Andrea Rubbert

Macrophage-tropic HIV and SIV envelope proteins induce a signal through the CCR5 chemokine receptor.

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) enter target cells by forming a complex between the viral envelope protein and two cell-surface membrane receptors: CD4 and a 7-span transmembrane chemokine receptor (reviewed in refs 1,2,3). Isolates of HIV that differ in cellular tropism use different subsets of chemokine receptors as entry cofactors: macrophage-tropic HIVs primarily use CCR5, whereas T-cell-tropic and dual-tropic isolates use CXCR4 (refs 1,2,3) receptors. HIV-mediated signal transduction through CCR5 is not required for efficient fusion and entry of HIV in vitro. Here we show that recombinant envelope proteins from macrophage-tropic HIV and SIV induce a signal through CCR5 on CD4+ T cells and that envelope-mediated signal transduction through CCR5 induces chemotaxis of T cells. This chemotactic response may contribute to the pathogenesis of HIV in vivo by chemo-attracting activated CD4+ cells to sites of viral replication. HIV-mediated signalling through CCR5 may also enhance viral replication invivo by increasing the activation state of target cells. Alternatively, envelope-mediated CCR5 signal transduction may influence viral-associated cytopathicity or apoptosis.

Natural killer cells from human immunodeficiency virus (HIV)-infected individuals are an important source of CC-chemokines and suppress HIV-1 entry and replication in vitro.

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.

Relevance of the Organic Cation Transporters 1 and 2 for Antiretroviral Drug Therapy in Human Immunodeficiency Virus Infection

Carrier-mediated transport across cell membranes is an important determinant of activity, resistance, and toxicity of chemotherapeutic agents including antiretroviral (ARV) drugs (ARDs). The organic cation transporters (OCTs) 1 and 2 have been implicated in the translocation of different cationic drugs but so far were insufficiently tested for interactions with ARDs. Here, we assessed among cationic drugs commonly used in human immunodeficiency virus (HIV) therapy inhibitors and substrates of OCTs, and analyzed the tissue distribution of OCTs and their expression in lymph nodes (LNs), the primary intracellular target of HIV and ARDs. Inhibitors were identified by measuring the attenuated uptake of the radiolabeled model substrate 1-methyl-4-phenylpyridinium into OCT-transfected human embryonic kidney-293 cells in the presence of ARDs. Substrates were identified by measuring OCT-specific intracellular accumulation using liquid chromatography/tandem mass spectrometry. Inhibitory drugs were (in order of increasing potency): nelfinavir < ritonavir < saquinavir < indinavir < trimethoprim < pentamidine, with consistently lower IC50 values determined for OCT1. Substrates with highest transport efficacy (Vmax/Km) were lamivudine (OCT1, 8 μl/mg protein/min; OCT2, 4.4 μl/mg protein/min) and zalcitabine (OCT1, 4.1 μl/mg protein/min; OCT2, 2.6 μl/mg protein/min). Using quantitative real-time polymerase chain reaction, a marked expression level of OCT1 was detected in human samples of liver, ovary, prostate, and testis, and of OCT2 in kidney, colon, heart, skeletal muscle, and testis. Expression of OCTs in LNs was low in HIV-negative control individuals but dramatically increased in HIV-infected persons. These data suggest that drug interactions about the OCTs may be relevant for the ARV therapy, in particular by influencing drug accession to infected tissues and hepatic or renal elimination.

CCR5 Signal Transduction in Macrophages by Human Immunodeficiency Virus and Simian Immunodeficiency Virus Envelopes.

ABSTRACT The 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.

Dendritic Cells Express and Use Multiple HIV Coreceptors

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.