INTRODUCTION

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The complex interactions of human cells with human immunodeficiency virus type 1 (HIV-1) include effects restricted to productive infection and other responses that extend beyond active viral replication. Among the events following viral exposure that may be unrelated to infection, the greatest effects have been attributed to the envelope glycoprotein, gp120. In early studies, gp120 was shown to kill rodent neurons through a Ca²⁺-dependent pathway (10). By binding CD4, gp120 was found to activate protein kinase p56^lck and thus induce translocation of NF-B into the nucleus (34). Recent studies revisiting cytopathogenicity have demonstrated that gp120 can initiate apoptosis in multiple cell types (48). In particular, primary macrophages exposed to gp120 display membrane tumor necrosis factor alpha (TNF-) and trigger gp120-dependent apoptosis in bystander cells through TNF receptors (18). The latter studies reveal an apparent paradox regarding macrophage-gp120 interactions. Macrophages display both major HIV-1 coreceptors CCR5, a -chemokine receptor, and CXCR4, an -chemokine receptor (45, 47). They are highly susceptible to viruses that utilize CCR5 for entry, but they are generally resistant to productive infection by laboratory-adapted virus species that are restricted to CXCR4 (32, 39). They respond to laboratory-adapted X4 HIV-1 and their envelope glycoproteins by Ca²⁺ uptake (27), by secretion of unidentified neurotoxins (16), by apoptosis (48), and by induction of apoptosis in neighboring cells (18). We showed that X4 viruses that do not replicate in macrophages still enter cells and undergo the early phases of virus replication (19, 36). More recent studies demonstrated that macrophage CXCR4 is competent to mediate virus entry and that some primary X4 HIV-1 species productively infect macrophages (40). R5 HIV-1 or envelope can also induce signal transduction, secretion of neurotoxins, and activation of ion channels in macrophages (3, 20, 48). These findings suggest that ligation of CD4 and CCR5 or CXCR4 on macrophages by HIV-1 envelope is not sufficient to predict subsequent completion of the viral life cycle or activation of cellular responses.

In the present work we focused upon acute effects of virus exposure to investigate potentially protective responses of macrophages to HIV-1 that can be dissociated from productive infection. Discrimination of effects unlinked to virus production was achieved by four approaches. First, we tested the effects of exposure of macrophages to six X4 HIV-1 species that do not productively infect macrophages (1, 11, 35, 41, 44), as well as six R5 species and one R5/X4 HIV-1 species that productively infect (8, 14, 15, 24, 26, 44). Second, we tested responses to isolated gp120 of both coreceptor phenotypes. Third, we monitored responses 6 to 24 h after virus exposure, which is well before the peak of infection of macrophages, about 2 weeks later. Finally, we evaluated responses in the presence and absence of inhibitors of HIV-1 infection. We measured production of a set of secreted proteins implicated in several phases of HIV-1 disease. Among these are certain -chemokines that block HIV-1 infection in vitro (27) and are elevated in some exposed but uninfected individuals (46). However, these factors also have been shown to stimulate HIV-1-infected T cells to enhanced viral replication in culture (22). In addition, we tested production of TNF-, one of the primary inflammatory cytokines produced by HIV-1-infected cells, which also can be elevated in the brains of some HIV-1-infected persons (17, 43). We have compared the abilities of multiple species of HIV-1 to induce primary human macrophages to produce macrophage inflammatory protein 1 (MIP-1), MIP-1, macrophage chemotactic protein (MCP-1), RANTES, and TNF-. In novel findings we report that within hours of exposure to HIV-1 or viral gp120, macrophages secreted very high levels of several chemokines and TNF-. Two of six R5 viruses and three of six X4 viruses tested failed to induce this response, and neutralizing antibodies or soluble CD4 failed to block this response, indicating that binding CD4 and either CCR5 or CXCR4 was insufficient for induction. By contrast, all the R5 HIV-1 species induced -chemokine synthesis at the peak of viral infection, as previously reported (37, 42). We conclude that a major immediate response of macrophages to either R5 or X4 HIV-1 exposure is secretion of high levels of -chemokines and TNF-. Such secretion may be seen as part of the innate immune response eliciting lymphocyte migration (13) to a viral source to establish an antigen-specific response prior to major viral spread.

