Determinant in Human Immunodeficiency Virus Type 1 for Efficient Replication under Cytokine-Induced CD4+ T-Helper 1 (Th1)- and Th2-Type Conditions

Cytokines are potent stimuli for CD4⁺-T-cell differentiation. Among them, interleukin-12 (IL-12) and IL-4 induce naive CD4⁺ T cells to become T-helper 1 (Th1) or Th2 cells, respectively. In this study we found that macrophage-tropic human immunodeficiency virus type 1 (HIV-1) strains replicated more efficiently in IL-12-induced Th1-type cultures derived from normal CD4⁺ T cells than did T-cell-line-tropic (T-tropic) strains. In contrast, T-tropic strains preferentially infected IL-4-induced Th2-type cultures derived from the same donor CD4⁺ T cells. Additional studies using chimeric viruses demonstrated that the V3 region of HIV-1 gp120 was the principal determinant for efficiency of replication. Cell fusion analysis showed that cells expressing envelope protein from a T-tropic strain effectively fused with IL-4-induced Th2-type culture cells. Flow cytometric analysis showed that the level of CCR5 expression was higher on IL-12-induced Th1-type culture cells, whereas CXCR4 was highly expressed on IL-4-induced Th2-type culture cells, although a low level of CXCR4 expression was observed on IL-12-induced Th1-type culture cells. These results indicate that HIV-1 isolates exhibit differences in the ability to infect CD4⁺-T-cell subsets such as Th1 or Th2 cells and that this difference may partly correlate with the expression of particular chemokine receptors on these cells. The findings suggest that immunological conditions are one of the factors responsible for inducing selection of HIV-1 strains.     

Opposite effects of IL-10 on the ability of dendritic cells and macrophages to replicate primary CXCR4-dependent HIV-1 strains

We investigated the effect of IL-10 on replication of primary CXCR4-dependent (X4) HIV-1 strains by monocyte-derived dendritic cells (DCs) and macrophages (M Phis). M Phis efficiently replicated CXCR4-dependent HIV-1 (X4 HIV-1) strains NDK and VN44, whereas low levels of p24 were detected in supernatants of infected DCs. IL-10 significantly increased X4 HIV-1 replication by DCs but blocked viral production by M Phis as determined by p24 levels and semiquantitative nested PCR. IL-10 up-regulated CXCR4 mRNA and protein expression on DCs and M Phis, suggesting that IL-10 enhances virus entry in DCs but blocks an entry and/or postentry step in M Phis. The effect of IL-10 on the ability of DCs and M Phis to transmit virus to autologous CD4(+) T lymphocytes was investigated in coculture experiments. DCs exhibited a greater ability than did M Phis to transmit a vigorous infection to CD4(+) T cells despite their very low replication capacity. IL-10 had no effect on HIV-1 replication in DC:T cell cocultures but markedly decreased viral production in M Phi:T cell cocultures. These results demonstrate that IL-10 has opposite effects on the replication of primary X4 HIV-1 strains by DCs and M Phis. IL-10 increases X4-HIV-1 replication in DCs but does not alter their capacity to transmit virus to CD4(+) T lymphocytes. These findings suggest that increased levels of IL-10 observed in HIV-1-infected patients with disease progression may favor the replication of X4 HIV-1 strains in vivo.     

Autophagy and CD4+ T lymphocyte destruction by HIV-1

The first step of HIV-1 infection is mediated by the binding of envelope glycoproteins (Env) to CD4 and two major coreceptors, CCR5 or CXCR4. The HIV-1 strains that use CCR5 are involved in primo-infection whereas those HIV-1 strains that use CXCR4 play a major role in the demise of CD4+ T lymphocytes and a rapid progression toward AIDS. Notably, binding of X4 Env expressed on cells to CXCR4 triggers apoptosis of uninfected CD4+ T cells. We now have just demonstrated that, independently of HIV-1 replication, transfected or HIV-1-infected cells that express X4 Env induce autophagy and accumulation of Beclin 1 in uninfected CD4+ T lymphocytes via CXCR4. Moreover, autophagy is a prerequisite to Env-induced apoptosis in uninfected bystander T cells, and CD4+ T cells still undergo an Env-mediated cell death with autophagic features when apoptosis is inhibited. To the best of our knowledge, these findings represent the first example of autophagy triggered through binding of virus envelope proteins to a cellular receptor, without viral replication, leading to apoptosis. Here, we proposed hypotheses about the significance of Env-induced Beclin 1 accumulation in CD4+ T cell death and about the role of autophagy in HIV-1 infected cells depending on the coreceptor involved.     

