SDF-1alpha regulates HIV-1-gp120-induced changes in CD79b surface expression and Ig production in activated human B cells

Binding of HIV-1 glycoprotein (gp120) to activated B cells of HIV-infected and HIV-uninfected subjects induces increased cell proliferation, cAMP generation, immunoglobulin (Ig) production and downregulation of the invariant chain, CD79b, of the B-cell receptor. We present evidence that the stromal cell-derived factor-1alpha (SDF-1alpha), itself a B-cell stimulant, reversed gp120-driven downregulation of CD79b in CD40- and IL-4-activated purified HIV-1 seronegative human peripheral blood B cells. SDF-1alpha augmented gp120-induced Ig production, downregulated CXCR4 receptor expression, and alone, exerted no effect on CD79b surface expression, reversed the gp120-induced downregulation of CD79b. These SDF-1alpha-modulated B-cell responses were specifically abrogated by an anti-SDF-1alpha antibody. These data suggest that SDF-1alpha plays an important regulatory role in the altered B-cell responses seen in HIV-1 infection. Further, these findings may enhance the understanding of the pathophysiology of HIV-1 infection and suggest a strategy utilizing SDF-1alpha or related molecules as an anti-HIV therapy.     

Inhibitory effect of the immunosuppressant FK506 on apoptotic cell death induced by HIV-1 gp120

Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 may play a central role in inducing immunoregulatory disorders after HIV infection. The apoptotic death of normal human peripheral blood mononuclear cells was induced by priming with gp120 followed by stimulation with an anti-T cell receptor (TCR) antibody. Tumor necrosis factor- produced by gp120-binding macrophages may be important to induce this cell death. Treatment of gp120-primed cells with an immunosuppressant (FK506) before TCR signaling inhibited apoptotic cell death, and this blocking effect of FK506 was concentration dependent. FK506 did not have any influence on cell growth and viability over the range of concentrations tested. These findings suggest that FK506 is a potentially useful drug in delaying the onset of AIDS after HIV infection.     

Characterization of human monoclonal antibodies selected with a hypervariable loop-deleted recombinant HIV-1(IIIB) gp120

Recombinant gp120 of the HIV-1(IIIB) isolate (BH10 clone) has been mutated to form the PR12 protein with the first 74 C-terminal amino acids and the V1, V2 and V3 hypervariable loops deleted. A variety of studies have shown that the CD4 binding domain (CD4bd) is very well exposed in PR12 in contrast to rgp120(LAI). Using PR12 for selection of human monoclonal antibodies (MAbs) from HIV-infected individuals, five MAbs were generated with specificities to the epitopes overlapping the CD4bd (1570A,1570C,1570D,1595 and 1599). The three MAbs, 1570A, C and D, generated from one HIV-infected individual, represent one MAb as determined by sequence analysis of the V(H)3 region. Since the epitopes overlapping the CD4bd exhibit variability among HIV-1 clades, the specificity of anti-CD4bd MAbs were distinguished by differing patterns of binding to recombinant envelope proteins derived from clade A, B, C, D and E viruses. The PR12-selected MAbs were also compared with a panel of gp120-selected anti-CD4bd MAbs and showed a different range of specificities. MAb 1599 is clade B specific, MAb 1595 reacts with the A, B and D clades, while MAb 1570 recognises the most conserved epitope, as it binds to all proteins. The results show that the exposure of different epitopes in the CD4bd of the PR12 protein allows this protein to serve as an immunogen and to induce anti-CD4bd antibodies.     

HIV-1 gp120 and immune network

It has been demonstrated that the immunodominant V3 loop of HIV-1 gp120 and its flanking regions bear sequence and structural homology to the framework and complementarity-determining regions of human immunoglobulins. It has been proposed that the Ig-like domain of gp120 might encode idiotypes and in this way permit HIV-1 entry into the immune regulatory network. This notion is strongly supported by results demonstrating that the anti-V3 loop and anti-Ig antibodies of healthy individuals share complementary structure and that V3 reactive antibodies are present in HIV-negative sera. This might be the mechanism by which HIV induces immunological abnormalities, and it should be taken into consideration in AIDS vaccine development.     