	MATERIALS AND METHODS

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Cells and viruses. Human monocytes were prepared from peripheral blood mononuclear cells of HIV-1- and hepatitis B virus-negative donors by countercurrent centrifugal elutriation. Monocytes were >98% pure by Ham56 and CD68 staining. Monocytes were allowed to adhere and differentiate to macrophages (MDM) at a concentration of 2.5 × 10⁵ cells/well in Dulbecco's Modified Eagle Medium (Sigma, St. Louis, Mo.) with 10% endotoxin-free, heat-inactivated human serum, 10% giant cell tumor conditioned medium (Sigma), 2 mM glutamine, and antibiotics. Cells were cultured for 7 days prior to infection or stimulation. The following HIV-1 species were used in this study: ADA (R5 [15]), BaL (R5 [14]), JR-CSF (R5 [24]), JR-FL (R5 [24]), Lai (X4 [33]), NDK (X4 [11]), NL4-3 (X4 [1]), NLHXADA-GP (R5 [44]), NLHXDADA-PG (X4 [44]), Yu2 (R5 [26]), Z6 (X4 [41]), IIIB (X4 [35]), and 89.6 (R5/X4 [8]). Viral stocks were prepared by either proviral DNA transfection of 293T cell (pYu2, pGP, pNDK, pNL4-3, pZ6, p89.6, pLai.2, and pPG), by cell-free virus infection of macrophages (ADA, BaL, JR-FL, and JR-CSF) or by culturing of chronically infected H9-HTLV-IIIB cells (35). ADA, JR-FL, and BaL were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program; ADA and BaL were also obtained from H. Gendelman. Viral stocks were concentrated by high-speed centrifugation (12,000 × g, 2 h, 4°C) and resuspended in phosphate-buffered saline to rule out medium effect for future experiments and were frozen at 80°C until use. Viral stocks were quantified for p24 antigen level using an HIV Ag kit (Coulter, Hialeah, Fla.) according to the manufacturer's instructions.

HIV-1 and gp120 exposure. MDM cells were differentiated for 7 days in differentiation medium prior to infection, and then medium was replaced with maintenance medium (DMEM with 10% endotoxin-free fetal bovine serum, 2 mM glutamine, and antibiotics). Cells were infected overnight with HIV-1 at a dose of 0.2 pg of p24 per cell or were cultured in the presence of purified HIV-1 envelope gp120 at a 20 nM concentration, and supernatants were sampled for assay of chemokines and TNF-. gp120s used for this experiment include BaL, CM235 (R5 [28]), IIIB (X4 [38]), MN (X4 [38]), and SF2 [R5/X4 [25]), which were obtained from the AIDS Research and Reference Reagent Program. Virus stocks and gp120 preparations were screened for endotoxin contamination using the E-TOXATE kit (Limulus Amebocyte Lysate; Sigma) and found to be negative (<0.06 EU per ml).

ELISA. -chemokines MIP-1, MIP-1, MCP-1, and RANTES and cytokine TNF- were measured by using the Quantikine enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, Minn.) according to the manufacturer's instructions. Macrophage cultures in triplicate in 24-well plates were exposed to either infectious species of HIV-1 or purified HIV-1 gp120, and culture supernatants were sampled for the ELISA assay. Experiments were repeated two to five times with cells derived from different donors. Macrophages were stimulated with 1 µg of lipopolysaccharide (LPS) (Sigma)/ml to mimic the maximum immune stimulation.

