Regulation of Human Immunodeficiency Virus Type 1 Infection, β-Chemokine Production, and CCR5 Expression in CD40L-Stimulated Macrophages: Immune Control of Viral Entry

Isolation and culturing of primary monocytes.

Human monocytes were recovered from peripheral blood mononuclear cells of HIV-1-, HIV-2-, and hepatitis B virus-seronegative donors after leukapheresis and then purified by countercurrent centrifugal elutriation (25). Monocytes were cultured as adherent monolayers (3.3 × 10⁶ cells/well in 6-well plates, 2.2 × 10⁶ cells/well in 12-well plates, and 1.1 × 10⁶ cells/well in 24-well plates) and differentiated for 7 days in Dulbecco modified Eagle medium (Sigma Chemical Co., St. Louis, Mo.) supplemented with 10% heat-inactivated pooled human serum, 50 μg of gentamicin (Sigma)/ml, 10 μg of ciprofloxacin (Sigma)/ml, and macrophage colony-stimulating factor (M-CSF; 1,000 U/ml; highly purified recombinant; a generous gift from Genetics Institute, Inc., Cambridge, Mass.). All tissue reagents were screened and found negative for endotoxin (<10 pg/ml) (Pyrotell Limulus amoebocyte lysate [LAL]; Associates of Cape Cod, Inc., Woods Hole, Mass.) and mycoplasma contamination (Gen-Probe II; Gen-Probe Inc., San Diego, Calif.).

Infection of MDM.

Seven days after plating, MDM were infected with HIV-1_ADA, HIV-1_JR-FL, or HIV-1_89.6 at a multiplicity of infection of 0.1 virus/target cell (25). Viral stocks were screened for mycoplasma and endotoxin using hybridization and Limulus amebocyte lysate assays, respectively. Culture media were half-exchanged every 2 to 3 days. RT activity was determined in triplicate samples of culture fluids as described below. One to seven days after infection, HIV-1_ADA-infected and replicate uninfected MDM were treated with soluble trimeric CD40L (a generous gift from Immunex Corporation, Seattle, Wash.).

Measurements of RT activity.

RT activity was determined in triplicate samples of cell culture fluids. For this assay, 10 μl of supernatant was incubated in a reaction mixture of 0.05% Nonidet P-40, 10 μg of poly(A)/ml, 0.25 μg of oligo(dT)/ml, 5 mM dithiothreitol, 150 mM KCl, 15 mM MgCl₂, and [³H]TTP in Tris-HCl buffer (pH 7.9) for 24 h at 37°C. Radiolabeled nucleotides were precipitated with cold 10% trichloroacetic acid on paper filters in an automatic cell harvester and washed with 95% ethanol. Radioactivity was estimated by liquid scintillation spectroscopy (37).

Detection of RT by the Lenti-RT activity assay.

Monocytes were cultured for 7 days prior to inoculation with the following HIV-1 strains: HIV-1_ADA, HIV-1_JR-FL, and HIV-1_89.6. Five days after inoculation with HIV-1, MDM were stimulated with CD40L (2 μg/ml) for 48 h. Culture fluids were collected, and the level of RT enzyme was determined using a Lenti-RT activity assay kit (Cavidi Tech, Uppsala, Sweden) in accordance with the manufacturer's instructions. Briefly, cell supernatants were incubated with bromo-dUTP, and incorporated bromo-UTP was detected by bromodeoxyuridine binding antibody conjugated to alkaline phosphatase. The level of RT in the sample was determined by colorimetric analysis of the alkaline phosphatase activity.

PCR analysis of HIV-1 DNA synthesis.

Monocytes (1.1 × 10⁶/ml) were cultured in 24-well plates (Costar Corp.) and infected with HIV-1 as described above. Prior to infection, the HIV-1 cell-free stocks were treated with DNase I for 30 min at 37°C (57). At 4, 8, 24, 48, and 96 h following viral exposure, samples were collected for RT analysis, and the residual medium was washed off with fresh phosphate-buffered saline (PBS; Sigma). The cells were then scraped into 0.5 ml of PBS. The resultant cell pellet was used for the extraction of cellular DNA with an Iso-quick nucleic acid extraction kit (ORCA Research Inc., Bothell, Wash.). The DNA was resuspended at a concentration of 2 × 10⁴ cell equivalents/μl. PCR was performed to identify early (primers for long terminal repeat [LTR] U3/R) and late (primers for LTR U3/gag) products of reverse transcription (79). A ratio comparing the levels of early and late viral cDNAs to the levels of mitochondrial DNA (an internal control) was then determined. Standard HIV-1 cDNAs were prepared by simultaneous amplification of serial twofold dilutions of DNA extracted from 8e5 cells harboring defective HIV-1 proviruses (14). Amplified products were run on a Southern blot, hybridized to radiolabeled oligonucleotide probes, and quantified on a PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.).

