might be attributable to IL-12-induced secretion of ß-chemokines into the medium.

View larger version (23K): [in this window] [in a new window]   FIGURE 5. Effects of IL-12 on ß-chemokine secretion by PBLs. PBLs from eight unrelated donors were cultured for 8 days in medium containing IL-2 in the absence (Med) or presence of IL-12 (10 ng/ml), after which the amounts of MIP-1, MIP-1ß, and RANTES were measured in the culture supernatants by quantitative ELISA.

We investigated whether MIP-1, MIP-1ß, or RANTES released from IL-12-treated PBLs were responsible for the inhibitory effect of IL-12 on CCR5 expression. PBLs were incubated with IL-2 for 8 days, washed, and then incubated overnight with conditioned medium from 8-day cultures of IL-12-treated PBLs from donor 3 (Fig. 5) in the absence or presence of neutralizing Abs to each of the three ß-chemokines. Abs to either MIP-1 or MIP-1ß reversed the inhibition of CCR5 expression induced by the conditioned medium, with the effect of the former Abs being most pronounced, whereas Abs to RANTES had no marked effect (Fig. 6). These results show that MIP-1 and MIP-1ß were primarily responsible for the inhibition of CCR5 expression induced by this culture supernatant. When the Abs to the three chemokines were combined, CCR5 expression recovered to a level higher than that apparent for PBLs incubated with IL-2-containing medium or with conditioned medium from cells not exposed to IL-12. This observation suggests that low concentrations of endogenous ß-chemokines limit CCR5 expression even in the absence of exogenous IL-12. RANTES also appears to contribute to inhibition of CCR5 expression when present at high concentrations, as indicated by the observation that the inhibitory effect of medium conditioned by IL-12-treated PBLs from donor 5 (Fig. 5) was reversed by Abs to RANTES (data not shown).

View larger version (15K): [in this window] [in a new window]   FIGURE 6. Effects of neutralizing Abs to ß-chemokines on the inhibition of CCR5 expression by conditioned medium from IL-12-treated PBLs. PBLs were cultured for 8 days in medium containing IL-2; they were then washed and incubated overnight with fresh IL-2-containing medium (Med) or with the supernatants from 8-day cultures of PBLs maintained in the absence (Sup(-)) or presence (Sup(+)) of IL-12. For the bottom four traces, the Sup(+) supernatant was preincubated for 1 h with neutralizing Abs (20 mg/ml) to RANTES (-RANTES), to MIP-1 (-MIP-1), to MIP-1ß (-MIP-1ß), or to the combination of all three Abs (-RMM). Cells were stained with mAb 5C7 to CCR5 or with an isotype control (IgG), and then subjected to FCA. Similar results were obtained with Sup(+) supernatant derived from PBLs of three different donors.

Inhibition of ß-chemokine secretion by Th2-type cytokines, IL-4 and IL-10, and by IFN- and IL-18

Some effects of IL-12, such as its anti-tumor activity (23), involve the induction of IFN-. IL-12 has been reported to stimulate the production of other cytokines from PBLs (24), including IL-4 and IL-10. To examine the contribution of cytokines previously associated with IL-12 effects, IFN-, IL-4, and IL-10 were tested on ß-chemokine production in the presence of IL-2. Because IL-18 is a potent IFN- inducer (25) and because TNF- has been reported to induce ß-chemokines from fibroblasts, these two cytokines were also tested.

As shown in Fig. 7, IL-12 is the only potent inducer of MIP-1 and MIP-1ß secretion in this set of cytokines, although TNF- did have a small stimulatory effect on MIP-1 and MIP-1ß. In contrast, IL-4, IL-10, IFN-, and IL-18 strongly inhibited MIP-1ß production and had less of an inhibitory effect on MIP-1 production. These data suggest that the Th2 cytokines, IL-4 and IL-10, and Th1 cytokine, IFN-, do not induce MIP-1 and MIP-1ß secretion from PBLs and are therefore unlikely to be indirectly mediating IL-12 effects on chemokine production.

View larger version (22K): [in this window] [in a new window]   FIGURE 7. Regulation of MIP-1 and MIP-1ß secretion by cytokines. PBLs were cultured in medium containing IL-2 (Med), or IL-2 with IL-4, IL-10, IL-12, IL-18, TNF- (20 ng/ml each) or with IFN- (100 ng/ml) for 8 days, and then supernatants were assayed for chemokines. Representative results from two donors are shown.