CD40-Mediated Induction of CD4 and CXCR4 on B Lymphocytes Correlates with Restricted Susceptibility to Human Immunodeficiency Virus Type 1 Infection: Potential Role of B Lymphocytes as a Viral Reservoir

Human immunodeficiency virus type 1 (HIV-1) replicates primarily in lymphoid tissues where it has ready access to activated immune competent cells. We used one of the major pathways of immune activation, namely, CD40-CD40L interactions, to study the infectability of B lymphocytes isolated from peripheral blood mononuclear cells. Highly enriched populations of B lymphocytes generated in the presence of interleukin-4 and oligomeric soluble CD40L upregulated costimulatory and activation markers, as well as HIV-1 receptors CD4 and CXCR4, but not CCR5. By using single-round competent luciferase viruses complemented with either amphotropic or HIV-derived envelopes, we found a direct correlation between upregulation of HIV-1 receptors and the susceptibility of the B lymphocytes to infection with dual-tropic and T-tropic strains of HIV-1; in contrast, cells were resistant to M-tropic strains of HIV-1. HIV-1 envelope-mediated infection was completely abolished with either an anti-CD4 monoclonal antibody or a peptide known to directly block CXCR4 usage and partially blocked with stromal cell-derived factor 1, all of which had no effect on the entry of virus pseudotyped with amphotropic envelope. Full virus replication kinetics confirmed that infection depends on CXCR4 usage. Furthermore, productive cycles of virus replication occurred rapidly yet under most conditions, without the appearance of syncytia. Thus, an activated immunological environment may induce the expression of HIV-1 receptors on B lymphocytes, priming them for infection with selective strains of HIV-1 and allowing them to serve as a potential viral reservoir.     

CXCR4-dependent infection of CD8+, but not CD4+, lymphocytes by a primary human immunodeficiency virus type 1 isolate

We recently isolated from an infant an X4-syncytium-inducing (SI) human immunodeficiency virus type 1 (HIV-1) variant (92US143-T8) that was able to infect CD8+ lymphocytes independently of CD4. Although it was CD4 independent, the 92US143-T8 isolate also maintained the ability to infect CD4+ cells. In the present study, we investigated the role of CXCR4 in the infection of CD4+ and CD8+ cells by this primary isolate. The expression of CXCR4 was down modulated in CD8+ lymphocytes after infection with the 93US143-T8 isolate. Infection of CD8+ lymphocytes by the 93US143-T8 isolate was prevented by treatment with AMD3100, a specific antagonist for CXCR4, indicating CXCR4-dependent infection. Interestingly, AMD3100 treatment had no inhibitory role in the infection of purified CD4+ lymphocytes by the same isolate. Furthermore, AMD3100 treatment failed to prevent infection of known CD4+ CXCR4+ T-cell lines (MT-2 and CEM) by the 93US143-T8 isolate. In fact, virus replication in the CD4+ cells was often enhanced in the presence of AMD3100. Viruses produced from the infected CD4+ cells in the presence of AMD3100 maintained an unchanged envelope genotype and an SI phenotype. For the first time, these results provide evidence of CXCR4-dependent infection of CD8+ lymphocytes by a primary HIV-1 isolate. This study also shows a different mode of infection for the CD4+ and CD8+ lymphocytes by the same HIV-1 variant. Finally, our findings suggest that a more careful evaluation is necessary before the random use of AMD3100 as a new entry inhibitor in patients harboring SI HIV-1 strains.     

Cyclic zinc-dithiocarbamate-S,S'-dioxide blocks CXCR4-mediated HIV-1 infection

To test the anti-human immunodeficiency virus type-1 (HIV-1) activity of 3,6,9,12-tetraazatetradecane-1,14-diylbis(zinc dithiocarbamate)-S,S'-dioxide (cyclic zinc-dithiocarbamate-S, S'-dioxide), MAGI and MAGIC-5 cells were used; the former express CXCR4 and the latter express both CXCR4 and CCR5, which are HIV-1 coreceptors. The compound markedly inhibited HIV-1 X4 (CXCR4-using) viral replication in both MAGI and MAGIC-5 cells. On the other hand, the replication of HIV-1 R5X4 (both CXCR4-and CCR5-using) in MAGI cells but not MAGIC-5 cells was inhibited by the compound. The compound was found to specifically inhibit HIV-1 (X4) envelope-mediated cell-to-cell fusion, binding of anti-CXCR4 monoclonal antibody (12G5) to CXCR4 expressed on the surface of cells, and calcium flux induced by stromal-derived factor-1alpha (SDF-1alpha) bound to CXCR4. The results suggest that the compound inhibited CXCR4-mediated HIV-1 infection by influencing to the HIV-1 coreceptor activity of CXCR4.     

