The chemokine receptors CCR5 and CXCR4 were found to function in vivo as the principal coreceptors for M-tropic and T-tropic human immunodeficiency virus (HIV) strains, respectively. Since many primary cells express multiple chemokine receptors, it was important to determine if the efficiency of virus-cell fusion is influenced not only by the presence of the appropriate coreceptor (CXCR4 or CCR5) but also by the levels of other coreceptors expressed by the same target cells. We found that in cells with low to medium surface CD4 density, coexpression of CCR5 and CXCR4 resulted in a significant reduction in the fusion with CXCR4 domain (X4) envelope-expressing cells and in their susceptibility to infection with X4 viruses. The inhibition could be reversed either by increasing the density of surface CD4 or by antibodies against the N terminus and second extracellular domains of CCR5. In addition, treatment of macrophages with a combination of anti-CCR5 antibodies or ␤-chemokines increased their fusion with X4 envelope-expressing cells. Conversely, overexpression of CXCR4 compared with CCR5 inhibited CCR5-dependent HIV-dependent fusion in 3T3.CD4.401 cells. Thus, coreceptor competition for association with CD4 may occur in vivo and is likely to have important implications for the course of HIV type 1 infection, as well as for the outcome of coreceptortargeted therapies.Most of the cells that were found to be targets for human immunodeficiency virus (HIV) infection in vivo (i.e., T cells, macrophages, and dendritic cells) express both CD4 and multiple chemokine receptors. Among the chemokine receptors that were shown in recent years to function as coreceptors for HIV type 1 (HIV-1) viral entry in vitro, CCR5 and CXCR4 emerged as the predominant coreceptors for primary isolates in vivo. The potential of a given chemokine receptor to function as an HIV-1 coreceptor may depend on multiple parameters such as its surface density (29), posttranslational modifications (11), and interactions with other membrane components such as CD4 and other chemokine receptors. Previously, we demonstrated that exposure of human cell lines to soluble T-tropic HIV-1 envelope at 37°C can induce the formation of a trimolecular complex between CD4, gp120, and the chemokine receptor CXCR4 that was evidenced by their coimmunoprecipitation with CD4 (22). In the promonocytic cell line U937, a low-level coprecipitation of CD4 and CXCR4 was seen prior to treatment with gp120, suggesting that some constitutive association between CD4 and chemokine receptors may exist in certain cells. Recently, in a study on human monocytes and macrophages, we found preexisting CD4-CCR5 and CD4-CXCR4 complexes in the absence of prior exposure to HIV-1 or soluble gp120 (sgp120), which correlated with the fusion potential of the cells with X4 and R5 (CXCR4-and CCR5-dependent HIV) envelope-expressing cells (22). In a separate study, using either murine 3T3.CD4ϩ cells infected with a recombinant vaccinia-CCR5 virus (vCCR5) or primary human monocytes and macrophages, copr...

No abstract

Studies of HIV-1-infected individuals on anti-retroviral therapies and of patients receiving lymphoablating treatments indicate that the thymus retains restorative capacity even in adults. The contributions of the thymic epithelial cells (TECs) to the regeneration of the thymus and the identity of epithelial cell progenitors were evaluated in murine models of transient thymic atrophy followed by a complete regeneration. Using microarray approach, we analyzed the pattern of gene expression in TECs sorted from mice that were depleted of thymocytes by steroid treatment or by irradiation. The initial analysis identified significant increases in the mRNA for cMyc, Trp63 and Tcf3 transcription factors known to be expressed in early epithelial cell progenitors in tissues other than the thymus. Immunohistochemistry showed that in involuted thymuses, the cMyc and Trp63 proteins were expressed in a subset of cortical thymic epithelial cells (cTECs) that were keratin 5 positive (K5(+)), typifying cTEC precursors. Importantly, confocal microscopy established that epithelial cells with the phenotype of putative TEC progenitors (i.e. K5(+)K8(+)) expressed the Trp63 protein and confirmed that K5(+)K8(+) TEC progenitors expanded significantly during atrophy and prior to the thymic regeneration. Thus, our data demonstrated for the first time that critical steps in the recovery of the adult thymus include expansion of TEC progenitors and elevated expression of Trp63, cMyc and Tcf3 transcription factors in the thymic stroma. These results suggest that TEC progenitors could be reactivated in the adult thymus and, therefore, reactivation of TEC progenitors could provide a new approach for thymic reconstitution.

The effect of interferon γ (IFN-γ) and interleukin 6 (IL-6) on infection of macrophages with human immunodeficiency virus type 1 (HIV-1) was investigated. By using a polymerase chain reaction–based viral entry assay and viral infectivity assay, it was demonstrated that IL-6 and IFN-γ augmented susceptibility of monocyte-derived macrophages (MDMs) to infection with T-cell tropic CXCR4-utilizing (X4) HIV-1 strains. Consistent with this finding, IFN-γ and IL-6 augmented fusion of MDMs with T-tropic envelope-expressing cells. The enhanced fusion of cytokine-treated MDMs with T-tropic envelopes was inhibited by the CXCR4 ligand, SDF-1, and by T22 peptide. IFN-γ and IL-6 did not affect expression of surface CXCR4 or SDF-1–induced Ca++ flux in MDMs. In contrast to the effect of IFN-γ on the infection of MDMs with X4 strains, IFN-γ inhibited viral entry and productive infection of MDMs with macrophage-tropic (M-tropic) HIV-1. Consistent with this finding, IFN-γ induced a decrease in fusion with M-tropic envelopes that correlated with a modest reduction in surface CCR5 and CD4 on MDMs. It was further demonstrated that macrophage inflammatory protein (MIP)-1α and MIP-β secreted by cytokine-treated MDMs augmented their fusion with T-tropic–expressing cells and inhibited their fusion with M-tropic envelope-expressing cells. These data indicate that proinflammatory cytokines, which are produced during opportunistic infections or sexually transmitted diseases, may predispose macrophages to infection with X4 strains that, in turn, could accelerate disease progression.

Prostaglandin E2 (PGE2) is induced in vivo by bacterial products including TLR agonists. To determine whether PGE2 is induced directly or via IL-1β, human monocytes and macrophages were cultured with LPS or with Pam3CSK4 in presence of caspase-1 inhibitor, ZVAD, or IL-1R antagonist, Kineret. TLR agonists induced PGE2 in macrophages exclusively via IL-1β-independent mechanisms. In contrast, ZVAD and Kineret reduced PGE2 production in LPS-treated (but not in Pam3CSK4-treated) monocytes, by 30–60%. Recombinant human IL-1β augmented COX-2 and mPGES-1 mRNA and PGE2 production in LPS-pretreated monocytes but not in un-primed or Pam3CSK4-primed monocytes. This difference was explained by the finding that LPS but not Pam3CSK4 induced phosphorylation of IRF3 in monocytes suggesting activation of the TRIF signaling pathway. Knocking down TRIF, TRAM, or IRF3 genes by siRNA inhibited IL-1β-induced COX-2 and mPGES-1 mRNA. Blocking of TLR4 endocytosis during LPS priming prevented the increase in PGE2 production by exogenous IL-1β. Our data showed that TLR2 agonists induce PGE2 in monocytes independently from IL-1β. In the case of TLR4, IL-1β augments PGE2 production in LPS-primed monocytes (but not in macrophages) through a mechanism that requires TLR4 internalization and activation of the TRIF/IRF3 pathway. These findings suggest a key role for blood monocytes in the rapid onset of fever in animals and humans exposed to bacterial products and some novel adjuvants.