We were next interested in determining whether the chemokines secreted in response to IL-12 were biologically active in triggering cells. As shown in Fig. 8A, control IL-2-cultured supernatant (S) did not stimulate cytosolic-free Ca²⁺ in monocytes or desensitize responses to MIP-1 or SDF-1 (30 nM each). A rapid and transient rise in intracellular Ca²⁺ concentration ([Ca²⁺]_i) was seen with IL-12 supernatant (Fig. 8B), which desensitized the response of monocytes to MIP-1, but not to SDF-1. These data indicate that IL-12 supernatant contains ligands for CCR1 and/or CCR5, but not ligands capable of stimulating through CXCR4. IL-12 supernatant also desensitized the response of monocytes to RANTES, but not to MCP-1 (data not shown). Complete desensitization of monocytes to IL-12 supernatant by MIP-1 was observed (Fig. 8C). Because MIP-1 desensitizes monocytes to MIP-1, MIP-1ß, and RANTES, but not to MCP-1, MCP-2, and MCP-3 (26), the above data suggest that IL-12 supernatant does not contain MCP-1, MCP-2, or MCP-3. A rapid and transient rise in [Ca²⁺]_i was also seen with IL-12 supernatant in macrophages, which desensitized the response of macrophages to MIP-1, but not to SDF-1ß (data not shown).

View larger version (53K): [in this window] [in a new window]   FIGURE 8. Calcium mobilization and desensitization. Monocytes loaded with fura-2 were stimulated sequentially with supernatant, MIP-1 and SDF-, and monitored for intracellular calcium. Reagents (15 µl) were added at 50, 150, and 220 s (arrowheads). A, IL-2 supernatant (S), MIP-1 (30 nM), and SDF-1 (30 nM). B, IL-12/IL-2 supernatant (IL-12S), MIP-1, and SDF-1. C, MIP-1, IL-12S, and SDF-1. The data shown for each of the conditions are representative of three independent measurements.

Because IL-12 down-modulated surface CCR5 expression through induction of MIP-1 and MIP-1ß, we next examined its impact on M-tropic HIV-1 infection of T cells. PBLs cultured in medium containing IL-2 produced 1697 pg/ml of p24, whereas PBLs cultured in medium containing both IL-12 and IL-2 before infection only produced 200 pg/ml of p24, an 88% reduction (Fig. 9).

View larger version (12K): [in this window] [in a new window]   FIGURE 9. Effects of IL-12 on HIV-1 infection in PBLs. PBLs were cultured in medium containing IL-2, or IL-12 plus IL-2 for 7 days. HIV-1 strain Ba-L was added to culture and left overnight. After extensive washing with PBS, PBLs were cultured in medium containing IL-2 for 7 days. Supernatant was assayed for p24 production by ELISA. The data shown are representative of two donors with similar results.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

CCR5 and CCR3 were identified as markers for Th1 and Th2 cells on the basis of their expression by PHA- and IL-2-stimulated clones polarized by treatment with IL-12 or IL-4, respectively (5, 6). In accordance with the reported data, our RT-PCR results showed increased CCR5 mRNA in PBLs cultured with IL-12- plus IL-2-containing medium, a condition standard for Th1 differentiation. However, because of the dramatically elevated production of ß-chemokines in our culture conditions, ligand-induced endocytosis of CCR5 results in down-modulation of the receptor from the cell surface. Conditions where Th1 clones continue to express CCR5 (5) suggest to us that cells in long-term culture may not continuously secrete CCR5-specific chemokines or that prior exposure to mitogenic (PHA) stimulation alters receptor modulation. Overall, our data indicate that surface CCR5 expression is not a stable marker for Th1 differentiation in primary cell culture. Our results are consistent with another study on murine Th1 cells and Th2 cells (28) where only Th1 cells, and not Th2 cells, expressed high levels of MIP-1, MIP-1ß and MCP-1.