Detection of MIP-1α, MIP-1β, RANTES, and TNF-α by an ELISA.

Monocytes were cultured for 7 days prior to infection with HIV-1. One to seven days after infection with HIV-1, MDM were stimulated with CD40L (2 μg/ml) for 6, 24, or 48 h. Culture fluids were collected for chemokine and cytokine determinations. Chemokine production was assayed using Quantikine ELISA kits (R & D Systems, Minneapolis, Minn.) in accordance with the manufacturer's instructions. Chemokine and cytokine levels were normalized to cell numbers by measuring cell viability (51).

MTT reduction assay.

Cell cytotoxicity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction (51). Cells were incubated with 100 μl of MTT solution for 20 min at 37°C. The extent of MTT conversion to formazan by mitochondrial dehydrogenase, indicating cell viability, was determined by measuring the optical density (OD) at 490 nm using a microplate reader. The ratio of OD from treated cells to the OD from control cells reflected the percentage of surviving cells and was used to standardize cytokine and chemokine production determined by the ELISA.

FACS analysis.

MDM were cultured for 7 days at a density of 2.2 × 10⁶ cells/ml. After 48 h in the presence or absence of CD40L (2 μg/ml), alone or in combination with CD40L antibody (M91; 20 μg/ml) or a cocktail of chemokine antibodies (anti-RANTES [5 μg/ml], anti–MIP-1β [5 μg/ml], and anti–MIP-1α [5 μg/ml]; R & D Systems), cells were washed with a 3% fetal bovine serum–PBS solution and then incubated with the following fluorescence-labeled antibodies: CCR5-PE, CD4-PE, and CD14-FITC (Pharmingen, Torrance, Calif.) (41). After 30 min of antibody incubation, cells were washed twice in a 3% fetal bovine serum–PBS solution and then fixed with 1% paraformaldehyde. Expression of cell surface antigens was assessed by immunofluorescence flow cytometry (FACSCalibur; Becton Dickinson) and analyzed with CellQuest software.

Intracellular calcium measurements.

MDM cultured on glass coverslips for 7 days were treated with human recombinant trimeric CD40L (2 μg/ml) for 24 h. Control and CD40L-treated MDM were washed and incubated with 5 μM fura II-AM (Molecular Probes, Inc., Eugene, Oreg.) for 30 min at 37°C in Ringer's solution of the following composition: 148 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 1.6 mM Na₂HPO₄, 1.5 mM CaCl₂, and 5 mM d-glucose. The cells were washed twice and then incubated again for 20 min in Ringer's solution to allow for intracellular dye cleavage. The coverslips were held by an Attofluor cell chamber (Molecular Probes, Inc.), and 10 to 15 cells were chosen for imaging at room temperature. Data were recorded as the fluorescence emitted at 510 nm following excitation at 340 and 380 nm using a PTI Deltascan system as previously described (81). The concentrations of Ca²⁺ were calculated as follows: [Ca²⁺] = K_d[(R − R_min)/(R_max − R)] × (380_min/380_max), where R_min and R_max are the fluorescence values in the absence (with 3 mM EGTA) and presence of saturating Ca²⁺ (3 mM), respectively; K_d was 224 nM using PTI calcium imaging software.

Production of HIV-1 reporter viruses and the viral entry assay.