CCL2 increases X4-tropic HIV-1 entry into resting CD4+ T cells

During human immunodeficiency virus type 1 (HIV-1) infection, there is a strong positive correlation between CCL2 levels and HIV viral load. To determine whether CCL2 alters HIV-1 infection of resting CD4(+) T cells, we infected purified resting CD4(+) T cells after incubation with CCL2. We show that CCL2 up-regulates CXCR4 on resting CD4(+) T cells in a CCR2-dependent mechanism, and that this augmentation of CXCR4 expression by CCL2 increases the ability of these cells to be chemoattracted to CXCR4 using gp120 and renders them more permissive to X4-tropic HIV-1 infection. Thus, CCL2 has the capacity to render a large population of lymphocytes more susceptible to HIV-1 late in the course of infection.     

Human immunodeficiency virus type 1 (HIV-1)-induced GRO-alpha production stimulates HIV-1 replication in macrophages and T lymphocytes

We examined the early effects of infection by CCR5-using (R5 human immunodeficiency virus [HIV]) and CXCR4-using (X4 HIV) strains of HIV type 1 (HIV-1) on chemokine production by primary human monocyte-derived macrophages (MDM). While R5 HIV, but not X4 HIV, replicated in MDM, we found that the production of the C-X-C chemokine growth-regulated oncogene alpha (GRO-alpha) was markedly stimulated by X4 HIV and, to a much lesser extent, by R5 HIV. HIV-1 gp120 engagement of CXCR4 initiated the stimulation of GRO-alpha production, an effect blocked by antibodies to CXCR4. GRO-alpha then fed back and stimulated HIV-1 replication in both MDM and lymphocytes, and antibodies that neutralize GRO-alpha or CXCR2 (the receptor for GRO-alpha) markedly reduced viral replication in MDM and peripheral blood mononuclear cells. Therefore, activation of MDM by HIV-1 gp120 engagement of CXCR4 initiates an autocrine-paracrine loop that may be important in disease progression after the emergence of X4 HIV.     

Enhanced inhibition of human immunodeficiency virus type 1 by Met-stromal-derived factor 1beta correlates with down-modulation of CXCR4

CXCR4 is a chemokine receptor used by some strains of HIV-1 as an entry coreceptor in association with cell surface CD4 on human cells. In human immunodeficiency virus type 1 (HIV-1)-infected individuals, the appearance of viral isolates with a tropism for CXCR4 (T tropic) has been correlated with late disease progression. The presumed natural ligands for CXCR4 are SDF-1alpha and SDF-1beta, which are proposed to play a role in blocking T-tropic HIV-1 cell entry. Here, we demonstrate that addition of an N-terminal methionine residue to SDF-1beta (Met-SDF-1beta) results in a dramatically enhanced functional activity compared to that of native SDF-1beta. Equivalent concentrations of Met-SDF-1beta are markedly more inhibitory for T-tropic HIV-1 replication than SDF-1beta. A comparison of the biological activities of these two forms of SDF-1beta reveals that Met-SDF-1beta induces a more pronounced intracellular calcium flux yet binds with slightly lower affinity to CXCR4 than SDF-1beta. Down-modulation of CXCR4 is similar after exposure of cells to either chemokine form for 2 h. However, after a 48-h incubation, the surface expression of CXCR4 is much lower for cells treated with Met-SDF-1beta. The enhanced blocking of T-tropic HIV-1 by Met-SDF-1beta appears to be related to prolonged CXCR4 down-modulation.     