We have previously shown that cytokines affect HIV-1 entry and replication in monocytes/macrophages through modulation of CCR5 expression. M-CSF and GM-CSF increased HIV-1 entry and production through up-regulation of CCR5, whereas IL-4 and IL-13 reduced HIV-1 production through down-modulation of CCR5 (11). Our present data indicate that CCR5 expression is regulated differentially in macrophages and T lymphocytes. Thus, whereas IL-4 and IL-13 directly reduce CCR5 expression on macrophages, these cytokines slightly increased CCR5 expression by T cells. In addition, unlike their effects on macrophages (11), M-CSF and GM-CSF did not induce CCR5 expression by T cells (data not shown). The effect of a specific cytokine on chemokine receptor expression therefore depends both on the type of cytokine receptor present on the target cell and on the array of chemokines induced by the cytokine. On the basis of our results, we propose that IL-12 selectively enhances Th1 cell responses through the following two mechanisms: 1) a direct effect on cell differentiation associated with Th1 cytokine expression, and 2) stimulation of T cells to produce chemokines that attract Th1 cells through CCR5 engagement. The ability of IL-12 to stimulate chemokine production may have relevance to other effects of IL-12 such as in stimulating anti-tumor immune responses and as a Th1 adjuvant for prophylactic and therapeutic vaccines.

Cytokine induction of chemokines has been the focus of several recent reports. Perera et al. (29) reported that IL-15 induces low levels of chemokines in T lymphocytes. Fehniger et al. (30) reported that IL-18 or IL-15 in combination with IL-12 induces cytokines and ß-chemokines in human NK cells. Our results showing the secretion of chemokines from PBLs in response to IL-12 plus IL-2 in the absence of prior Ag sensitization suggest a possible role for T cell-derived ß-chemokines in early responses to infection.

IL-12 promotes lymphocyte replication (31, 32, 33), induces the production of ß-chemokines, and down-regulates the expression of CCR5 on CD4⁺ T cells. Given that ß-chemokines play a pivotal role in protection against HIV-1 infection (34, 35) and that CCR5 is the primary coreceptor for M-tropic HIV-1 (36, 37, 38, 39, 40), IL-12 may contribute to control of HIV infection through its effects on chemokines and their receptors. Our results showing that M-tropic HIV-1 replication is inhibited in T cells cultured in medium containing IL-12 plus IL-2 (compared with that containing IL-2 alone), in conjunction with results showing that IL-12 also inhibited SI virus infection of T cells (41), suggest that a combination of IL-2 and IL-12 may be useful as an adjunct to current antiviral therapies for HIV infection. Whether combination of IL-2, IL-12, and IL-13 can simultaneously control HIV-1 infection of both T cells and macrophages warrants further investigation.

The ability of IL-12 to stimulate chemokine production may have relevance to other effects of IL-12 such as in stimulating antitumor immune responses and as an adjuvant for prophylactic and therapeutic vaccines. The observation that combinations of IL-12 and IL-2 are more effective in controlling tumor growth than either cytokine alone (42, 43) raises the possibility that ß-chemokine induction may be involved in the antitumor activity of these cytokines.

	Acknowledgments

 
We thank V. Calvert for the preparation of human PBLs and monocytes, and Tamas Oravecz and Selice Chay for advice and help on scan. The mAbs 5C7 and 2D7 were obtained through the AIDS Research and Reference Reagent Program of the Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

	Footnotes

 
¹ Address correspondence and reprint requests to Drs. Jinhai Wang or Michael A. Norcross, Laboratory of Cell and Viral Regulation, Division of Therapeutic Proteins, Center for Biologics Evaluation and Research, Food and Drug Administration, National Institutes of Health, Building 29B, Room 4E12, HFM 535, 8800 Rockville Pike, Bethesda, MD 20892. E-mail addresses:

² Abbreviations used in this paper: M-tropic, macrophage-tropic; rhIL-2, recombinant human IL-2; MIP, macrophage-inflammatory protein; SDF, stromal cell-derived factor; FCA, flow cytometric analysis; MFI, mean fluorescence intensity.

