Michele Navarra

gp120 induces cell death in human neuroblastoma cells through the CXCR4 and CCR5 chemokine receptors.

Abstract: To infect target cells, the human immunodeficiency virus (HIV) type I (HIV‐1) must engage not only the well‐known CD4 molecule, but it also requires one of several recently described coreceptors. In particular, the CXCR4 (LESTR/fusin) receptor allows fusion and entry of T‐tropic strains of HIV, whereas CCR5 is the major coreceptor used by primary HIV‐1 strains that infect macrophages and CD4+ T‐helper cells (M‐tropic viruses). In addition, the α chemokine SDF1α and the β chemokines MIP1α, MIP1β, and RANTES, natural ligands of CXCR4 and CCR5, respectively, are potent soluble inhibitors of HIV infection by blocking the binding between the viral envelope glycoprotein gp120 and the coreceptors. Approximately two‐thirds of individuals with acquired immunodeficiency syndrome (AIDS) show neurologic complications, which are referred to a syndrome called AIDS dementia complex or HIV‐1‐associated cognitive/motor complex. The HIV‐1 coat glycoprotein gp120 has been proposed as the major etiologic agent for neuronal damage, mediating both direct and indirect effects on the CNS. Furthermore, recent findings showing the presence of chemokine receptors on the surface of different cell types resident in the CNS raise the possibility that the association of gp120 with these receptors may contribute to the pathogenesis of neurological dysfunction. Here, we address the possible role of α and β chemokines in inhibiting gp120‐mediated neurotoxicity using the human neuroblastoma CHP100 cell line as an experimental model. We have previously shown that, in CHP100 cells, picomolar concentrations of gp120 produce a significant increase in cell death, which seems to proceed through a Ca2+ ‐ and NMDA receptor‐dependent cascade. In this study, we gained insight into the mechanism(s) of neurotoxicity elicited by the viral glycoprotein. We found that CHP100 cells constitutively express both CXCR4 and CCR5 receptors and that stimulation with phorbol 12‐myristate 13‐acetate down‐regulates their expression, thus preventing gp120‐induced cell death. Furthermore, all the natural ligands of these receptors exerted protective effects against gp120‐mediated neuronal damage, although with different efficiencies. These findings, together with our previous reports, suggest that the neuronal injury observed in HIV‐1 infection could be due to direct (or indirect) interactions between the viral protein gp120 and chemokine and/or NMDA receptors.

Cell signaling pathways in the mechanisms of neuroprotection afforded by bergamot essential oil against NMDA-induced cell death in vitro

The effects of bergamot essential oil (BEO; Citrus bergamia, Risso) on excitotoxic neuronal damage was investigated in vitro.

Death of cultured human neuroblastoma cells induced by HIV-1 gp120 is prevented by NMDA receptor antagonists and inhibitors of nitric oxide and cyclooxygenase

The cytotoxic effects of the human immunodeficiency virus type 1 (HIV-1) coat protein gp120 were studied in human CHP100 neuroblastoma cell cultures. Incubation of neuroblastoma cultures with gp120 (1 pM-10 nM) induces cell death which is not concentration-related. The significant cell death evoked by 10 pM gp120 was prevented by neutralization of the viral protein with a monoclonal anti-gp120 (IgG) antibody. In addition, gp120-induced cytotoxicity was inhibited by [DL-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid] (CGP37849; 100 microM), [(+/-)-3R*, 4as*, 6R*, 8aR*-6-(phosphonomethyl) decahydro-isoquinoline-3-carboxylic acid] (LY274614; 100 microM), MK801 (dizocilpine; 200 nM) and 7-chloro kynurenic acid (100 microM), selective antagonists of the NMDA receptor complex; by contrast, (6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 100 microM), a non-NMDA antagonist, was ineffective. Prevention of the lethality elicited by the HIV-1 coat protein was also obtained by incubating neuroblastoma cells with gp120 in Ca(2+)-free medium. The lethal effects induced by gp120 involve activation of L-arginine-nitric oxide (NO) pathway since these were prevented by haemoglobin (10 microM), a NO-trapping agent, and by D-arginine (1 mM), the less active enantiomer of the endogenous precursor of NO synthesis. Cytoprotection was also afforded by N omega-nitro-L-arginine methyl ester (L-NAME; 200 microM), an inhibitor of NO synthase, and this was reversed by L-arginine (1 mM). Interestingly, indomethacin and flufenamic acid (10 microM), two inhibitors of cyclooxygenase, protected neuroblastoma cells from death induced by gp120. Furthermore, indomethacin prevented the neuroblastoma cell death evoked by exposure of cultures to sodium nitroprusside (SNP; 0.2-1.6 mM), a NO donor. Finally significant cytotoxic effects were observed after incubation of neuroblastoma cells with prostaglandin E2 (0.1-10 microM). In conclusion, the present data suggest that death of human CHP100 neuroblastoma cells in culture produced by gp120 involves NO and PGE2 production.

