Decline in CD4+ cell numbers reflects increase in HIV-1 replication

Changes in CD4+ cell numbers were studied in relation to the presence of HIV-1 antigen (HIV-1-Ag) in serum from homosexual men followed prospectively. During 30 months of follow-up the mean CD4+ cell number (x 10(9) per liter) was stable in 134 at entry HIV-1 antibody (HIV-1-Ab) seropositives, who remained HIV-1-Ag negative (from 0.59 to 0.62) and declined in 38 at entry HIV-1-Ab seropositives who were persistently HIV-1-Ag positive (from 0.43 to 0.34). In sera of 9 of 65 HIV-1-Ab seroconverters HIV-1-Ag was detected only once, 3 months before or concomitantly with antibody seroconversion. Another 11 men became persistently HIV-1-Ag positive with antibody seroconversion or 2-6 weeks thereafter. A decline in CD4+ cell numbers was seen between 6 months before and the moment of HIV-1-Ab seroconversion, independently of duration and level of antigen expression. This indicates initial HIV-1 replication in both HIV-1-Ag negatives and positives. Following antibody seroconversion, HIV-1-Ag negatives had higher CD4+ cell numbers than HIV-1-Ag positives. Similarly to those who were HIV antigenemic from entry of the study, the HIV-1-Ab seroconverters who concomitantly with seroconversion or shortly thereafter became HIV-1 antigenemic showed a steady and significant (p = 0.01) decline in CD4+ cell numbers. In those who remained HIV-1-Ag negative after antibody seroconversion, CD4+ cell numbers were stable during follow-up.(ABSTRACT TRUNCATED AT 250 WORDS)     

HIV-1 gp120- and gp160-induced apoptosis in cultured endothelial cells is mediated by caspases

The immune dysfunction and cell destruction that occur in the human immunodeficiency virus (HIV)-infected host appear to result from the direct cytopathic effects of viral infection and the effects of viral proteins on uninfected bystander cells. Recently, the alpha-chemokine receptor CXCR4 has been reported to mediate apoptosis in neuronal cells and in CD4(+) and CD8(+) T cells after its binding to HIV-1 envelope proteins. In the current study, it was observed that human umbilical vein endothelial cells (HUVEC) undergo apoptosis after their treatment with the HIV-1 envelope proteins gp120/160. Anti-CXCR4 monoclonal antibody decreased HIV-1 gp120/160-induced apoptosis, suggesting that the CXCR4 chemokine receptor mediates the apoptotic effects of these HIV envelope glycoproteins. Further studies revealed that caspases play an important role in this process because the pretreatment of cells with a general caspase enzyme inhibitor decreased the extent of HUVEC apoptosis induced by gp120/160. In addition, it was found that caspase-3 was activated on HIV-1 gp120/160 treatment of these cells. It was also observed that gp120/160 treatment slightly increased the expression of the pro-apoptotic molecule Bax. These results suggest that HIV-1 envelope glycoproteins can disrupt endothelial integrity through the interaction with CXCR4, thereby facilitating virus transit out of the bloodstream and contributing to the vascular injury syndromes seen in acquired immunodeficiency syndrome. (Blood. 2000;96:1438-1442)     

Antigen-driven CD4+ T cell and HIV-1 dynamics: residual viral replication under highly active antiretroviral therapy

Antigen-induced stimulation of the immune system can generate heterogeneity in CD4+ T cell division rates capable of explaining the temporal patterns seen in the decay of HIV-1 plasma RNA levels during highly active antiretroviral therapy. Posttreatment increases in peripheral CD4+ T cell counts are consistent with a mathematical model in which host cell redistribution between lymph nodes and peripheral blood is a function of viral burden. Model fits to patient data suggest that, although therapy reduces HIV replication below replacement levels, substantial residual replication continues. This residual replication has important consequences for long-term therapy and the evolution of drug resistance and represents a challenge for future treatment strategies.     

Propagation of CD4+ T cells specific for HIV type 1 envelope gp120 from chronically HIV type 1-infected subjects