HIV-1 reporter viruses were produced as previously described (29). Briefly, envelope (Env)-defective recombinant HIV-1 luciferase reporter viruses were generated by cotransfection of 293T cells with 20 μg of pNL4–3env-LUC and 4 μg of plasmids encoding different HIV-1 Env proteins or pSVMLVenv using the calcium phosphate method (30). Pseudotyped HIVs were collected 48 h after transfection and assayed for RT enzyme activity. pNL4–3env-LUC, which encodes full-length Env-defective NL4–3 HIV-1 proviral DNA and expresses the luciferase reporter gene, was constructed as described previously (30). Complementation of Env-defective HIV-1 with HIV-1 Env expression plasmids in trans allows a single round of infection to occur and infected cells to be detected by the expression of luciferase. The Env plasmids used were YU-2env (HIV-1; CCR5 strain), HXB2env (HIV-1; CXCR4 strain), and MLVenv (amphotropic murine leukemia virus).

For the viral entry assay, 293T cells were transfected with plasmids expressing either CD4, CCR5, or CXCR4, alone or in combination. The transfected cells were plated on 24-well plates (10⁵ cells/well) 24 h after transfection. The cells were incubated for 1 h at 37°C in macrophage-conditioned medium (MCM; diluted 1:10) from control MDM (Con MCM) or CD40L MCM prior to the addition of pseudotyped HIV-1 reporter viruses (50,000 cpm of viruses per well). The cell lysates were prepared 48 h after infection and assayed for luciferase activity (counts per second).

Statistical tests.

Data were reported as means and standard deviations (SD) of the mean. The data were normalized to cell numbers using the MTT assay for cell viability. The normalized values were used to perform statistical analyses using the analysis of variance, followed by the two-tailed Student t test for paired observations. To account for any donor-specific differences, experiments were performed with MDM derived from multiple donors. All assays were performed a minimum of three times with MDM obtained from three independent donors. Each assay was done in triplicate.

CD40L inhibits virus production in HIV-1-infected MDM.

To examine the effects of CD40L on ongoing HIV-1 infection and virus production in MDM, freshly elutriated human monocytes were plated and then allowed to differentiate for 7 days in medium containing M-CSF. The resulting MDM were infected with HIV-1_ADA and then activated with soluble trimeric CD40L (2 μg/ml). Using this experimental system, our initial studies revealed that a single treatment with CD40L led to the inhibition of productive HIV-1 infection in the infected MDM. However, this effect was not sustainable over extended periods of time (data not shown).

In an attempt to maintain constant levels of CD40L within our cultures, the ligand was added at 3-day intervals, beginning 1 day following virus inoculation. Compared to untreated, HIV-1-infected controls, HIV-1-infected MDM treated with CD40L (2 μg/ml) showed decreased levels of virus production, as measured by RT activity (Fig. 1A; from 20 × 10⁵ to 2 × 10⁵ cpm/ml). This CD40L-mediated inhibition was consistently observed at days 3, 6, and 9 following virus inoculation (Fig. 1A).

To substantiate the inhibitory effects of CD40L on virus production, increasing doses of CD40L (ranging from 0.02 to 3.0 μg/ml) were used. CD40L-mediated inhibition of HIV-1 was shown to be dose dependent, with doses of greater than or equal to 1 μg/ml causing the most dramatic decreases in virus production, as measured by RT activity (Fig. 1B). Similar results were obtained using MDM from different donors (n ≥ 3). The specificity of the effect of CD40L on HIV-1 infection was determined by use of neutralizing antibodies to CD40L, such as M90 (Fig. 1C) and M91 (see Fig. 6A and 7A).

To further confirm the significance of the inhibitory effect of CD40L on HIV-1, a panel of HIV-1 strains, including the macrophage-tropic (M-tropic) strains HIV-1_ADA and HIV-1_JR-FL and the dual-tropic strain HIV-1_89.6, were used to infect MDM. Five days after inoculation, MDM were stimulated with CD40L (2 μg/ml). Supernatants were collected 48 h after activation and analyzed for RT activity using the Lenti-RT activity assay kit as described in Materials and Methods. MDM infected with HIV-1_ADA and then treated with CD40L exhibited a 64% decrease (from 224 ± 4.52 to 81 ± 32.68 pg/ml) in RT levels compared to untreated HIV-1_ADA-infected MDM (Fig. 2). Similarly, activation of MDM infected with HIV-1_JR-FL or HIV-1_89.6 also led to decreased virus production, with an 88% decrease (from 94 ± 12.31 to 11 ± 5.87 pg/ml) in the JR-FL-infected cultures and an 87% decrease (from 199 ± 28.85 to 26 ± 2.22 pg/ml) in the 89.6-infected cultures (Fig. 2). Similar results were confirmed by both radioactive labeling and alkaline phosphatase RT activity assays using supernatants collected from four different MDM donors.