Treatment with inhibitors of HIV-1 replication. Fully differentiated macrophages were pretreated with 1 µM 2',3'-dideoxycytidine (ddC) (Sigma) for 12 h followed by virus infection (0.2 pg of p24/cell) in the presence of ddC. Supernatants were collected for the MIP-1 assay at 6 h. Alternatively, fully differentiated macrophages were pretreated with 20-µg anti-CCR5 (45523, 2D7, and 45549) and anti-CXCR4 (44717, 44708, and 12G5) (except 2D7; 10 µg/ml) at 4°C for 2 h, and then cells were infected with ADA and IIIB (0.04 pg of p24/cell) in the presence of antibodies; at 3 h supernatants were harvested for the MIP-1 assay. For the soluble CD4 neutralization assay, viruses were preincubated with 10 µg of sCD4/ml for 30 min at 37°C, and then cells were infected with viruses (0.2 pg of p24/cell) in the presence of sCD4; supernatants were collected at 6 h for the MIP-1 assay.

Treatment with biochemical inhibitors and cytotoxicity assay. Intracellular Ca²⁺ chelator MAPTAM [1,2-bis(o-amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraacetoxymethyl ester] (Calbiochem, San Diego, Calif.) with concentrations up to 6 µM and translational inhibitor cycloheximide (Sigma) with various concentrations (from 10 to 100 µM) were used to pretreat macrophages prior to exposure to ADA and human T-lymphotropic virus type IIIB (HTLV-IIIB). Macrophages were exposed to viruses for 5 h, and culture supernatants were assayed for MIP-1 by ELISA. Cytotoxic effects of MAPTAM and cycloheximide were measured by lactate dehydrogenase (LDH) release using the CytoTox 96 Assay (Promega, Madison, Wisc.) following the manufacturer's instructions. The extent of cytotoxicity was calculated using the following formula: percent cytotoxicity = 100(c  a)/(b  a), where a is spontaneous LDH release from control macrophage, b is maximum LDH release from lysis buffer-treated macrophage, and c is LDH release from virus-exposed macrophages with various concentrations of MAPTAM or cycloheximide.

Electrophoresis and immunoblot. Purified gp120s (0.5 µg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (4 to 15% precast gradient gel; Bio-Rad, Hercules, Calif.) and transferred by electroblotting to nitrocellulose filters (10 V, 30 min). Blots were incubated with sheep anti-gp120 (1:10,000; NIH AIDS Research and Reference Reagent Program) for 1 h at room temperature. Blots were then washed and incubated with horseradish peroxidase-conjugated anti-sheep immunoglobulin G (1:5,000; DAKO, Carpinteria, Calif.) for 1.5 h. Immunoreactive proteins were visualized with luminofor solution (100 mM Tris-HCl [pH 8.5], 2.5 mM Luminol, 400 µM p-Coumaric acid, 1:1,800 dilution of 30% hydrogen peroxide).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

HIV-1 induction of -chemokine and TNF- by primary macrophages is strain specific, rapid, and unlinked to productive infection. To investigate activation of macrophages, we selected a panel of HIV-1 species, some of which replicate in macrophages and others of which do not. Consistent with previous studies, the R5 viruses ADA, BaL, JR-CSF, JR-FL, NLHXADA-GP, YU-2, and R5/X4 89.6 all productively infected macrophages, while the X4 viruses HTLV-IIIB, Lai, NL4-3, NLHXADA-PG, NDK, and Z6 did not (data not shown). We then tested supernatants from some infected macrophages for levels of MIP-1, a -chemokine essential for certain antiviral immune responses (9) which shares with HIV-1 its cell surface receptor, CCR5 (2). Supernatants were collected 24 h after viral exposure to assay immediate macrophage responses and 2 weeks after exposure to assay responses near the peak of viral expression. Within 24 h, ADA, BaL, JR-CSF, HTLV-IIIB, and NDK induced high levels of MIP-1 production by macrophages, from 14,000 to more than 45,000 pg per ml (Fig. 1A). JR-FL, YU-2, NL4-3, and Z6 failed to induce macrophage responses. By contrast, 2 weeks after exposure, all viruses that productively infect macrophages, ADA, BaL, JR-CSF, JR-FL, and YU-2, induced MIP-1, but no X4 virus induced a response (Fig. 1B). Dual-tropic 89.6 also induced MIP-1 at the peak of infection (data not shown). The latter responses are consistent with previous reports (37, 42). However the immediate response of macrophages to HIV-1 exposure is novel. In addition, the panel of viral species competent to elicit this response does not conform to previous classifications based on tropism or coreceptor utilization. We repeated HIV-1 exposure to macrophages from several different donors and measured chemokine levels 24 h after treatment (Fig. 1C). With one exception, the classification of virus species was reproducible. The R5 species ADA, NLHXADA-GP, and X4 species HTLV-IIIB and Lai were active; R5 YU-2 and X4 NL4-3 and Z6 were inactive. X4 NDK was moderately active in MIP-1 induction. However, cells from two donors responded to ADA and HTLV-IIIB by MIP-1 production but failed to respond to BaL. We are investigating the source of this discrepancy.