HIV-specific CD4+ T cell responses, in particular to the HIV envelope antigen gp120, are often undetectable in the peripheral blood of HIV-infected individuals. The failure to detect these cells poses a significant impediment to studying the T cell populations that are considered to be essential for controlling HIV infection and has led to speculation that these cells are entirely depleted during HIV infection. This study was designed to test whether gp120-specific CD4+ T cells exist in HIV-infected subjects and can be expanded from peripheral blood mononuclear cells by in vitro stimulation with the gp120 antigen, allowing better characterization of these cells. Although gp120-specific T cell responses were barely observed in patient cells ex vivo before antigenic stimulation, CD4+ T cells specific for gp120 were successfully propagated from the blood of each asymptomatic chronically HIV-infected subject studied. The dominant epitopes recognized by gp120-specific CD4+ T cells from these HIV-infected subjects were mapped to well-conserved sites in the C1 and C2 domains of gp120. Two CD4+ T cell lines recognizing these two regions were subsequently established. The CD4+ T cell lines proliferated and produced interferon gamma in response to the specific epitopes, and the responses were MHC class II restricted. These T cell lines also exhibited cross-reactivity with gp120 from T cell line-adapted HIV-1 strains IIIB and MN, as well as with gp120 from primary isolates SF33 (subtype B), CA1 (subtype A), and CA10 (subtype A/E). The data demonstrate that CD4+ T cells specific for gp120 are not entirely depleted from the peripheral blood of chronically HIV-infected subjects; these cells are present in low numbers but can be expanded after antigenic stimulation in vitro.     

HIV-1 Protein gp120 Induces Mouse Lung Fibroblast-to-Myofibroblast Transdifferentiation via CXCR4 Activation

BackgroundIndividuals with HIV have ～2-fold increased risk of developing pulmonary fibrosis. The mechanism(s) by which this occurs has yet to be determined. HIV-1 protein gp120 activates CXCR4 in the lymphocyte, promoting a variety of intracellular signaling pathways including those common to TGFβ1 associated with lung fibroblast-to-myofibroblast transdifferentiation. We hypothesized that gp120 promotes pulmonary fibrotic changes via activation of CXCR4 in the lung fibroblast.MethodsMouse primary lung fibroblasts (PLFs) were cultured ± gp120, then analyzed for α-SMA expression and stress fiber formation. In parallel, PLFs were cultured ± gp120 ± AMD3100 (a CXCR4 antagonist), and α-SMA, pan and phospho-Akt, and total and phospho-MAPK (or ERK1/2) protein expression was quantified. Finally, lungs and PLFs from wild-type and HIV-1 transgenic mice were analyzed for hydroxyproline and α-SMA content.Resultsgp120 treatment increased α-SMA expression and myofibroblast differentiation in PLFs. gp120 treatment activated phosphorylation of ERK1/2, but not PI3K-Akt. Pretreatment with AMD3100 inhibited gp120-induced ERK1/2 phosphorylation and gp120-induced α-SMA expression. In parallel, there was a significant increase in hydroxyproline content in lungs from older HIV-1 transgenic mice and a >3-fold increase in α-SMA expression in PLFs isolated from HIV-1 transgenic mice.Conclusionsgp120 induces α-SMA expression and fibroblast-to-myofibroblast transdifferentiation by activating the CXCR4-ERK1/2 signaling pathway in mouse PLFs. Lungs of older HIV-1 transgenic mice contain higher hydroxyproline content and their PLFs have a striking increase in α-SMA expression. These results suggest a mechanism by which individuals with HIV are at increased risk of developing pulmonary fibrotic changes as they age.     

CCR5/CD4/CXCR4 oligomerization prevents HIV-1 gp120IIIB binding to the cell surface