Secretory products from CD40L-stimulated MDM inhibit productive HIV-1 infection.

Having demonstrated that the direct addition of CD40L to cultures of infected MDM could inhibit virus production and that antibodies to CD40L could block this effect, we next examined the effects of secretory products from CD40L-activated MDM on productive HIV-1 infection. For this work, MCM collected from uninfected (Con MCM), CD40L-treated (CD40L MCM), HIV-1_ADA-infected (HIV-1 MCM), or HIV-1_ADA-infected and CD40L-treated (HIV-1/CD40L MCM) cells was placed on replicate cultures of infected MDM, and virus production was measured as RT activity. Figure 3 demonstrates that CD40L MCM caused an 80% reduction (from 30 × 10⁵ to 6 × 10⁵ cpm/ml) in RT activity when added to replicate cultures of MDM infected with HIV-1_ADA. Interestingly, transfer of HIV-1/CD40L MCM also caused a 73% reduction (from 30 × 10⁵ to 8 × 10⁵ cpm/ml) in RT activity. The addition of neutralizing antibodies to CD40L (M90; 8 μg/ml) to MCM before addition to replicate MDM cultures did not block the inhibitory effects of either CD40L MCM (data not shown) or HIV-1/CD40L MCM (Fig. 3). However, in MDM directly treated with CD40L, neutralizing antibodies blocked the inhibitory effects of CD40L on HIV-1 (Fig. 1C). These data suggest that secretory factors produced in response to CD40L-mediated activation of MDM inhibit HIV-1 replication.

CD40L and CD40L MCM inhibit viral DNA synthesis in HIV-1-inoculated MDM.

To determine at what stage CD40L or secretory products from CD40L-activated MDM affect HIV-1 infection in MDM, we examined the effects of these factors on the earliest stages of the HIV-1 life cycle. For this work, MDM were pretreated for 1 h with medium alone, CD40L MCM, or antibody to CD4 (BL4; 10 μg/ml) or for 24 h with zidovudine (AZT; 5 μM) and then inoculated with DNase-treated HIV-1_ADA stocks. Four hours after inoculation, MDM were washed and then retreated with medium alone, CD40L (2 μg/ml), CD40L MCM, CD4 antibody, or AZT. At 4, 8, 24, 48, and 96 h following inoculation with virus, cells were fractured and DNA was isolated for measurement of viral nucleic acid synthesis. DNA PCR was performed to identify early (LTR U3/R) and late (LTR U3/gag) viral cDNA products of reverse transcription (79). A ratio comparing the levels of early and late viral cDNAs to the levels of mitochondrial DNA (an internal control) was then determined.

The Southern blot results from a representative experiment are shown in Fig. 4A. In HIV-1_ADA-infected cells, treatment with CD40L (2 μg/ml) or with CD40L MCM led to decreased synthesis of both early and late viral cDNAs. At 8 h postinfection (Fig. 4C), cells treated with CD40L showed a 72% reduction in viral DNA synthesis, while treatment with CD40L MCM induced a 56% decrease in the synthesis of viral DNA. At 48 h following virus inoculation (Fig. 4D), CD40L- and CD40L MCM-treated cells demonstrated 44 and 48% reductions in early viral DNA synthesis, respectively. Inhibition of both early and late viral gene products was also observed at 24 h postinoculation (data not shown). In MDM pretreated with AZT or CD4 antibody (BL4), which was used as a positive control, viral DNA synthesis was suppressed (Fig. 4A, C, and D). Similar results were obtained using MDM from three different donors. The results suggest that CD40L and secretory products from CD40L-stimulated MDM inhibit early events in the HIV-1 life cycle.

CD40L induces β-chemokine and TNF-α production in MDM.

The experiments described above suggest that CD40L inhibits HIV-1 infection in MDM through a macrophage-derived soluble factor(s). In an attempt to identify the factor(s) responsible for such an effect, we next examined the effects of CD40L on MDM secretory function. Since a role for chemokines in the regulation of HIV-1 infection has been proposed by others (40, 74), we measured proinflammatory cytokine and chemokine production in CD40L-stimulated MDM. For this work, we assayed culture supernatants from uninfected, HIV-1-infected, CD40L-stimulated, and HIV-1-infected and CD40L-stimulated MDM for the presence of β-chemokines (MIP-1α, MIP-1β, and RANTES) and proinflammatory cytokines (TNF-α) with an ELISA.