View larger version (18K): [in this window] [in a new window]   FIG. 1.   R5 and X4 HIV-1 species activate macrophages to produce -chemokine MIP-1. MDM cells were infected with R5 (ADA, BaL, JR-CSF, JR-FL, and Yu2) and X4 (IIIB, NDK, NL4-3, and Z6) HIV-1 species, and supernatants were harvested for assay of MIP-1 at 24 h (A) and 14 days (B) after infection. (C) MDM cells from different donors were infected with the indicated HIV-1 species, and 24 h after infection, cell supernatants were harvested for assay of MIP-1 levels. Numbers in parenthesis are numbers of different donors (numerator) or independently prepared viruses (denominator). (D) Macrophages were infected with the HIV-1 strain ADA, IIIB, Yu2, or PG at the doses indicated, and 24 h after infection, supernatants were harvested for assay of MIP-1. Data represent means ± standard deviations for multiple experiments in triplicate (A, B, and D). Box plot (C) symbols indicate 50% (line inside the box), 75% (box extents), 90% (capped bars), and 95% (symbol marks above or below capped bars) data.

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Inhibitors of HIV-1 infection do not affect induction of MIP-1. (A) Macrophages were cultured in the presence or absence of 1 µM ddC and then exposed to infectious ADA, YU-2, HTLV-IIIB, Z6, or LPS or mock infected as described in Materials and Methods. Supernatants were harvested for the MIP-1 assay after 6 h. (B) ADA, YU-2, HTLV-IIIB, and Z6 species were pretreated with soluble CD4 prior to exposure to macrophages as described in Materials and Methods. Supernatants were harvested for the MIP-1 assay after 6 h. (C) Macrophages were pretreated with monoclonal antibodies to CCR5 or CXCR4 and then exposed to ADA or HTLV-IIIB as described in Materials and Methods. Supernatants were harvested for the MIP-1 assay after 3 h.

View larger version (19K): [in this window] [in a new window]   FIG. 3.   R5 and X4 HIV-1 species activate macrophages to produce -chemokines and TNF-. Macrophages were infected with R5 (ADA, GP, and Yu2) and X4 (IIIB, NDK, NL4-3, and Z6) HIV-1 species. After 16 h, supernatants were collected for assay of -chemokines, MIP-1, MIP-1, RANTES, and MCP-1, and the cytokine TNF-. LPS was used as a positive control for immune activation. (A) MIP-1, MIP-1, and TNF- levels. (B) RANTES and MCP-1 levels. Data represent means ± standard deviations for experiments in triplicate.

R5 and X4 HIV-1 gp120 induces -chemokine and TNF- production by macrophages. Macrophages were exposed to purified, glycosylated recombinant gp120 from two R5, two X4, and one X4/R5 HIV-1 species. Cell supernatants were collected after 16 h and assayed for the levels of MIP-1, MIP-1, RANTES, and TNF- (Fig. 4A and B). HTLV-IIIB and BaL gp120 induced levels of each factor comparable to that induced by LPS, but CM235, MN, and SF2 gp120 failed to induce the factors measured. The strain specificity observed using intact HIV-1 was reproduced using purified HIV-1 gp120. Macrophages responded to BaL virus and envelope and to HTLV-IIIB virus and envelope. The dose responses to HTLV-IIIB and BaL were very similar, indicating comparable activities between X4 and R5 gp120 in these inductive events (Fig. 4C). The gp120 proteins tested migrated similarly, indicating that none was degraded (Fig. 4D). These findings suggest that the immediate response of macrophages to HIV-1 exposure is induced through binding of gp120 to cell surface receptors. However, the inability of anti-CCR5 and -CXCR4 antibodies and soluble CD4 to block the response indicates that these receptors may not be involved. With this negative information in hand, it is premature to speculate on the nature of the cell surface receptors involved.