Circulating gp120 alters the blood-brain barrier permeability in HIV-1 gp120 transgenic mice

The mechanism underlying invasion of the central nervous system by HIV-1 is unclear. We recently demonstrated blood–brain barrier changes in a model of HIV-1 gp120 transgenic mice. To test whether this alteration was intrinsic to the brain endothelium of transgenic mice or depended on circulating gp120, we used brain endothelial cultures from gp120 transgenic and non-transgenic mice and exposed them to serum from gp120 transgenic or non-transgenic mice. We measured permeability to albumin as a marker of functional endothelial integrity. A significant increase in permeability (up to 47%) was observed in transgenic and non-transgenic cultures exposed to serum samples from transgenic but not to those from non-transgenic mice. This permeability was neutralized after immunoabsorption of sera with anti-gp120 monoclonal antibody. These findings demonstrate that the blood–brain barrier alteration in HIV-1 gp120 transgenic mice is due to circulating gp120.     

gp120mAb对gp120抑制大鼠海马脑片CA1区LTP作用的研究

目的探讨人类免疫缺陷病毒Ⅰ型(HIV-1)的包膜糖蛋白gp120特异抗体gp120mAb对gp120引起大鼠海马脑片CA1区的突触传递及可塑性变化的影响.方法应用离体脑片记录技术,记录大鼠海马CA1区的兴奋性突触后电位(EPSP),研究gp120mAb对gp120抑制高频电刺激Schaffer侧支引起的鼠海马长时程增强效应(LTP)作用的影响.结果gp120对高频电刺激(HFS,100 Hz,1 000 ms×2,串间隔20秒,共2次)Schaffer侧支引起的大鼠海马CA1区LTP产生抑制作用,而对其基础EPSP没有影响.用浓度为200 pmol/L的gp120灌流脑片,可引起LTP的维持发生抑制.这种抑制作用可被gp120特异抗体gp120mAb(50 ng/ml)所拮抗.结论gp120mAb可能是通过拮抗gp120抑制海马CA1区的LTP诱发和维持而参与艾滋病痴呆(HIV-1 associated dementia,HAD)的形成.     

RANTES, MDC and SDF-1alpha, prevent the HIVgp120-induced food and water intake decrease in rats

Human immunodeficiency virus (HIV)-wasting syndrome might be facilitated by the HIVgp120 affecting the immunological system. We studied the effect (subchronic administration: 5 days) of HIVgp120, and a few immune-response mediators: regulated upon activation normal T-cell expressed and presumably secreted (RANTES), stromal derived factor-1alpha (SDF-1alpha), macrophage-derived chemokine (MDC), and their combination, on food and water intake in rats, motor control and pain perception. Eighty male adult Wistar rats received an intracerebroventricular (icv) administration of: vehicle 5 microl/day or 0.92 nmol daily of HIVgp120IIIB, RANTES, SDF-1alpha, or MDC, and the combination of RANTES+HIVgp120IIIB, SDF-1alpha+HIVgp120IIIB, or MDC+HIVgp120IIIB. Food and water intake was measured every day during administration, and 24 and 48 h after the last administration. Rats were also weighed the first and the last day of experiment in order to detect the impact of these treatments in the body weight. HIVgp120IIIB significantly decreased food and water intake. These rats gain less weight than the control (vehicle) and chemokines-treated subjects with exception of those treated with SDF-1alpha that also gain less weight. In addition, HIVgp120 deteriorated motor control. HIVgp120IIIB effects on food and water intake, and motor control were prevented by these chemokines. HIVgp120+RANTES, HIVgp120+SDF-1alpha, and SDF-1alpha alone induced hyperalgesia. Results suggest an interaction between HIVgp120 and the chemokine system to generate the HIV-wasting syndrome, the motor abnormalities and changes in pain perception.     

