Department of Medicine and Cancer Research Institute, Division of Hematology/Oncology, University of California, School of Medicine, San Francisco, CA 94143-1270, USA1
Author for correspondence: Jay Levy.Fax +1 415 476 8365.
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Abstract |
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Our present studies with a large panel of chemokines indicate that most had no effect on HIV infection. RANTES, MIP-1 and MIP-1ß, as well as MCP-2, and to a lesser extent, lymphotactin, significantly inhibited replication of certain primary isolates of HIV-1. MCP-3 and MDC enhanced virus replication. In addition, we found that the chemokine-mediated antiviral effect of RANTES and MCP-2 could be prevented, in part, by the addition of another chemokine with different biological activity.
For these studies CD4+ cells from seronegative donors were isolated from FicollHypaque-separated PBMC using anti-CD4 antibody-coupled immunomagnetic beads (Dynal) (Mackewicz et al., 1991 ). More than 95% were CD4+/CD3+ cells (less than 1% CD8+ cells) as assessed by flow cytometry (Landay et al., 1993
). Cell cultures were grown in RPMI 1640 medium (BioWhittaker) supplemented with 10% heat-inactivated (56 °C, 30 min) foetal calf serum (Gemini Bioproducts), 100 U/ml recombinant IL-2 (Glaxo-Wellcome), 2 mM l-glutamine and 1% antibiotics.
The four primary HIV-1 isolates used (designated SV, NB, EM and ALA-22) were obtained from clinically healthy HIV-infected subjects. The biological phenotype of SV, NB and ALA-22 was non-syncytium-inducing as determined in MT-2 cells. These viruses have been cultivated in the laboratory for less than 2 months and only in PBMC. Their infection is blocked by RANTES, MIP-1 and MIP-1ß. The SF2 strain of HIV-1, isolated from a patient with candidiasis (Levy et al., 1984
), and the primary isolate, EM, have a tropism for T-cell lines in vitro. They are syncytium-inducing (SI), non-macrophage-tropic viruses resistant to the inhibitory effects of the ß-chemokines MIP-1
, MIP-ß and RANTES (Mackewicz et al., 1996
). The TCID50 of each virus was determined in PBMC as described (McDougal et al., 1985
).
The purified CD4+ T-cells were stimulated with phytohaemagglutinin (PHA, 3 µg/ml; Sigma) for 3 days, washed and infected with 1040 TCID50 of HIV-1 per 106 cells as described (Barker et al., 1998 ). After incubation for 2 h, cells were washed, and plated at 5x105 cells per well in duplicate or triplicate in 48-well plates (Becton Dickinson). Varying concentrations of recombinant chemokines were added and the cultures were passaged every 23 days for up to 1 week. Cultures were replenished with fresh medium and chemokine at each passage. The culture fluids were monitored for virus replication by measurement of reverse transcriptase (RT) activity (Hoffman et al., 1985
).
The recombinant chemokines studied were used at concentrations ranging from 50 ng to 5000 ng/ml and were provided by Michael Luther, Glaxo-Wellcome. All were obtained from R&D Systems, except for NAP-2, MIG, PF4 and lymphotactin, which were obtained from Peprotech. The recombinant MDC proteins evaluated came from both R&D Systems and Peprotech. The range of concentrations of chemokines tested for anti-HIV activity corresponded approximately to 0·5100 ED50 of chemotactic activity (as noted by the company providing the reagents). Results were analysed for statistical significance by the MannWhitney U-test.
Fig. 1 shows the effect of 0·55 µg/ml of a large panel of commercially available recombinant chemokines. For most of the cytokines, the effect did not vary over a large range of concentrations (50 ng5 µg). They caused a <20% difference in HIV replication when compared with the control-infected culture, regardless of the virus used (Fig. 1
). Besides RANTES, MIP-1
and MIP-1ß, only MCP-2 and lymphotactin produced an inhibitory effect within the range of concentrations tested (Fig. 2
). With MCP-2 and NSI viruses, up to a 90% decrease in virus replication was observed with
1 µg/ml, and 70% with
0·25 µg/ml (Figs 1
and 2
). No substantial inhibitory effect of this chemokine on SI viruses (Fig. 2
) was noted. Lymphotactin at a concentration of 1 µg/ml or more reduced the replication of both NSI and SI viruses by at least 50% (Fig. 2
). In some experiments, Gro-ß inhibited infection by SI viruses, but this finding was not consistently seen.
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Several chemokines share receptors (Adams & Lloyd, 1997 ) and therefore may compete for the same receptor. By other mechanisms, the effect of one chemokine may dominate over another. In evaluating this possibility, RANTES at a concentration (5 ng/ml) that suppressed replication of NSI viruses (SV or NB) by 70% was mixed with MCP-3 (1000 ng/ml); the inhibition of virus production was abrogated. In another study, MCP-3 (2000 ng/ml) prevented inhibition of virus replication by a low concentration of RANTES (20 ng/ml). At higher concentrations of RANTES (80 ng/ml) this effect of MCP-3 was not observed. In addition, whereas MCP-2 (5001000 ng/ml) inhibited replication of the NSI virus, ALA-22, the addition of MCP-1 (1000 ng/ml) prevented this activity.