Effect of the resection of the sciatic nerve on the Th1/Th2 balance in the synovia of the ankle joint of adjuvant arthritic rats

Inflamed synovia of the ankle joint after 2–4 weeks of adjuvant injection receives dense sensory innervation. To study the role of sensory nerves on the local inflammation, the relative expression of T helper 1 and 2 lymphocyte (Th1 and Th2) markers was investigated on both axotomized adjuvant arthritic (AA) rats, whose sciatic nerves were resected before adjuvant injection, and on sham-operated ones. Immunohistochemical expressions of CXC chemokine receptor 3 (CXCR3) and CC chemokine receptor 4 (CCR4) were examined and compared with those of Th1 cytokine (interferon-gamma, IFN-gamma), Th2 cytokine (interleukin-4, IL-4), and anti-T cell antibody (W3/25). Double-positive cells for IFN-gamma/CXCR3 and for IL-4/CCR4 were greater than 90% and greater than 95%, respectively. The reciprocal combinations, IL-4/CXCR3 and IFN-gamma/CCR4, however, yielded less than 10% and less than 5% of double-positive cells. CXCR3 and CCR4 thus appear to be available as markers for Th1/Th2 subsets in the synovia of AA rats. Using these markers, it became clear that the percentage of Th1 cells to total Th cells was higher than that of Th2 cells in axotomized AA rats at weeks 2–4, whereas in sham-operated AA rats, the percentage of Th1 cells to total Th cells was higher than that of Th2 cells at week 2 and the latter exceeded the former at week 4. Our observations strongly suggested the presence of the anti-inflammatory action of sensory nerves in rats with adjuvant arthritis.     

Role of N-glycans and SDF-1alpha on the coassociation of CD4 with CXCR4 at the plasma membrane of monocytic cells and blood lymphocytes

CXCR4 is a coreceptor, along with CD4, for human immunodeficiency virus type 1 (HIV-1). Trimolecular complexes between HIV-1 glycoprotein (gp)120, CD4 and CXCR4 constitute a prerequisite for HIV entry. We studied whether CD4 is associated with CXCR4 on CD4+ CXCR4+ cells. Using the conformation-dependent anti-CXCR4 mAb 12G5, CD4 was coimmunoprecipitated with CXCR4 from the membrane of U937 cells which support HIV-1(LAI) efficient infection, and from that of peripheral blood lymphocytes (PBL). CD4 association with CXCR4 increased upon PBL coculture for 5 days with autologous monocytes, decreased upon treatment of the cells or the CD4-CXCR4 complex with either N-glycanase or stromal cell derived factor-1alpha (SDF-1alpha) and was abolished by incubation of the cells with both, N-glycanase and SDF-1alpha. This indicates that glycans are partly involved in CD4 association with CXCR4 and may partly explain the inhibitory effect of SDF-1alpha on HIV infection.     

Inhibitory and enhancing effects of IFN-gamma and IL-4 on SHIV(KU) replication in rhesus macaque macrophages: correlation between Th2 cytokines and productive infection in tissue macrophages during late-stage infection

HIV-1 is dual-tropic for CD4+ T lymphocytes and macrophages, but virus production in the macrophages becomes manifest only during late-stage infection, after CD4+ T cell functions are lost, and when opportunistic pathogens begin to flourish. In this study, the SHIV/macaque model of HIV pathogenesis was used to assess the role of cytokines in regulating virus replication in the two cell types. We injected complete Freund's adjuvant (CFA) intradermally into SHIV(KU)-infected macaques, and infused Schistosoma mansoni eggs into the liver and lungs of others. Tissues examined from these animals demonstrated that macrophages induced by CFA did not support viral replication while those induced by S. mansoni eggs had evidence of productive infection. RT-PCR analysis showed that both Th1 (IL-2 and IFN-gamma) and Th2 cytokines (IL-4 and IL-10) were present in the CFA lesions but only the Th2 cytokines were found in the S. mansoni lesions. Follow-up studies in macaque cell cultures showed that whereas IFN-gamma caused enhancement of virus replication in CD4+ T cells, it curtailed viral replication in infected macrophages. In contrast, IL-4 enhanced viral replication in infected macrophages. These studies strongly suggest that cytokines regulate the sequential phases of HIV replication in CD4 T cells and macrophages.     

C-C chemokine receptor 4 expression defines a major subset of circulating nonintestinal memory T cells of both Th1 and Th2 potential