Treatment of uninfected MDM with CD40L (2 μg/ml) alone induced a 16-fold increase in RANTES (from 13 ± 3 pg/ml in control cells to 219 ± 5 pg/ml in CD40L-treated MDM) (Fig. 5A), a 4-fold increase in MIP-1α (from 134 ± 2 pg/ml to 543 ± 35 pg/ml) (Fig. 5B), a 5-fold increase in MIP-1β (from 79 ± 2 pg/ml to 410 ± 81 pg/ml) (Fig. 5C), and a 4-fold increase in TNF-α (from 81 ± 1 pg/ml to 333 ± 65 pg/ml) (Fig. 5D). In our experimental system, HIV-1 infection alone induced only a modest increase in these factors, a finding that is consistent with previously published results (63). In contrast, HIV-1-infected and CD40L-activated cells consistently produced the highest levels of these factors (Fig. 5). Indeed, CD40L treatment of HIV-1-infected MDM led to enhanced β-chemokine and TNF-α production, inducing a 13-fold increase in RANTES (from 26 ± 7 pg/ml to 340 ± 18 pg/ml), a 4-fold increase in MIP-1α (from 304 ± 20 pg/ml to 1,300 ± 384 pg/ml), a 22-fold increase in MIP-1β (from 111 ± 2 pg/ml to 2,500 ± 70 pg/ml), and a nearly 8-fold increase in TNF-α (from 116 ± 4 pg/ml to 900 ± 90 pg/ml), when compared to the levels produced by HIV-1-infected MDM at day 4 postinfection. The effects of CD40L on β-chemokine and TNF-α production were demonstrated to be dose dependent (data not shown).

These data suggested that the reduction in productive viral infection induced by CD40L (Fig. 1A) might be linked to the enhanced production of β-chemokines and TNF-α. To test this hypothesis, we examined the effects of blocking antibodies to individual cytokines and chemokines on the CD40L-mediated inhibition of HIV-1 infection. RANTES and TNF-α were selected as the primary candidates for this work, as both have been linked to the inhibition of HIV-1 infection in many cell types (4, 7, 9, 32, 41, 72).

Antibodies to RANTES and TNF-α reverse CD40L-mediated inhibition of HIV-1 infection.

To determine whether the ability of CD40L to inhibit HIV-1 was linked to its ability to enhance β-chemokine production, we examined the effect of neutralizing antibodies to RANTES (Fig. 6A) on virus production in CD40L-treated MDM. CD40L-induced inhibition of HIV-1 infection was blocked by antibodies to both CD40L (M91; 10 μg/ml) (Fig. 6A) and RANTES (5 μg/ml) (Fig. 6A). Other antibodies to CD40L (M90; 8 μg/ml) also blocked the production of RANTES in both uninfected (from 180 to 37 pg/ml) and infected (from 370 to 57 pg/ml) cells treated with CD40L (Fig. 6B). Used as a positive control for this work, RANTES (500 ng/ml) was administered to MDM 1 h prior to virus inoculation and added again (200 ng/ml) every 3 days. Treatment of MDM with RANTES caused an 80% decrease in RT activity compared to that in untreated controls (from 15 × 10⁵ to 3 × 10⁵ cpm/ml). Importantly, this response was also blocked by the addition of antibodies to RANTES (5 μg/ml). Moreover, treatment with MIP-1β or monocyte chemotactic protein 1 (MCP-1) (at doses ranging from 100 to 500 ng/ml) also inhibited HIV-1 infection. MIP-1β caused a 60% decrease in HIV-1 infection compared to results for untreated controls (from 15 × 10⁵ to 6 × 10⁵ cpm/ml), and MCP-1 induced a 72% decrease in RT activity (from 15 × 10⁵ to 4 × 10⁵ cpm/ml). Although the inhibitory effects of both MIP-1β and MCP-1 were blocked by the addition of their respective neutralizing antibodies (2 μg/ml), antibodies to these chemokines had no significant effect on the CD40L-mediated inhibition of HIV-1 infection in MDM (data not shown).