Antibody repertoire against HIV-1 gp120 triggered in nude and normal mice by GM-CSF/gp120 immunization

Granulocyte-macrophage colony stimulating factor (GM-CSF) facilitates the induction of primary immune responses by activating and recruiting antigen-presenting cells (APC), which efficiently present antigen determinants to Th cells. We have derived a functional GM-CSF/gp120 chimeric protein that, following immunization in soluble, adjuvant-independent form in normal mice, triggers highly specific, high affinity anti-gp120 antibodies. In contrast, nude mice respond with mutated, polyreactive, low affinity antibodies that mature further and increase in affinity in T cell-reconstituted nude mice. Anti-gp120 antibody production in nude mice is mediated principally by GM-CSF/gp120-triggered IL-4 production, since neutralizing anti-IL-4 abrogates the in vivo response. The anti-gp120 antibody response in normal, nude and T cell-reconstituted nude mice is encoded at a remarkably high frequency by the VH81X and VH7183 genes, a family used notably during fetal life and, when expressed at the adult stage, associated with autoimmune disease. We conclude that HIV gp120 binds and selects a subpopulation of developing B cells expressing a set of VH genes associated with immunodeficiency and autoimmunity.     

Potency of HIV-1 envelope glycoprotein gp120 antibodies to inhibit the interaction of DC-SIGN with HIV-1 gp120

The interaction of DC-SIGN with gp120 provides an attractive target for intervention of HIV-1 transmission. Here, we have investigated the potency of gp120 antibodies to inhibit the DC-SIGN-gp120 interaction. We demonstrate that although the V3 loop is not essential for DC-SIGN binding, antibodies against the V3 loop partially inhibit DC-SIGN binding, suggesting that these antibodies sterically hinder DC-SIGN binding to gp120. Polyclonal antibodies raised against non-glycosylated gp120 inhibited both low and high avidity DC-SIGN-gp120 interactions in contrast to polyclonal antibodies raised against glycosylated gp120. Thus, glycans present on gp120 may prevent the generation of antibodies that block the DC-SIGN-gp120 interactions. Moreover, the polyclonal antibodies against non-glycosylated gp120 efficiently inhibited HIV-1 capture by both DC-SIGN transfectants and immature dendritic cells. Therefore, non-glycosylated gp120 may be an attractive immunogen to elicit gp120 antibodies that block the binding to DC-SIGN. Furthermore, we demonstrate that DC-SIGN binding to gp120 enhanced CD4 binding, suggesting that DC-SIGN induces conformational changes in gp120, which may provide new targets for neutralizing antibodies.     

The many facets of SDF-1alpha, CXCR4 agonists and antagonists on hematopoietic progenitor cells

Stromal cell-derived factor-1alpha (SDF-1alpha) has pleiotropic effects on hematopoietic progenitor cells (HPCs). We have monitored podia formation, migration, proliferation, and cell-cell adhesion of human HPC under the influence of SDF-1alpha, a peptide agonist of CXCR4 (CTCE-0214), a peptide antagonist (CTCE-9908), and a nonpeptide antagonist (AMD3100). Whereas SDF-1alpha induced migration of CD34(+) cells in a dose-dependent manner, CTCE-0214, CTCE-9908, and AMD3100 did not induce chemotaxis in this concentration range albeit the peptides CTCE-0214 and CTCE-9908 increased podia formation. Cell-cell adhesion of HPC to human mesenchymal stromal cells was impaired by the addition of SDF-1alpha, CTCE-0214, and AMD3100. Proliferation was not affected by SDF-1alpha or its analogs. Surface antigen detection of CXCR4 was reduced upon treatment with SDF-1alpha or AMD3100 and it was enhanced by CTCE-9908. Despite the fact that all these molecules target the same CXCR4 receptor, CXCR4 agonists and antagonists have selective effects on different functions of the natural molecule.     

Ethanol potentiates HIV-1 gp120-induced apoptosis in human neurons via both the death receptor and NMDA receptor pathways