CCR4, a chemokine receptor for macrophage-derived chemokine (MDC) and thymus and activation-regulated chemokine (TARC), has been implicated as a preferential marker for Th2 lymphocytes. Following in vitro polarization protocols, most Th2 lymphocytes express CCR4 and respond to its ligands TARC and MDC, whereas Th1 lymphocytes express CXC chemokine receptor 3 and CCR5 (but not CCR4). We show in this study that CCR4 is a major receptor for MDC and TARC on T lymphocytes, as anti-CCR4 mAbs significantly inhibit the migration of these cells to MDC and TARC. CCR4 is also highly expressed in most single-positive CD4(+) thymocytes and on a major fraction of blood nonintestinal (alpha(4)beta(7)(-)) memory CD4 lymphocytes, including almost all skin memory CD4(+) cells expressing the cutaneous lymphocyte Ag (CLA), but weakly or not expressed in other subsets in thymus and blood. Interestingly, major fractions of circulating CCR4(+) memory CD4 lymphocytes coexpress the Th1-associated receptors CXC chemokine receptor 3 and CCR5, suggesting a potential problem in using these markers for Th1 vs Th2 lymphocyte cells. Moreover, although production of Th2 cytokines in blood T cells is associated with CCR4(+) CD4 lymphocytes, significant numbers of freshly isolated circulating CCR4(+) memory CD4 lymphocytes (including both CLA(+) and CLA(-) fractions) readily express the Th1 cytokine IFN-gamma after short-term stimulation. Our results are consistent with a role for CCR4 as a major trafficking receptor for systemic memory T cells, and indicate that the patterns and regulation of chemokine receptor expression in vivo are more complex than indicated by current in vitro models of Th1 vs Th2 cell generation.     

Dendritic cells preferentially transfer CXCR4-using human immunodeficiency virus type 1 variants to CD4+ T lymphocytes in trans

Human immunodeficiency virus type 1 (HIV-1) preferentially utilizes the CCR5 coreceptor for target cell entry in the acute phase of infection, while later in disease progression the virus switches to the CXCR4 coreceptor in approximately 50% of patients. In response to HIV-1 the adaptive immune response is triggered, and antibody (Ab) production is elicited to block HIV-1 entry. We recently determined that dendritic cells (DCs) can efficiently capture Ab-neutralized HIV-1, restore infectivity, and transmit infectious virus to target cells. Here, we tested the effect of Abs on trans transmission of CCR5 or CXCR4 HIV-1 variants. We observed that transmission of HIV-1 by immature as well as mature DCs was significantly higher for CXCR4- than CCR5-tropic viral strains. Additionally, neutralizing Abs directed against either the gp41 or gp120 region of the envelope such as 2F5, 4E10, and V3-directed Abs inhibited transmission of CCR5-tropic HIV-1, whereas Ab-treated CXCR4-tropic virus demonstrated unaltered or increased transmission. To further study the effects of coreceptor usage we tested molecularly cloned HIV-1 variants with modifications in the envelope that were based on longitudinal gp120 V1 and V3 variable loop sequences from a patient progressing to AIDS. We observed that DCs preferentially facilitated infection of CD4⁺ T lymphocytes of viral strains with an envelope phenotype found late in disease. Taken together, our results illustrate that DCs transmit CXCR4-tropic HIV-1 much more efficiently than CCR5 strains; we hypothesize that this discrimination could contribute to the in vivo coreceptor switch after seroconversion and could be responsible for the increase in viral load.     

Preferential use of CXCR4 by R5X4 human immunodeficiency virus type 1 isolates for infection of primary lymphocytes

Coreceptor specificity of human immunodeficiency virus type 1 (HIV-1) strains is generally defined in vitro in cell lines expressing CCR5 or CXCR4, but lymphocytes and macrophages are the principal targets in vivo. CCR5-using (R5) variants dominate early in infection, but strains that use CXCR4 emerge later in a substantial minority of subjects. Many or most CXCR4-using variants can use both CXCR4 and CCR5 (R5X4), but the pathways that are actually used to cause infection in primary cells and in vivo are unknown. We examined several R5X4 prototype and primary isolates and found that they all were largely or completely restricted to CXCR4-mediated entry in primary lymphocytes, even though lymphocytes are permissive for CCR5-mediated entry by R5 strains. In contrast, in primary macrophages R5X4 isolates used both CCR5 and CXCR4. The R5X4 strains were also more sensitive than R5 strains to CCR5 blocking, suggesting that interactions between the R5X4 strains and CCR5 are less efficient. These results indicate that coreceptor phenotyping in transformed cells does not necessarily predict utilization in primary cells, that variability exists among HIV-1 isolates in the ability to use CCR5 expressed on lymphocytes, and that many or most strains characterized as R5X4 are functionally X4 in primary lymphocytes. Less efficient interactions between R5X4 strains and CCR5 may be responsible for the inability to use CCR5 on lymphocytes, which express relatively low CCR5 levels. Since isolates that acquire CXCR4 utilization retain the capacity to use CCR5 on macrophages despite their inability to use it on lymphocytes, these results also raise the possibility that a CCR5-mediated macrophage reservoir is required for sustained infection in vivo.