To further investigate the role of MDM secretory products in the CD40L-mediated inhibition of productive HIV-1 infection, we examined the relationship between decreased viral infection and cytokine production. TNF-α was selected as a representative cytokine, as it has been reported to diminish HIV-1 infection in macrophages through the production of β-chemokines (4, 32, 41). To determine whether the inhibitory effects of CD40L on HIV-1 replication were mediated by TNF-α, MDM were infected for 7 days with HIV-1_ADA and then treated with CD40L (2 μg/ml) or TNF-α (20 ng/ml). Supernatants were collected 6, 24, 48, and 72 h after activation and analyzed for RT activity (Fig. 7A) or TNF-α production (Fig. 7B). As shown in Fig. 7A, the reduction in HIV-1 infection induced by CD40L was partially blocked by the addition of antibodies to CD40L (from 6 × 10⁵ to 10 × 10⁵ cpm/ml). Importantly, the addition of neutralizing antibodies to TNF-α (2 μg/ml), when administered in conjunction with CD40L 7 days after infection, blocked the inhibitory effects of CD40L on HIV-1 (from 6 × 10⁵ to 17 × 10⁵ cpm/ml) (Fig. 7A). Used as a positive control for this work, TNF-α alone caused a 45% decrease in RT activity compared to that in untreated controls (from 15 × 10⁵ to 8 × 10⁵ cpm/ml) (P < 0.01). This response was also blocked by the addition of neutralizing antibodies to TNF-α (2 μg/ml) (from 8 × 10⁵ to 16 × 10⁵ cpm/ml) (Fig. 7A).

As shown in Fig. 7B, the ability of CD40L to induce TNF-α production was blocked by preincubation with neutralizing antibodies to CD40L (from 1.2 to 0.07 ng/ml). Importantly, antibodies to both CD40L and TNF-α blocked the CD40L-induced production of RANTES in both uninfected (Fig. 7C) and infected (data not shown) MDM. Interestingly, TNF-α (20 ng/ml) induced the production of RANTES (Fig. 7C) and MIP-1α and MIP-1β (data not shown). This effect was blocked by the addition of neutralizing antibodies to TNF-α (Fig. 7C). Moreover, stimulation of MDM with CD40L led to an early (1 to 4 h postactivation) increase in TNF-α production, followed by a later (4 to 8 h poststimulation) increase in β-chemokine levels (data not shown). Together, these data suggest that increased levels of TNF-α may contribute to the inhibitory effects of CD40L through further induction of β-chemokines.

CD40L-mediated activation alters CCR5 expression on MDM.

To further investigate the link between CD40L-mediated inhibition of HIV-1 infection and enhanced β-chemokine production, we examined the effects of CD40L activation on the expression of the β-chemokine receptor CCR5. MDM were treated with CD40L (2 μg/ml) for 48 h. The expression of CD14 (a monocyte marker), CCR5 (a β-chemokine receptor), and CD4 on both untreated and treated MDM was determined by FACS analysis. Macrophage populations were identified by forward- and side-scatter analysis and by CD14 immunoreactivity. The mean fluorescence intensity of CCR5 and CD4 expression on cells positive for both parameters was then determined.

Our experiments demonstrated that treatment with CD40L diminished CCR5 surface expression on MDM compared to untreated MDM. While on average CCR5 expression was decreased by 30% following CD40L treatment (Fig. 8A), the reduction in CCR5 expression ranged from 20 to 60%, depending on the donor (n = 5). This downregulation in CCR5 expression was shown to be specific, as antibodies to CD40L (M91; 20 μg/ml) were able to reverse the effect (Fig. 8C). Importantly, similar results were also observed with HIV-1-infected macrophages, where treatment with CD40L caused a 33 to 60% decrease in CCR5 expression (data not shown). In addition, MDM treated with the positive control RANTES (500 ng/ml), MIP-1β (500 ng/ml), MIP-1α (500 ng/ml), or TNF-α (100 ng/ml) also showed downregulation in CCR5 expression compared to control cells. RANTES induced a 30% reduction in CCR5 cell surface expression, MIP-1β and MIP-1α led to a 50% decrease, and TNF-α caused a 30% reduction (data not shown). Interestingly, a cocktail of chemokine antibodies (anti-RANTES [5 μg/ml], anti–MIP-1β [5 μg/ml], and anti–MIP-1α [5 μg/ml]) was also able to reverse the downregulation in CCR5 expression induced by treatment with CD40L (Fig. 8C). These results are consistent with published reports (4, 6, 41) showing that β-chemokines and TNF-α downregulate CCR5 expression. Importantly, these data provide further support for the hypothesis that soluble factors induced by CD40L-mediated activation of MDM lead to downregulation of the HIV-1 coreceptor, CCR5.