Neuronal loss is a hallmark of AIDS dementia syndromes. Human immunodeficiency virus type I (HIV-1)-specific proteins may induce neuronal apoptosis, but the signal transduction of HIV-1 gp120-induced, direct neuronal apoptosis remains unclear. Ethanol (EtOH) is considered to be an environmental co-factor in AIDS development. However, whether EtOH abuse in patients with AIDS increases neuronal dysfunction is still uncertain. Using pure, differentiated, and post-mitotic NT2.N-derived human neurons, we investigated the mechanisms of HIV-1 and/or EtOH-related direct neuronal injury and the molecular interactions between HIV-1-specific proteins and EtOH. It was demonstrated that NT2.N neurons were susceptible to HIV-1 Bal (R5-tropic strain) gp120-induced direct cell death. Of importance, EtOH induced cell death in human neurons in a clinically-relevant dose range and EtOH strongly potentiated HIV-1 gp120-induced neuronal injury at low and moderate concentrations. Furthermore, this potentiation of neurotoxicity could be blocked by N-methyl-D-aspartate (NMDA) receptor subunit 2B (NR2B) antagonists. We analyzed human genomic profiles in these human neurons, using Affymetrix genomics technology, to elucidate the apoptotic pathways involved in HIV-1- and EtOH-related neurodegeneration. Our findings indicated significant over-expression of selected apoptosis functional genes. Significant up-regulation of TRAF5 gene expression may play an essential role in triggering potentiation by EtOH of HIV-1 gp120-induced neuronal apoptosis at early stages of interaction. These studies suggested that two primary apoptotic pathways, death receptor (extrinsic) and NMDA receptor (intrinsic)-related programmed cell-death pathways, are both involved in the potentiation by EtOH of HIV-1 gp120-induced direct human neuronal death. Thus, these data suggest rationally-designed, molecular targets for potential anti-HIV-1 neuroprotection.     

Recombination property of the HIV-1 gp120 gene

Using a chimeric primer consisting of the nucleotide sequence derived from the HIV-1 envelope gene coding for the second conserved region of gp120, and the highly conserved sequence derived from the human immunoglobulin gene coding for the VHIII domain, it has been identified in sera of AIDS patients HIV-1 field isolates carrying the complete and active Chi recombination hot spot (GCTGGTGG). The recombination between the HIV-1 gene coding for the central portion of gp120 and the bacterial gene coding for the clp protease was also demonstrated in vivo. These results point out serious concern that vectored AIDS vaccine candidates carrying the HIV-1 env gene on viral and bacterial vectors could become the source of potentially new infectious diseases rather than an effective instrument for AIDS prevention.     

Simultaneous induction of apoptotic and survival signaling pathways in macrophage-like THP-1 cells by Shiga toxin 1

Shiga toxins have been shown to induce apoptosis in many cell types. However, Shiga toxin 1 (Stx1) induced only limited apoptosis of macrophage-like THP-1 cells in vitro. The mechanisms regulating macrophage death or survival following toxin challenge are unknown. Differentiated THP-1 cells expressed tumor necrosis factor receptors and membrane-associated tumor necrosis factor alpha (TNF-alpha) and produced soluble TNF-alpha after exposure to Stx1. However, the cells were refractory to apoptosis induced by TNF-alpha, although the cytokine modestly increased apoptosis in the presence of Stx1. Despite the partial resistance of macrophage-like THP-1 cells to Stx1-mediated killing, treatment of these cells with Stx1 activated a broad array of caspases, disrupted the mitochondrial membrane potential (DeltaPsi(m)), and released cytochrome c into the cytoplasm. The DeltaPsi(m) values were greatest in cells that had detached from plastic surfaces. Specific caspase inhibitors revealed that caspase-3, caspase-6, caspase-8, and caspase-9 were primarily involved in apoptosis induction. The antiapoptotic factors involved in macrophage survival following toxin challenge include inhibitors of apoptosis proteins and X-linked inhibitor of apoptosis protein. NF-kappaB and JNK mitogen-activated protein kinases (MAPKs) appeared to activate survival pathways, while p38 MAPK was involved in proapoptotic signaling. The JNK and p38 MAPKs were shown to be upstream signaling pathways which may regulate caspase activation. Finally, the protein synthesis inhibitors Stx1 and anisomycin triggered limited apoptosis and prolonged JNK and p38 MAPK activation, while macrophage-like cells treated with cycloheximide remained viable and showed transient activation of MAPKs. Collectively, these data suggest that Stx1 activates both apoptotic and cell survival signaling pathways in macrophage-like THP-1 cells.     

HIV-1 gp120 receptor on CD4-negative brain cells activates a tyrosine kinase.

