ABSTRACTThe capacity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelopes to transduce signals through chemokine coreceptors on macrophages was examined by measuring the ability of recombinant envelope proteins to mobilize intracellular calcium stores. Both HIV and SIV envelopes mobilized calcium via interactions with CCR5. The kinetics of these responses were similar to those observed when macrophages were treated with MIP-1β. Distinct differences in the capacity of envelopes to mediate calcium mobilization were observed. Envelopes derived from viruses capable of replicating in macrophages mobilized relatively high levels of calcium, while envelopes derived from viruses incapable of replicating in macrophages mobilized relatively low levels of calcium. The failure to efficiently mobilize calcium was not restricted to envelopes derived from CXCR4-utilizing isolates but also included envelopes derived from CCR5-utilizing isolates that fail to replicate in macrophages. We characterized one CCR5-utilizing isolate, 92MW959, which entered macrophages but failed to replicate. A recombinant envelope derived from this virus mobilized low levels of calcium. When macrophages were inoculated with 92MW959 in the presence of MIP-1α, viral replication was observed, indicating that a CC chemokine-mediated signal provided the necessary stimulus to allow the virus to complete its replication cycle. Although the role that envelope-CCR5 signal transduction plays in viral replication is not yet understood, it has been suggested that envelope-mediated signals facilitate early postfusion events in viral replication. The data presented here are consistent with this hypothesis and suggest that the differential capacity of viral envelopes to signal through CCR5 may influence their ability to replicate in macrophages.

Binding of HIV type 1 (HIV-1) envelope glycoproteins to the surface of a CD4(+) T cell transduces intracellular signals through the primary envelope receptor, CD4, and a coreceptor, either CCR5 or CXCR4. Furthermore, envelope-CD4(+) cell interactions increase rates of apoptosis in peripheral blood mononuclear cells (PBMCs). We demonstrate that in primary T lymphocytes, recombinant HIV-1 envelope proteins induce the activation of caspase-3 and caspase-6, which belong to a family of cysteine proteases that, upon activation, promote programmed cell death. Envelope-mediated activation of caspase-3 and caspase-6 depended on envelope-CD4 receptor interactions; CCR5-utilizing as well as CXCR4-utilizing envelopes elicited this response. Focal adhesion kinase (FAK) is a substrate of both caspase-3 and caspase-6, and inactivation of FAK by these caspases promotes apoptosis. En-velope treatment of lymphocytes led to the cleavage of FAK in a manner consistent with caspase-mediated cleavage.

Binding of HIV-1 envelope glycoproteins to the surface of a CD4+ cell transduces intracellular signals through the primary envelope receptor, CD4, and/or the envelope coreceptor, a seven-transmembrane chemokine receptor. Macrophage-tropic strains of HIV-1 preferentially use CCR5 as an entry coreceptor, whereas T cell-tropic strains use CXC chemokine receptor-4 for entry. Intracellular signals transduced by HIV-1 envelope may have immunopathogenic consequences, including anergy, syncytium formation, apoptosis, and inappropriate cell trafficking. We demonstrate here that a recombinant envelope protein derived from an M-tropic isolate of HIV-1 can transduce CD4-dependent as well as CCR5-dependent intracellular signals in primary human CD4+ T cells. Novel HIV-induced intracellular signals that were identified include tyrosine phosphorylation of focal adhesion kinase (FAK) and CCR5, which are involved in cell adhesion and chemotaxis, respectively. HIV envelope-induced cellular association of FAK and CCR5 was also demonstrated, suggesting that ligation of CD4 and CCR5 leads to the formation of an activation complex composed of FAK and CCR5. Activation of this signaling pathway by HIV-1 envelope may be an important pathogenic mechanism of dysregulated cellular activation and trafficking during HIV infection.

Despite exposure to multiple strains of both macrophage (M)-tropic and T cell (T)-tropic HIV, primary infection is largely restricted to relatively homogeneous M-tropic virus. Since dendritic cells (DCs) play a pivotal role in the early events of HIV infection, several studies have focused on the role of DCs in this restriction. It has been proposed that DCs are more efficiently infected with M-tropic versus T-tropic viruses; however, the infectability of DCs and the relevance of their infectability for inducing productive infection is controversial. It has also been suggested that variability in DC expression of coreceptors for M-tropic versus T-tropic virus could explain the restriction in the transmitting virus. Using HIV-pulsed DCs from individuals with a homozygous deletion in the CCR5 gene as a human "knockout" model, we demonstrate that infection of DCs per se is not necessary to promulgate infection in CD4+ T cells. The data also suggest that transmission of HIV to CD4+ T cells is not dependent on DC coreceptor expression.

Cells of the dendritic lineage are thought to be among the first cells infected after mucosal exposure to HIV. In this study, we have identified the presence of multiple chemokine receptors on dendritic cells (DC) that may function as coreceptors for HIV entry. DC effectively used CCR5 for entry of macrophage (M)-tropic isolates. CCR3, the eotaxin receptor, initially identified on eosinophils, is expressed on DC and may be used as an entry coreceptor by certain dual-tropic strains. CXCR4 was not expressed on DC, although SDF-1 induced a calcium flux and DC could be infected by T cell line (T)-tropic HIV. Our findings provide evidence for the presence of a non-CXCR4 SDF-1 receptor on DC that is used mainly by T-tropic strains of HIV. DC from individuals homozygous for a 32-bp deletion of the CCR5 gene are also infectable with M-tropic strains of HIV-1, and this infection is inhibited by stromal cell-derived factor (SDF)1, suggesting that this receptor can also be used by M-tropic HIV for entry. Delineation of the spectrum of coreceptor usage on DC may offer new approaches to interfere with the initiation and propagation of HIV infection.

Macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES (regulated on activation, normal T cell expressed and secreted), which are the natural ligands of the CC-chemokine receptor CCR5, inhibit replication of MT-2- negative strains of HIV-1 by interfering with the ability of these strains to utilize CCR5 as a coreceptor for entry in CD4(+) cells. The present study investigates the capacity of natural killer (NK) cells isolated from HIV-infected individuals to produce CC-chemokines and to suppress HIV replication in autologous, endogenously infected cells as well as to block entry of MT-2-negative HIV into the CD4(+) T cell line PM-1. NK cells freshly isolated from HIV-infected individuals had a high number of mRNA copies for MIP-1alpha and RANTES. NK cells produced significant amounts of RANTES, MIP-1alpha, and MIP-1beta constitutively, in response to stimulation with IL-2 alone and when they were performing their characteristic lytic activity (K562 killing). After CD16 cross-linking and stimulation with IL-2 or IL-15 NK cells produced CC-chemokines to levels comparable to those produced by anti-CD3-stimulated CD8(+) T cells. Furthermore, CD16 cross-linked NK cells suppressed (49-97%) viral replication in cocultures of autologous CD8/NK-depleted PBMC to a degree similar to that of PHA or anti-CD3-stimulated CD8(+) T cells. In 50% of patients tested, NK-mediated HIV suppression could be abrogated by neutralizing antibodies to MIP-1alpha, MIP-1beta and RANTES; in contrast, CD8(+) T cell-mediated suppression was not significantly overcome upon neutralization of CC-chemokines. Supernatants derived from cultures of CD16 cross-linked NK cells stimulated with IL-2 or IL-15 dramatically inhibited entry of a MT-2-negative strain of HIV, BaL, in the CD4(+)CCR5(+) PM-1 T cell line. These data suggest that activated NK cells may be an important source of CC-chemokines in vivo and may suppress HIV replication by CC-chemokine-mediated mechanisms in addition to classic NK-mediated lytic mechanisms.

Dendritic cells (DC) are the first immunocompetent cells to encounter antigen at areas of inflammation in mucous membranes1, which are the major sites where the initiation of HIV infection occurs. HIV enters a mucous membrane and interacts with Langerhans cells (LC)/DC resulting in binding of the virus to the cell with or without infection. The cell then migrates and delivers virus to the paracortical region of the draining lymphoid tissue: LC/DC also provide activation stimuli to CD4+ T cells which become infected leading to replication and spread of virus2,3. Recently, in vivo data in the macaque model have clarified certain pathogenic events associated with primary simian immunodeficiency virus (SIV) infection. SIV was placed in the vaginal vault and infected cells were then identified and followed using in situ PCR technology. DC in the lamina propria of the cervicovaginal mucosa were found to contain SIV DNA 2 days after exposure to virus. Infected cells were observed in the sub-capsular and paracortical regions of the draining lymph nodes; this series of events mirrors the course that DCs take upon receiving a signal to migrate from the tissues to lymphoid organs4. Thus, in an animal model of HIV, DC appeared to be responsible for bringing virus from the site of inoculation to the paracortical T cell regions of the draining lymphoid organs leading to viral replication and systemic spread of infection.

Chemokines were originally characterized by their ability to direct migration and induce activation of selected leukocyte populations. The beta-chemokines MIP-1 alpha, MIP-beta, and RANTES have been implicated in the suppression of viral replication by CD8+ T cells from HIV-infected individuals. The present study was undertaken to evaluate the effect of beta-chemokines on HIV replication in cocultures of dendritic cells (DCs) and CD4+ T cells, and an in vitro model of the lymphoid microenvironment. In the acute infection system, where DCs from uninfected individuals are pulsed with HIV and cocultured with autologous CD4+ T cells, no inhibition of replication of monocytotropic or T cell tropic viral isolates by MIP-1 alpha, MIP-1 beta, and RANTES, alone or in combination, was observed. In contrast, in an endogenous infection system, where the DCs and CD4+ T cells were obtained from HIV-infected subjects, addition of recombinant beta-chemokines suppressed HIV replication. However, neutralizing antibodies to beta-chemokines did not affect the suppressive activity of CD8+ T cells from HIV-infected donors in either system, suggesting that CD8+ T cell-mediated suppression is not due exclusively to beta-chemokines. Furthermore, no significant differences in secretion of MIP-1 alpha, MIP-1 beta, and RANTES by purified CD8+ T cells were noted in uninfected versus HIV-infected donors, regardless of the stage of disease. These results indicate that HIV suppression by CD8+ T cells derived from HIV-infected donors is a multifactorial phenomenon and not limited to the action of MIP-1 alpha, MIP-1 beta, and RANTES.