CCR5 and CXCR4, the respective cell surface coreceptors of R5 and X4 HIV-1 strains, both form heterodimers with CD4, the principal HIV-1 receptor. Using several resonance energy transfer techniques, we determined that CD4, CXCR4, and CCR5 formed heterotrimers, and that CCR5 coexpression altered the conformation of both CXCR4/CXCR4 homodimers and CD4/CXCR4 heterodimers. As a result, binding of the HIV-1 envelope protein gp120_IIIB to the CD4/CXCR4/CCR5 heterooligomer was negligible, and the gp120-induced cytoskeletal rearrangements necessary for HIV-1 entry were prevented. CCR5 reduced HIV-1 envelope-induced CD4/CXCR4-mediated cell-cell fusion. In nucleofected Jurkat CD4 cells and primary human CD4⁺ T cells, CCR5 expression led to a reduction in X4 HIV-1 infectivity. These findings can help to understand why X4 HIV-1 strains infection affect T-cell types differently during AIDS development and indicate that receptor oligomerization might be a target for previously unidentified therapeutic approaches for AIDS intervention.For HIV-1 to enter a target cell, the viral envelope glycoprotein gp120 must interact with a set of cell surface molecules that include the primary receptor, CD4 (1), and a chemokine receptor (CCR5 or CXCR4) that acts as a coreceptor (2, 3). These molecules form CD4/chemokine receptor complexes, as deduced from coprecipitation data for CXCR4 or CCR5 with CD4 (4–8).Most HIV-1 variants isolated from newly infected individuals use CCR5 and CD4 to enter host cells; these M-tropic R5 strains are predominant in acute and asymptomatic phases of HIV infection. CD4⁺ T helper type 1 (Th1) cells, which express high CCR5 levels (9, 10), are implicated in maintaining asymptomatic status (11, 12). The “viral shift” from R5 to T-tropic X4 HIV-1 strains correlates with AIDS progression (13, 14). X4 strains infect mainly CD4⁺ Th2 cells, which express little CCR5 and whose CXCR4 levels resemble those of Th1 cells (15, 16), which suggests that cell susceptibility to HIV-1 infection depends on the CD4/coreceptor ratio and on receptor levels during cell activation and/or differentiation (17). CXCR4 and CCR5 are present as homodimers and heterodimers at the plasma membrane (18–20). In addition, gp120-mediated CD4/CXCR4 and CD4/CCR5 association and clustering is reported (21–23). Nonetheless, little is known of how CCR5 expression influences the CD4/CXCR4 interaction, or of the molecular basis that underlies the differences in X4 strains infection relative to CCR5 levels at the cell surface.Here, we identify CD4/CXCR4/CCR5 oligomers at the cell membrane, even in the absence of ligands. CCR5 expression in these complexes modifies the heterodimeric CD4/CXCR4 conformation and blocks gp120_IIIB binding, without altering binding of the CXCR4 ligand CXCL12 and its subsequent signaling. gp120_IIIB-triggered LIMK1 activation, cofilin dephosphorylation, and the actin cytoskeleton rearrangement necessary for cell-cell fusion were impeded in CD4/CXCR4/CCR5-expressing cells. The data obtained using recombinant gp120_IIIB glycoprotein were confirmed by experiments showing that X4 HIV-1 infection of Jurkat and primary T cells is regulated by CCR5 expression.     

Characterization of recombinant gp120 and gp160 from HIV-1: binding to monoclonal antibodies and soluble CD4

We compared four preparations of recombinant HIV-1 envelope glycoprotein: mammalian (Chinese hamster ovary cells) gp120 (Celltech); baculovirus gp120 from American Biotechnologies Inc. (ABT) and from MicroGeneSys (MGS); and baculovirus gp160 (Institute of Virology, Oxford, UK). Each envelope glycoprotein binds to a neutralizing monoclonal antibody (MAb) directed against the V3 loop, confirming the integrity of this type-specific neutralization epitope. MGS gp120 binds abnormally well to a MAb which recognizes an epitope preferentially exposed on denatured gp120. Consistent with this finding, MGS gp120 binds to soluble CD4 (sCD4) with an affinity 50-100-fold lower than that of Celltech gp120. The affinity of Celltech gp120 from sCD4 is 2.3 nM, indistinguishable from that of gp120 extracted from HIV-1 virions. Baculovirus gp120 (ABT) and gp160 also have a high affinity for sCD4. A significant proportion of anti-gp120 antibodies in HIV-positive human sera recognize epitopes that are dependent on the mammalian glycosylation pattern, and a human HIV-positive serum inhibits the binding of mammalian gp120 to sCD4 five- to 10-fold more potently than it inhibits baculovirus gp120 binding to sCD4.     

HIV-1 gp120 induces autophagy in cardiomyocytes via the NMDA receptor

Background

HIV-1 envelope glycoprotein gp120 (gp120) is considered as one of the major virulent proteins responsible for the involvement of the cardiovascular system. Autophagy as a form of self maintenance plays important roles in cell survival and death. HIV-1 gp120 is reported to induce autophagy in a variety of cells. However, the effect of gp120 on autophagy in cardiomyocytes has not been reported. This study aimed to test our hypothesis that gp120 could induce autophagy in cardiomyocytes.

Methods

Rat cardiomyocyte H9c2 cells were treated with gp120 (100 ng/ml) in vitro for 4 h, 1 day and 7 days. The autophagy related proteins were analyzed by Western blot and the autophagosomes were analyzed by confocal microscopy.

Results

The autophagic proteins and autophagosomes were markedly increased in the H9c2 cells after 4 h of gp120 treatment. Furthermore, gp120 induced autophagic proteins and autophagosomes were significantly inhibited by the N-methyl-d-aspartic acid (NMDA) receptor inhibitor MK801, c-Jun N-terminal kinase (JNK) inhibitor SP600125, and the class III phosphoinositide 3-kinase (PI3K) inhibitor 3-methyladenine (3-MA), while there was no change in the cells pretreated with the CXCR4 antagonist AMD3100. In addition, no apparent cell death was observed in the cardiomyocytes treated with gp120 for up to 7 days.

Conclusions

In summary, our research demonstrated for the first time that HIV-1 gp120 could induce autophagy of cardiomyocytes and the NMDA receptor, JNK and class III PI3K were involved in this process. This observation provides a new insight into the mechanisms of in the cardiovascular involvement during HIV-1 infection.     