Received for publication June 8, 1999. Accepted for publication September 8, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Trinchieri, G.. 1995. Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity. Annu. Rev. Immunol. 13:251.[Medline]
2. Kobayashi, M., L. Fitz, M. Ryan, R. M. Hewick, S. C. Clark, S. Chan, R. Loudon, F. Sherman, B. Perussia, G. Trinchieri. 1989. Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes. J. Exp. Med. 170:827.[Abstract/Free Full Text]
3. Manetti, R., P. Parronchi, M. G. Giudizi, M. P. Piccinni, E. Maggi, G. Trinchieri, S. Romagnani. 1993. Natural killer cell stimulatory factor (interleukin 12 [IL-12]) induces T helper type 1 (Th1)-specific immune responses and inhibits the development of IL-4-producing Th cells. J. Exp. Med. 177:1199.[Abstract/Free Full Text]
4. Chan, S. H., B. Perussia, J. W. Gupta, M. Kobayashi, M. Pospisil, H. A. Young, S. F. Wolf, S. Young, S. C. Clark, G. Trinchieri. 1991. Induction of interferon production by natural killer cell stimulatory factor: characterization of the responder cells and synergy with other inducers. J. Exp. Med. 173:869.[Abstract/Free Full Text]
5. Loetscher, P., M. Uguccioni, L. Bordoli, M. Baggiolini, B. Moser, C. Chizzolini, J. M. Dayer. 1998. CCR5 is characteristic of Th1 lymphocytes. Nature 391:344.[Medline]
6. Sallusto, F., C. R. Mackay, A. Lanzavecchia. 1997. Selective expression of the eotaxin receptor CCR3 by human T helper 2 cells. Science 277:2005.[Abstract/Free Full Text]
7. Sallusto, F., D. Lenig, C. R. Mackay, A. Lanzavecchia. 1998. Flexible programs of chemokine receptor expression on human polarized T helper 1 and 2 lymphocytes. J. Exp. Med. 187:875.[Abstract/Free Full Text]
8. Zella, D., O. Barabitskaja, J. M. Burns, F. Romerio, D. E. Dunn, M. G. Revello, G. Gerna, Jr M. S. Reitz, R. C. Gallo, F. F. Weichold. 1998. Interferon increases expression of chemokine receptors CCR1, CCR3, and CCR5, but not CXCR4 in monocytoid U937 cells. Blood 91:4444.[Abstract/Free Full Text]
9. Bleul, C. C., L. Wu, J. A. Hoxie, T. A. Springer, C. R. Mackay. 1997. The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes. Proc. Natl. Acad. Sci. USA 94:1925.[Abstract/Free Full Text]
10. Jourdan, P., C. Abbal, N. Nora, T. Hori, T. Uchiyama, J. P. Vendrell, J. Bousquet, N. Taylor, J. Pene, H. Yssel. 1998. IL-4 induces functional cell-surface expression of CXCR4 on human T cells. J. Immunol. 160:4153.[Abstract/Free Full Text]
11. Wang, J., G. Roderiquez, T. Oravecz, M. A. Norcross. 1998. Cytokine regulation of human immunodeficiency virus type 1 entry and replication in human monocytes/macrophages through modulation of CCR5 expression. J. Virol. 72:7642.[Abstract/Free Full Text]
12. Di Marzio, P., J. Tse, N. R. Landau. 1998. Chemokine receptor regulation and HIV type 1 tropism in monocyte-macrophages. AIDS Res. Hum. Retroviruses 14:129.[Medline]
13. Foli, A., W. Saville, M. W. Baseler, R. Yarchoan. 1995. Effects of the Th1 and Th2 stimulatory cytokines interleukin-12 and interleukin-4 on human immunodeficiency virus replication. Blood 85:2114.[Abstract/Free Full Text]
14. Perales, M. A., P. R. Skolnik, J. Lieberman. 1996. Effect of interleukin-12 on in vitro HIV Type 1 replication depends on clinical stage. AIDS Res. Hum. Retroviruses 12:659.[Medline]
15. Connors, M., J. A. Kovacs, S. Krevat, J. C. Gea-Banacloche, M. C. Sneller, M. Flanigan, J. A. Metcalf, R. E. Walker, J. Falloon, M. Baseler, et al 1997. HIV infection induces changes in CD4⁺ T-cell phenotype and depletions within the CD4⁺ T-cell repertoire that are not immediately restored by antiviral or immune-based therapies. Nat. Med. 3:533.[Medline]