Importantly, the addition of CD40L (2 μg/ml) to cultures of MDM led to a 40% reduction in CD4 cell surface expression. This downregulation in CD4 was specific, as antibodies to CD40L (M91; 20 μg/ml) were able to reverse the effect (Fig. 8D). In contrast, a cocktail of chemokine antibodies (anti-RANTES [5 μg/ml], anti–MIP-1β [5 μg/ml], and anti–MIP-1α [5 μg/ml]) had no significant effect on the CD40L-induced downregulation of CD4 expression (Fig. 8D). The profiles shown in Fig. 8 are representative of the trends observed using MDM from five donors. These data suggest that the inhibitory effects of CD40L on productive HIV-1 infection in MDM may be mediated, at least in part, through the production of soluble factors, which in turn decrease CCR5 cell surface expression.

To further confirm the role of CD40L in regulation of CCR5, we determined the level of CCR5 activity on both untreated and CD40L-treated MDM using calcium imaging analysis. MIP-1α (1 μg/ml), a natural ligand for CCR5, was used to assay for chemokine receptor-mediated increases in intracellular calcium levels (Fig. 9). ATP (100 μM) was used as a control, as it affects intracellular calcium levels through CCR5-independent pathways (Fig. 9). In MDM pretreated with CD40L (2 μg/ml for 24 h), the calcium response induced by application of the CCR5 ligand MIP-1α was reduced (Fig. 9B) in comparison to the response evoked in untreated MDM (Fig. 9A). In contrast, ATP-induced calcium responses remained unchanged (Fig. 9A and B). This observation was confirmed in three separate experiments performed with three sets of MDM donors. The average of these data is shown in Fig. 9C, where the intracellular calcium response induced by MIP-1α was significantly higher (P < 0.01) in untreated MDM (0.965 ± 0.037; n = 3) than in CD40L-treated MDM (0.834 ± 0.043). These data are expressed as ratios of the absorbances at 340 and 380 nm. This finding, in conjunction with the results of the FACS analysis, demonstrates that the levels and activity of CCR5 on MDM are decreased after treatment with CD40L.

Secretory factors from CD40L-stimulated MDM inhibit M-tropic HIV-1 entry.

To determine whether factors produced by CD40L-treated MDM could block the entry of HIV-1, Con MCM or CD40L MCM (CD40L was used at 2 μg/ml) was placed on CCR5- and CD4-, CXCR4- and CD4-, CCR5-, CXCR4-, or CD4-transfected 293T cells. Viral entry was detected by measuring HIV-1 luciferase reporter virus activity. For this work, transfected 293T cells were incubated for 1 h at 37°C with either Con MCM or CD40L MCM and then infected with Env-defective recombinant HIV-1 luciferase reporter viruses pseudotyped with either YU2, a representative M-tropic CCR5 Env protein, or HXB2, a T-lymphocyte-tropic CXCR4 Env protein (29). The amphotropic murine leukemia virus (A-MLV) envelope was used as a control for specificity. The efficiency of viral entry was determined by measuring luciferase activity (counts per second) 48 h after infection. Results were obtained from triplicate determinations using MCM collected from four MDM donors.

As shown in Fig. 10A, CD40L MCM inhibited infection with HIV-1 luciferase reporter viruses containing YU2 Env by 40%, compared to the results for cells treated with Con MCM. In contrast, entry of a reporter virus containing HXB2 Env was not inhibited by the addition of CD40L MCM (Fig. 10C). Neither Con MCM nor CD40L MCM inhibited infection by virus pseudotyped with nonspecific A-MLV Env (Fig. 10B and 10D) or Env-defective HIV-1 reporter virus (data not shown). In addition, the direct addition of fresh medium or soluble CD40L alone had no inhibitory effects in any of the cell systems described above. These data suggest that the observed inhibition of viral entry is due to soluble factors produced by CD40L-activated MDM.