View larger version (27K): [in this window] [in a new window]   FIG. 4.   R5 and X4 HIV-1 envelope glycoprotein gp120 activates macrophages to produce -chemokines and TNF-. Macrophages were incubated overnight with 20 nM recombinant gp120 from R5 clones BaL and CM235, X4 clones IIIB and MN, and the R5X4 clone SF2, and supernatants were harvested for the chemokine-cytokine assay. (A) MIP-1 and MIP-1 levels. (B) RANTES and TNF- levels. Data represent means ± standard deviations for triplicate experiments. (C) Macrophages were incubated with IIIB and BaL gp120 at the doses indicated, and after 16 h, the supernatant was harvested to assay MIP-1 levels. Data shown are representative of experiments in duplicate. (D) Western blot of gp120. The migration of molecular mass markers of 220 and 97 kDa is indicated.

Biochemical requirements for induction of chemokine synthesis. The secretion of -chemokines by macrophages in response to HIV-1 exposure may result from new synthesis of the proteins or from release of proteins from intracellular pools. To determine whether the chemokines measured in macrophage culture media were newly synthesized, cells were preincubated with graded doses of the translational inhibitor cycloheximide prior to exposure to ADA or HTLV-IIIB. After 5 h, MIP-1 levels and cell viability were measured (Fig. 5A and B). Cycloheximide was not toxic at any dose employed; however, it inhibited the production of MIP-1 even at a 10 µM concentration. We conclude that HIV-1 exposure induces new synthesis of MIP-1 protein in macrophages.

View larger version (15K): [in this window] [in a new window]   FIG. 5.   Inhibition of HIV-1-induced MIP-1 production by treatment of macrophages with the translational inhibitor cycloheximide or the calcium chelator MAPTAM. Macrophages were treated for 1 h with the indicated doses of cycloheximide or MAPTAM prior to HIV-1 infection. Supernatants were harvested 5 h after infection for assay of toxicity and MIP-1 levels. (A) MIP-1 produced by cycloheximide-treated macrophages. (B) Cell death of cycloheximide-treated macrophages. (C) MIP-1 produced by MAPTAM-treated macrophages. (D) Cell death of MAPTAM-treated macrophages. Percent cytotoxicity was calculated as described in Materials and Methods. Data represent means ± standard deviations for experiments performed in triplicate.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Our novel results show that within hours of exposure and independent of virus replication, primary human macrophages respond to HIV-1 and its envelope glycoprotein by new synthesis of several physiologically important proteins. This response is viral strain specific, but it is unrelated to coreceptor phenotype and does not appear to involve binding of CD4, CCR5, or CXCR4.

Because HIV-1 has appropriated cellular receptors that are intimately involved in immune responses, considerable research has been devoted to elucidation of the effects of HIV-1 binding on cellular activity. As early as 1989, it was recognized that HIV induction of cytokine synthesis by macrophages could be dissociated from productive infection and attributed to binding of cell surface receptors (5, 21, 30). Our studies confirm and extend these findings to demonstrate that a panel of three -chemokines and TNF- are induced rapidly and coordinately by HIV-1 and its envelope glycoprotein. The ability to activate macrophages using X4 HIV-1 strains unable to infect them, as well as the inability to block R5 HIV-1-mediated activation by inhibition of virus replication, indicate that HIV-1 initiates a program of activation and cellular gene expression in primary macrophages independently of productive infection. A different pathway of activation, linked to HIV-1 expression, may be involved in the synthesis of -chemokines at the peak of productive HIV-1 infection of macrophages, as previously reported (4, 29, 37, 42) and as shown here. However, the only viral product required for the rapid induction observed here is gp120.