HIV‐1 coat protein gp120 stimulates interleukin‐1β secretion from human neuroblastoma cells: evidence for a role in the mechanism of cell death

The role of the pro‐inflammatory cytokine interleukin‐1β (IL‐1β) in the mechanism of cell death induced by the human immunodeficiency virus type 1 (HIV‐1) recombinant coat glycoprotein, gp120 IIIB, has been studied in the human CHP100 neuroblastoma cell line maintained in culture. Death of neuroblastoma cells typically elicited by 10 pM gp120 or by human recombinant IL‐1β (10 ng ml−1) has been minimized by the antagonist of IL‐1 receptor, i.e. IL‐1ra (0.5 and 50 ng ml−1, respectively), an endogenous molecule that antagonizes most of the biological actions of IL‐1β, or by an antibody (5 and 50 ng ml−1) which blocks the human IL‐1 receptor type I (IL‐1RI). ELISA experiments have established that gp120 enhances immunoreactive IL‐1β levels in the culture medium and this is prevented by exposure to the IL‐1 converting enzyme (ICE) inhibitor t‐butoxycarbonyl‐L‐aspartic acid benzyl ester‐chloromethylketone [Boc‐Asp(OBzl)‐CMK] used at a concentration (2.5 μM) which significantly (P<0.001) reduces cell death. Death of CHP100 cells induced by gp120 is also prevented by acetyl‐Tyr‐Val‐Ala‐Asp‐chloromethylketone (Ac‐YVAD‐CMK; 10 – 100 μM), a second inhibitor of ICE, supporting the concept that the viral protein stimulates the conversion of the 31 kDa pro‐IL‐1β in to the 17 kDa mature cytokine which is then secreted to cause death. In conclusion, our present data demonstrate that gp120 stimulates the secretion of IL‐1β which then triggers CHP100 neuroblastoma cell death via stimulation of IL‐1 receptor type I.

Cholesterol-dependent modulation of the toxicity of HIV-1 coat protein gp120 in human neuroblastoma cells.

The human immunodeficiency virus type 1 (HIV‐1) coat glycoprotein gp120 binds to its (co)receptors and orchestrates cell entry by the direct fusion of viral and target cell membranes. Here, we modulated membrane fluidity of human neuroblastoma CHP100 cells by modulating their cholesterol content, and investigated the ability of gp120 to induce cell death in comparison with the untreated cells. We show that in normal CHP100 cells gp120 induces necrosis by: (i) increased cyclooxygenase and 5‐lipoxygenase activity, and metabolites generated thereof (prostaglandin E2 and leukotriene B4, respectively); (ii) increased membrane lipoperoxidation; and (iii) increased mitochondrial uncoupling. These events were triggered by a rapid increase in intracellular calcium, and in cholesterol‐depleted cells engaged CXCR4 chemokine receptors. The intracellular calcium chelator EGTA‐AM protected CHP100 cells almost completely against the toxic effects of gp120. However, gp120‐induced necrosis and related biochemical changes were negligible in cholesterol‐enriched, and significantly enhanced in cholesterol‐depleted, CHP100 cells exposed to the viral glycoprotein under the same experimental conditions. Taken together, these results suggest that membrane fluidity may control the neurotoxic effects of HIV‐1 glycoprotein gp120.

Evidence for a role of protein tyrosine kinases in cell death induced by gp120 in CHP100 neuroblastoma cells

HIV-1 coat protein gp120 is able to kill neuronal cells in culture. Here we address the possible role of protein tyrosine kinases (PTKs) in gp120-induced neurotoxicity using the CHP100 human neuroblastoma cell line as experimental model. For this purpose, the effect of specific PTK inhibitors like genistein, herbimycin A and lavendustin A was evaluated on CHP100 cell death elicited by the viral protein. Here we report that genistein (1-10 microM) significantly reduced the cytotoxic effects induced by gp120 (10 pM). The same protective action was offered by a pre-treatment with herbimycin A (0.1-1 microM) or lavendustin A (1-10 microM). Conversely, daidzein (1-100 microM), a genistein analogue devoid of PTK inhibitory properties, failed to reduce CHP100 cell death caused by gp120. These findings suggest that PTKs can be involved in the signal transduction cascade by which the glycoprotein induces neurotoxicity.