Human immunodeficiency virus (HIV-1) infection in the human brain leads to characteristic neuropathological changes, which may result indirectly from interactions of the envelope glycoprotein gp120 with neurons and/or glial cells. We therefore investigated the binding of recombinant gp120 (rgp120) to human neural cells and its effect on intracellular signalling. Here we present evidence that rgp120, besides binding to galactocerebroside or galactosyl-sulfatide, specifically binds to a protein receptor of a relative molecular mass of approximately 180,000 Da (180 kDa) present on the CD4-negative glioma cells D-54, but not on Molt4 T lymphocytes. Binding of rgp120 to this receptor rapidly induced a tyrosine-specific protein kinase activity leading to tyrosine phosphorylation of 130- and 115-kDa proteins. The concentration of intracellular calcium was not affected by rgp120 in these cells. Our data suggest a novel signal transducing HIV-1 gp120 receptor on CD4-negative glial cells, which may contribute to the neuropathological changes observed in HIV-1-infected brains.     

beta1 integrin antagonism on adherent, differentiated human neuroblastoma cells triggers an apoptotic signaling pathway

Integrin receptors mediate several functions including prevention of matrix detachment-induced apoptosis (anoikis) of several adherent cell types. We report here that antagonists of beta1 integrins trigger an apoptotic signaling pathway in adherent differentiated LAN-5 human neuroblastoma cells, a cell line which represents a model system for the study of human neurons. The pathway is characterized by cytochrome c release into the cytoplasm, and activation of caspase-9 and caspase-3, 4-6h after treatment; cleavage products of caspase-8 and caspase-2 were not detectable in the cells. Coordinate inactivation of cell survival pathways, including cleavage of focal adhesion kinase, decreased expression of protein kinase B, and reduced phosphorylation of the pro-apoptotic protein, Bad, also characterized the signaling pathway. These events occurred in adherent cells; DNA fragmentation and detachment followed as late events 18-24h after addition of beta1 integrin antagonists. zDEVD-fmk, an irreversible inhibitor of caspase-3-like enzymes, and cytochalasin D, an actin depolymerizing agent, blocked caspase-3 cleavage and delayed cell death. In contrast to these results, undifferentiated, adherent and dividing LAN-5 cells did not die in response to beta1 integrin antagonists.These studies identify a distinct apoptotic pathway which is triggered by antagonists of beta1 integrins on differentiated adherent neuronal cells.     

趋化因子SDF-1 对淋巴细胞白血病细胞的作用

基质细胞衍生因子-1(stromakcell-derivedfactor-1,SDF-1)是一种由骨髓基质细胞释放的趋化因子,是趋化因子CXC亚家族的成员之一.近年研究表明SDF-1不仅与正常机体造血功能的维持及造血干/祖细胞的回髓定位和植入有密切的关系,同时与各种白血病细胞的移动和功能之间也存在复杂的相互作用.本文重点就SDF-1与淋巴细胞白血病细胞之间的相互作用及其意义对国外的研究现状作一综述.     

HIV-1 gp120 chemokine receptor-mediated signaling in human macrophages

The chemokine receptors CCR5 and CXCR4 serve as the cellular receptors in conjunction with CD4 for HIV-1 entry and infection of target cells. Although the virus has subverted these molecules for its own use, their natural function is to respond to activation and migration signals delivered by extracellular chemokines. A principal research objective of our laboratory is to understand the consequences of virus-chemokine receptor interactions for cellular function, as well as for entry and infection. We hypothesized that CXCR4-using (X4) and CCR5-using (R5) HIV-1 strains might elicit signals through the chemokine receptors that result in aberrant function and/or regulate virus entry or postentry steps of infection. We have focused on primary human macrophages, which express both CXCR4 and CCR5, because macrophages are a principal target for HIV-1 in vivo, in appropriate macrophage activation appears to play a major role in the pathogenesis of certain sequelae of AIDS, such as HIV encephalopathy, and macrophage infection is regulated at several steps subsequent to entry in ways that are linked to envelope-receptor interactions. This review summarizes our recent findings regarding the mechanisms of chemokine-receptor signaling in macrophages, the role of viral envelope glycoproteins in eliciting macrophage signals, and how these activation pathways may participate in macrophage infection and affect cell functions apart from infection.