The cellular response we report is viral strain specific, and the specificity resides in HIV-1 gp120. We do not yet have enough information to define the structural basis of the specificity. Both R5 and X4 HIV-1 are active; however, they appear to employ receptor(s) different from CCR5 or CXCR4, since not all R5 or X4 viruses could activate cells and the response was maintained in the presence of antibodies to CCR5 or CXCR4. Examining the strain specificity, ADA and NLHXADA-GP induced chemokine synthesis while NL4-3 did not. NLHXADA-GP carries the ADA V3 region, embedded in the HXB-2 envelope on the background of the NL4-3 viral genome (44). These isolated results point to the ADA R5 V3 region as a principal determinant of the response. However, HTLV-IIIB (HXB-2) gp120 also induced chemokine responses in our hands, while NLHXADA-PG virus, a construct nearly identical to NLHXADA-GP but carrying HXB-2 V3, failed to induce a response. On that basis, it appears that activity in chemokine induction depends both on the V3 region and on sequences in envelope outside it.

There is precedent for activation of macrophages by both R5 and X4 HIV-1 species. Fantuzzi and colleagues recently reported that R5 and X4 gp120 activate -chemokine synthesis by macrophages through a CD4-independent route, results consistent with those reported here (12). In addition, gp120 of both phenotypes has been shown to activate several ion channels in primary macrophages (27). Ca²⁺ mobilization induced by gp120 was reported in the latter study, in a study of R5 HIV-1 activation of macrophages (3), and is implicated in the chemokine synthesis we describe. However, the strain specificity we observe only partially overlaps that previously reported (12, 27). HTLV-IIIB envelope activated macrophages in previous studies (12, 27) and in those we describe. Although we found that the HIV-1 JR-FL reagent was able to induce MIP-1 at the peak of virus infection, it did not induce a rapid response, unlike results of the previous studies (12, 27). Some of these differences may be technical. We employ a growth factor mixture to induce macrophage differentiation; the previous studies allow differentiation through adherence. Liu et al. used 10-fold more gp120 than was employed here to activate ion channels through CCR5 and CXCR4 (27). Finally, the levels of MIP-1 and RANTES induced in our system were more than 10-fold higher than those observed by Fantuzzi et al.

Synthesis of -chemokines by macrophages can be activated by other stimuli. Macrophages responded to contact with cells expressing CD40L, an activator expressed on the surface of T cells, by production of -chemokines at levels and with kinetics comparable to that described here (23). Another study reported that the HIV-1 protein Nef can activate macrophages to produce MIP-1 and MIP-1 but not RANTES (42), all of which we observed to be synthesized in response to HIV-1 gp120. The latter study also showed that the levels of MIP-1 and MIP-1 in macrophage supernatants, about 10- to 50-fold lower than we report, were sufficient to activate lymphocyte chemotaxis (42). Extrapolating from that study, it is likely that the higher levels of MIP-1 and MIP-1, as well as the other -chemokines produced in our experimental system in response to HIV-1, would also induce lymphocyte migration to activated macrophages.

The -chemokines have complex roles in HIV-1 infection. Previous studies have demonstrated that they inhibit R5 HIV-1 infection in culture and that their levels are elevated in HIV-1-infected persons who appear to control their infection (6, 7, 46). However, -chemokine exposure can also increase viral replication in vitro in T cells producing X4 virus (22). We speculate that the TNF- and -chemokine responses we describe are relevant in their physiological activities. In an animal model of virally induced myocarditis, MIP-1 was the pivotal factor initiating antiviral responses, including cytotoxic-T-cell migration (9). We consider that the rapid response of macrophages to HIV-1 exposure we describe in vitro may constitute an innate immune response that is protective in vivo, summoning activated T cells, including HIV-specific cytotoxic T cells, to eliminate HIV-1-infected cells prior to major spread of the virus through tissue. Further studies are required to illuminate the activities of -chemokines and other cellular products of macrophages activated by HIV-1.