16. Jr Davey, R. T., D. G. Chaitt, S. C. Piscitelli, M. Wells, J. A. Kovacs, R. E. Walker, J. Falloon, M. A. Polis, J. A. Metcalf, H. Masur, et al 1997. Subcutaneous administration of interleukin-2 in human immunodeficiency virus type 1-infected persons. J. Infect. Dis. 175:781.[Medline]
17. Wu, L., W. A. Paxton, N. Kassam, N. Ruffing, J. B. Rottman, N. Sullivan, H. Choe, J. Sodroski, W. Newman, R. A. Koup, C. R. Mackay. 1997. CCR5 levels and expression pattern correlate with infectability by macrophage-tropic HIV-1, in vitro. J. Exp. Med. 185:1681.[Abstract/Free Full Text]
18. Wu, L., G. LaRosa, N. Kassam, C. J. Gordon, H. Heath, N. Ruffing, H. Chen, J. Humblias, M. Samson, M. Parmentier, et al 1997. Interaction of chemokine receptor CCR5 with its ligands: multiple domains for HIV-1 gp120 binding and a single domain for chemokine binding. J. Exp. Med. 186:1373.[Abstract/Free Full Text]
19. Moore, J. P.. 1997. Coreceptors: implications for HIV pathogenesis and therapy. Science 276:51.[Free Full Text]
20. Amara, A., S. L. Gall, O. Schwartz, J. Salamero, M. Montes, P. Loetscher, M. Baggiolini, J. L. Virelizier, F. Arenzana-Seisdedos. 1997. HIV coreceptor down-regulation as antiviral principle: SDF-1-dependent internalization of the chemokine receptor CXCR4 contributes to inhibition of HIV replication. J. Exp. Med. 186:139.[Abstract/Free Full Text]
21. Raport, C. J., J. Gosling, V. L. Schweickart, P. W. Gray, I. F. Charo. 1996. Molecular cloning and functional characterization of a novel human CC chemokine receptor (CCR5) for RANTES, MIP-1ß, and MIP-1. J. Biol. Chem. 271:17161.[Abstract/Free Full Text]
22. Samson, M., O. Labbe, C. Mollereau, G. Vassart, M. Parmentier. 1996. Molecular cloning and functional expression of a new human CC-chemokine receptor gene. Biochemistry 35:3362.[Medline]
23. Nastala, C. L., H. D. Edington, T. G. McKinney, H. Tahara, M. A. Nalesnik, M. J. Brunda, M. K. Gately, S. F. Wolf, R. D. Schreiber, W. J Storkus. 1994. Recombinant IL-12 administration induces tumor regression in association with IFN- production. J. Immunol. 153:1697.[Abstract]
24. Windhagen, A., D. E. Anderson, A. Carrizosa, R. E. Williams, D. A. Hafler. 1996. IL-12 induces human T cells secreting IL-10 with IFN-. J. Immunol. 157:1127.[Abstract]
25. Okamura, H., S. I. Kashiwamura, H. Tsutsui, T. Yoshimoto, K. Nakanishi. 1998. Regulation of interferon- production by IL-12 and IL-18. Curr. Opin. Immunol. 10:259.[Medline]
26. Uguccioni, M., M. D’Apuzzo, M. Loetscher, B. Dewald, M. Baggiolini. 1995. Actions of the chemotactic cytokines MCP-1, MCP-2, MCP-3, RANTES, MIP-1 and MIP-1ß on human monocytes. Eur. J. Immunol. 25:64.[Medline]
27. Schrum, S., P. Probst, B. Fleischer, P. F. Zipfel. 1996. Synthesis of the CC-chemokines MIP-1, MIP-1ß, and RANTES is associated with a type 1 immune response. J. Immunol. 157:3598.[Abstract]
28. Bradley, L. M., V. C. Asensio, L. K. Schioetz, J. Harbertson, T. Krahl, G. Patstone, N. Woolf, I. L. Campbell, N. Sarvetnick. 1999. Islet-specific Th1, but not Th2, cells secrete multiple chemokines and promote rapid induction of autoimmune diabetes. J. Immunol. 162:2511.[Abstract/Free Full Text]
29. Perera, L. P., C. K. Goldman, T. A. Waldmann. 1999. IL-15 induces the expression of chemokines and their receptors in T lymphocytes. J. Immunol. 162:2606.[Abstract/Free Full Text]