These studies indicate that, in addition to the chemokines already known to have anti-HIV properties, MCP-2 and to a lesser extent, lymphotactin, showed consistent inhibition of HIV replication (Figs 1 and 2
). As with MIP-1
, MIP-1ß and RANTES, the effect of MCP-2 (at
1 µg/ml) was limited to NSI isolates. These results support findings recently reported (Gong et al., 1998
). Lymphotactin was effective in reducing virus replication of both NSI and SI isolates. However, high doses (
1 µg) of lymphotactin were necessary to show a substantial (55%) decrease in virus production (Fig. 2
).
CCR5 and CXCR4 are the primary coreceptors for macrophage-tropic and T-cell-tropic HIV-1 strains, respectively (for a review see Berger, 1997 ). In addition, certain virus strains can use the CC-chemokine receptors CCR-1, CCR2b and CCR3 (Berger, 1997
; Frade et al., 1997
). All the MCP recognize CCR2b (the chemokine receptor to which CCR5 is most closely related: 76% identity) (Rucker et al., 1996
), whereas CCR3 serves as a receptor for RANTES, MIP-1
, MIP-1ß, eotaxin, MCP-2, MCP-3 and MCP-4 (Adams & Lloyd, 1997
). Nevertheless, despite the overlap of receptor usage among the MCPs, only MCP-2, probably because it can bind to CCR5 (Gong et al., 1998
), showed a reproducible inhibitory effect on replication of the NSI isolates evaluated (Figs 1
and 2
).
Our results also indicated that, at certain concentrations, MCP-3 and MDC can enhance virus replication in CD4+ cells (Figs 1 and 2
). These findings contrast with a previous report showing inhibitory activity of MCP-3 on replication of SI isolates of HIV-1 in PBMC (Schols et al., 1997
), and MDC on both NSI and SI HIV isolates (Pal et al., 1997
). We did not detect any inhibition of HIV production by either of these chemokines. Recently, MDC was shown to be a ligand for CCR-4 but not CCR-1, CCR-3 or CCR-5 (Yoshida et al., 1998
). Thus, MDC would not be expected to compete for HIV binding to a virus coreceptor. However, these different observations may be due to the experimental conditions used (e.g. PBMC instead of CD4+ cells), the various concentrations of chemokine evaluated or the source of the chemokine. Nevertheless, the recombinant MDC we used came from two sources (R&D Systems and Peprotech) and had chemotactic activity. The reported antiviral activity of MDC has also not been confirmed by other studies in which recombinant MDC was used (Lee et al., 1998
). The maturation of MDC, resulting in NH2-terminal modification, could influence its receptor binding and activation profile. This possibility could account for the conflicting results obtained with the recombinant MDC versus the purified natural product originally used (Pal et al., 1997
).
The mechanisms underlying chemokine-mediated anti-HIV activity may involve competition for receptor occupancy and/or down-regulation of the receptor (Trkola et al., 1998 ). Conceivably, MCP-2 could exert its inhibitory activity on NSI isolates by competitive binding to CCR5, which does not occur with the other MCPs; lymphotactin-mediated inhibition of NSI and SI strains could involve mechanisms different from competition for the receptors. In terms of enhancement, the increase in HIV replication observed with the addition of some chemokines (Figs 1
and 2
) may result from upregulation of certain virus receptors as reported recently (Dolei et al., 1998
). These possibilities are under study.
Chemokines may have opposite regulatory effects and therefore specific combinations of chemokines could result in qualitatively different effects on HIV replication than that observed when single chemokines are used alone. In this regard, we have found that chemokines, devoid of any inhibitory activity on HIV, may affect inhibition induced by other chemokines. In particular, the concentration of RANTES or MCP-2 required to produce a certain level of inhibition must be increased when these chemokines are present in combination with MCP-3 and MCP-1, respectively.
This study presents further information on the potential role of chemokines in blocking HIV infection. Most chemokines do not appear to substantially affect the levels of HIV replication in CD4+ T-cells. Moreover, levels of those chemokines that inhibit HIV infection were higher than expected under normal physiological conditions. Nevertheless, we cannot exclude the possibility that these chemokines play some role in HIV pathogenesis, perhaps via inflammatory responses or by interfering with HIV adaptation to certain coreceptors. Further understanding of these cytokines might assist in the development of novel antiviral strategies that are capable of blocking infection by both macrophage-tropic and T-cell line-tropic isolates.
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Acknowledgments |
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References |
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Received 19 March 1999;
accepted 25 May 1999.