Microglia are the cells primarily responsible for viral load in the CNS, where they probably play a role in the development of HIVD, and because of poor penetration of antiretrovirals into the CNS, they may serve as a potential “sanctuary” site for the virus (4
). Since virus replication can differ between microglia and MDM (44
), understanding the virus-cell biology in microglia is critical for the development of strategies to treat HIVD. The discovery that chemokine receptors act as HIV coreceptors has been a major advance in delineating tropism, and the role of these receptors in microglial entry and replication has been the subject of recent publications (16
). Here we have shown that three major chemokine receptors implicated in HIV entry, CCR5, CCR3, and CXCR4, are expressed on adult microglial cells, albeit at different levels (Fig. ). Two of these, CCR5 and CXCR4, are functional chemokine receptors, as measured by their abilities to mediate signal transduction after stimulation with their respective ligands. In these experiments we noted robust Ca2+
responses with chemokine concentrations within the same range as (or lower than) those used for stimulation of MDM (20a
). Although we did not detect eotaxin-mediated calcium signaling, it is formally possible that CCR3 could mediate a signal in the absence of changes in intracellular calcium levels, as such signaling has been reported with other GPCRs (45
). While our primary interest in chemokine receptors on human microglia relates to their role in HIV infection, these and future experiments will help us understand the roles of chemokine receptors in a number of inflammatory conditions involving microglia (2
Our results demonstrating that SDF-1α, MIP-1β, and RANTES induced internal calcium fluxes in microglia are not entirely unexpected, insomuch as MDM and microglia share many of the same phenotypes, with some notable differences (44
). Two groups have demonstrated MDM signaling in response to treatment with SDF-1α (20a
), and a recent report by Herbein and colleagues demonstrated that MDM signal in response to the CCR5 ligands MIP-1β and RANTES (20a
). However, there are other differences between microglia and MDM, including the time course of expression of chemokine receptors and differences in the replication potential among different isolates. Furthermore, whereas the responses of MDM and microglia were qualitatively similar, we cannot make any quantitative statements based on our data.
Depending on the assay, several viruses isolated from individuals with HIVD can use CCR5, CCR3, and, to some extent, CXCR4 as coreceptors for entry into cells transfected with plasmids expressing these receptors (43
). Theoretically, HIV could utilize any of these chemokine receptors for entry into microglial cells, and He et al. have proposed that CCR3 and CCR5 function as coreceptors for fetal microglia (19
). Using antibodies, we demonstrated that CCR5 is the predominant coreceptor involved in HIVD virus infection of adult microglia, with antibodies against either CCR3 or CXCR4 having only a modest or no effect on viral replication. Results with pseudotyped or wild-type viruses were quite congruent with each other and previous data (43
). It is possible that the isolates utilize the CCR3 or CXCR4 on microglia inefficiently simply because of the low number of chemokine receptor molecules on the cell surface or, alternatively, because CCR3 and CXCR4 are not presented in the right context. For example, there may be an optimal CD4/coreceptor ratio that can be achieved only with a coreceptor that is expressed at high levels, such as CCR5 (25
). Another possibility is that a virion could simultaneously use two coreceptors complexed together, e.g., CCR5 and CCR3, and that under some circumstances antibodies against the coreceptor present in the lower concentration (CCR3) could partially block entry. This would explain why we see some inhibition with anti-CCR3 antibodies but have not been able to infect microglia with a pseudotyped virus that uses CCR3 but not CCR5 (20b
). Therefore, CCR3 and CXCR4 may play roles in microglial entry, but for an infection of microglia to occur at relevant levels, HIV must use CCR5, which is both necessary and sufficient for infection.
Additionally, we found that envelopes from several HIVD isolates can mediate infection of cells transfected with other GPCRs previously described as HIV coreceptors. But they do so with apparently reduced efficiency in comparison with CCR5, at least within the constraints of this assay system, which did not quantify the level of expression of each of the coreceptors on the transfected cell surface. This area will need further clarification when antibodies against these alternative coreceptors become available.
We have previously suggested that direct amplification of envelope genes from HIV-infected brains may clarify the potential role of these other coreceptors, since it does not introduce the selection bias associated with viral isolation (43
). For infection of microglia, it is also quite possible that CCR3, CXCR4, and other coreceptors are expressed at higher levels in the CNS of individuals with HIVD and that under those circumstances they play a more significant role in HIV entry.
In contrast to microglia, which can be infected soon after isolation (43a
), undifferentiated monocytes are relatively resistant to infection on day 1 after isolation (46
). Monocytes have low levels of CCR5 until cultured, whereas microglia have high levels of CCR5 soon after purification from brain tissue (Fig. ). These high levels of CCR5 may explain why the CNS is infected early during the course of HIV infection, at a time when most viruses use CCR5 as a coreceptor, and why virus is present in the brains of many patients with or without HIVD (23
). Chemokine receptor levels may also contribute to the differences we have previously noted between replication in MDM and in microglia (44
). Given the detectable levels of CXCR4 present in microglia, it is somewhat surprising that more CXCR4-using viruses have not been isolated from brains, particularly since these isolates are particularly prominent in the late stages of HIV disease, when HIVD is more prevalent. In MDM, blocks to replication beyond the entry step have been identified for some HIV strains (42
), and further experimentation may clarify this issue in microglia. Alternatively, parenchymal microglia may become infected only after the CNS perivascular population of macrophages/microglia has been infected (26
). This perivascular population, which may be phenotypically different with regard to HIV infection, may act as a filter, allowing only viruses using a certain coreceptor repertoire to infect the parenchymal microglia, which are used in our experimental system (18