30. Fehniger, T. A., M. H. Shah, M. J. Turner, J. B. VanDeusen, S. P. Whitman, M. A. Cooper, K. Suzuki, M. Wechser, F. Goodsaid, and M. A. Caligiuri. Differential cytokine and chemokine gene expression by human NK cells following activation with IL-18 or IL-15 in combination with IL-12: implications for the innate immune response. J. Immunol. 162:4511.
31. Hayes, M. P., J. Wang, M. A. Norcross. 1995. Regulation of interleukin-12 expression in human monocytes: selective priming by interferon- of lipopolysaccharide-inducible p35 and p40 genes. Blood 86:646.[Abstract/Free Full Text]
32. Perussia, B., S. H. Chan, A. D’Andrea, K. Tsuji, D. Santoli, M. Pospisil, D. Young, S. F. Wolf, G. Trinchieri. 1992. Natural killer (NK) cell stimulatory factor or IL-12 has differential effects on the proliferation of TCR-ß⁺, TCR-⁺ T lymphocytes, and NK cells. J. Immunol. 149:3495.[Abstract]
33. Gately, M. K., B. B. Desai, A. G. Wolitzky, P. M. Quinn, C. M. Dwyer, F. J. Podlaski, P. C. Familletti, F. Sinigaglia, R. Chizonnite, U. Gubler. 1991. Regulation of human lymphocyte proliferation by a heterodimeric cytokine, IL-12 (cytotoxic lymphocyte maturation factor). J. Immunol. 147:874.[Abstract]
34. Zagury, D., A. Lachgar, V. Chams, F. S. Fall, J. Bernard, J. F. Zagury, B. Bizzini, A. Gringeri, E. Santagostino, J. Rappaport, et al 1998. C-C chemokines, pivotal in protection against HIV type 1 infection. Proc. Natl. Acad. Sci. USA 95:3857.[Abstract/Free Full Text]
35. Cocchi, F., A. L. DeVico, A. Garzino-Demo, S. K. Arya, R. C. Gallo, P. Lusso. 1995. Identification of RANTES, MIP-1, and MIP-1ß as the major HIV-suppressive factors produced by CD8⁺ T cells. Science 270:1811.[Abstract/Free Full Text]
36. Choe, H., M. Farzan, Y. Sun, N. Sullivan, B. Rollins, P. D. Ponath, L. Wu, C. R. Mackay, G. LaRosa, W. Newman, et al 1996. The ß-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 85:1135.[Medline]
37. Alkhatib, G., C. Combadiere, C. C. Broder, Y. Feng, P. E. Kennedy, P. M. Murphy, E. A. Berger. 1996. CC CKR5: a RANTES, MIP-1, MIP-1ß receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 272:1955.[Abstract]
38. Dragic, T., V. Litwin, G. P. Allaway, S. R. Martin, Y. Huang, K. A. Nagashima, C. Cayanan, P. J. Maddon, R. A. Koup, J. P. Moore, W. A. Paxton. 1996. HIV-1 entry into CD4⁺ cells is mediated by the chemokine receptor CC-CKR-5. Nature 381:667.[Medline]
39. Deng, H., R. Liu, W. Ellmeier, S. Choe, D. Unutmaz, M. Burkhart, P. Di Marzio, S. Marmon, R. E. Sutton, C. M. Hill, et al 1996. Identification of a major co-receptor for primary isolates of HIV-1. Nature 381:661.[Medline]
40. Doranz, B. J., J. Rucker, Y. Yi, R. J. Smyth, M. Samson, S. C. Peiper, M. Parmentier, R. G. Collman, R. W. Doms. 1996. A dual-tropic primary HIV-1 isolate that uses fusin and the ß-chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors. Cell 85:1149.[Medline]
41. Salgame, P., M. X. Guan, A. Agahtehrani, E. E. Henderson. 1998. Infection of T cell subsets by HIV-1 and the effects of interleukin-12. J. Interferon Cytokine Res. 18:521.[Medline]
42. Wigginton, J. M., K. L. Komschlies, T. C. Back, J. L. Franco, M. J. Brunda, R. H. Wiltrout. 1996. Administration of interleukin-12 with pulse interleukin-2 and the rapid and complete eradication of murine renal carcinoma. J. Natl. Cancer Inst. 88:38.[Abstract/Free Full Text]
43. Addison, C. L., J. L. Bramson, M. M. Hitt, W. J. Muller, J. Gualdie, F. L. Graham. 1998. Intratumoral coinjection of adenoviral vectors expressing IL-2 and IL-12 results in enhanced frequency of regression of injected and untreated distal tumors. Gene Ther. 5:1400.[Medline]

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