Soluble HLA-G protein secreted by allo-specific CD4+ T cells suppresses the allo-proliferative response: a CD4+ T cell regulatory mechanism

We recently reported that the nonclassical HLA class I molecule HLA-G was expressed in the endomyocardial biopsies and sera of 16% of heart transplant patients studied. The aim of the present report is to identify cells that may be responsible for HLA-G protein expression during the allogeneic reaction. Carrying out mixed lymphocyte cultures in which the responder cell population was depleted either in CD4(+) or CD8(+) T cells, we found that soluble HLA-G5 protein but not the membrane-bound HLA-G isoform was secreted by allo-specific CD4(+) T cells from the responder population, which suppressed the allogeneic proliferative T cell response. This inhibition may be reversed by adding the anti-HLA-G 87G antibody to a mixed lymphocyte culture. That may indicate a previously uncharacterized regulatory mechanism of CD4(+) T cell proliferative response.     

HIV-1 specific CD8+ T cells with an effector phenotype and control of viral replication

Most people infected with HIV-1 cannot control viral replication despite the presence of virus-specific CD8+ T cells. It has been postulated that this inability is related to the failure of these cells to mature into fully differentiated effector cells. We tested this hypothesis by comparing the maturation phenotype of virus-specific CD8+ T cells in people who could control viral replication off anti-retroviral therapy with those who could not. In five patients with treated acute HIV-1-infection, structured treatment interruption (STI) induced control of viral replication was associated with expansion of virus-specific CD8+ T cells with a fully differentiated effector phenotype. These effector cells were also expanded in treatment-naive chronically infected individuals who spontaneously controlled viral replication, and augmented expression of perforin was noted in both settings. Our data show that full maturation of virus-specific CD8+ T cells is possible in the context of HIV-1-infection, and suggest that such maturation might be important in viral control.     

HIV-1 envelope T cell epitope "hotspots " among mice and humans and among CD4+ and CD8+ T cell subpopulations

HIV-1-specific T cell responses correlate with control of infection and disease, thus encouraging a full understanding of the peptides and antigen-processing mechanisms that govern T cell activation. We have previously demonstrated that CD4(+) T cell epitopes cluster nonrandomly within envelope protein "hotspot" regions. The current study was initiated to determine whether envelope-specific CD8(+) T cells might share epitope "hotspots" with the CD4(+) T cell population. Identification of CD8(+) T cell determinants by ELISPOT assays with peripheral blood mononuclear cells from four HIV-1-infected individuals, in conjunction with a survey of determinants in the Los Alamos database, revealed similarities among "hotspot" positions for CD4(+) and CD8T(+) cells within mice and humans. These results emphasized the important influence that envelope peptide position may have on antigen processing, and the consequent impact such processing may have on HIV-1-specific CD4(+) and CD8(+) T-cell activities.     

Loss of viral control in early HIV-1 infection is temporally associated with sequential escape from CD8+ T cell responses and decrease in HIV-1-specific CD4+ and CD8+ T cell frequencies

The outcome of human immunodeficiency virus type 1 (HIV-1) infection is related to the set-point plasma virus load (pVL) that emerges after primary HIV-1 infection (PHI). This set-point pVL generally remains stable but eventually increases with progression to disease. However, the events leading to loss of viremic control are poorly understood. Here, we describe an individual who presented with symptomatic PHI and subsequently progressed rapidly, after an initial period of 1 year during which viral replication was well controlled. Escalation of viral replication in this atypical case was preceded by the emergence of escape variants in many epitopes targeted by dominant CD8+ T cell responses and a marked decrease in HIV-1-specific CD4+ and CD8+ T cell frequencies. There were no changes in viral tropism, replication kinetics, or neutralizing antibody titers. These findings demonstrate the temporal relationship between viral escape from CD8+ T cell activity, decrease in HIV-1-specific T cell frequencies, and loss of control of viral replication.     

Galectin-1: a key effector of regulation mediated by CD4+CD25+ T cells

The naturally occurring population of dedicated regulatory T cells that coexpress CD4 and CD25 is known to play a key role in the maintenance of peripheral T-cell tolerance; however, their mechanism of action has remained obscure. Here we report that a member of the family of beta-galactoside-binding proteins, galectin-1, is overexpressed in regulatory T cells, and that expression is increased after activation. Most importantly, blockade of galectin-1 binding significantly reduced the inhibitory effects of human and mouse CD4+CD25+ T cells. Reduced regulatory activity was observed in CD4+CD25+ T cells obtained from galectin-1-homozygous null mutant mice. These results suggest that galectin-1 is a key effector of the regulation mediated by these cells.