CCR5-, DC-SIGN-dependent endocytosis and delayed reverse transcription after human immunodeficiency virus type 1 infection in human astrocytes.

We sought to determine the pathway of HIV-1 entry into human astrocytes and the fate of HIV-1 by detecting viral DNA and GFP-tagged HIV-1 in HIV-1-infected primary astrocytes. Immunochemistry and FACS analysis were used to assess the expression of DC-SIGN in human purified cultures of astrocytes. HIV-1 LTR was detected by PCR in infected cultures of human embryonic astrocytes at their third passage. GFP-Vpr-labeled R5 tropic HIV-1 was used to infect astrocytes, and was followed by confocal microscopy. Forty percent of astrocytes expressed DC-SIGN at the membrane level. Viral DNA was detected 5 days after infection in human astrocytes, but not in the presence of anti-CCR5 and anti-DC-SIGN mAbs. T20, NH4Cl, and bafilomycin had no effect on viral DNA detection. We found that 67% of the fluorescent GFP-Vpr-labeled R5 tropic HIV-1 viruses were present in the endosomes of astrocytes at 24 h, but not in the presence of anti-CCR5 or DC-SIGN mAbs. Bafilomycin and NH(4)Cl each increased the amount of fluorescent HIV-1 detected outside endosomes. Titers of p24 remained low from day 1 to day 5 postinfection, in the presence or absence of NH4Cl. Astrocytes express DC-SIGN and HIV-1 penetrates into these cells through CCR5- and/or DCSIGN- mediated endocytosis, via a pH-dependent pathway, with a delayed reverse transcription after infection without productive infection.

Effects of SDF-1alpha and gp120IIIB on apoptotic pathways in SK-N-SH neuroblastoma cells.

CXCR4, a chemokine receptor constitutively expressed in the brain, binds both ligands, the chemokine SDF-1alpha and the HIV envelope glycoprotein gp120(IIIB). There seem to be intracellular differences between the neuronal apoptosis induced by SDF-1alpha and that induced by gp120(IIIB), but the apoptotic pathways involved have not been compared in human neuronal cells. In this study, we characterized the apoptotic intracellular pathways activated by neurotoxic concentrations of SDF-1alpha and gp120(IIIB) in human neuroblastoma cells SK-N-SH. SDF-1alpha (10 nM) and gp120(IIIB) (2 nM) induced similar levels of apoptosis after 24 h of incubation (49 +/- 4% and 48 +/- 3%, respectively, of the neurons were apoptotic). SDF1alpha-induced apoptosis was completely abolished by the inhibition of Src phosphorylation by PP2. Exposure to SDF-1alpha (10 nM) triggered an increase in Src phosphorylation, with a maximum after 20 min of incubation (1.80 +/- 0.24 times higher than control, P = 0.01). NMDA calcium flux was enhanced only if cells were incubated with SDF-1alpha for 20 min before applying NMDA. By contrast, gp120(IIIB)-induced apoptosis was not affected by the inhibition of Src phosphorylation. Moreover, gp120(IIIB) enhanced NMDA calcium flux immediately, without modifying Src phosphorylation status. Finally, levels of phospho-JNK increased following exposure to gp120(IIIB) (by a factor of 1.46 +/- 0.4 at 120 min, P = 0.03), but not after exposure to SDF-1alpha. Thus, SDF-1alpha and gp120(IIIB) induced a similar level of neuronal apoptosis, but by activating different intracellular pathways. SDF-1alpha enhanced NMDA activity indirectly via Src phosphorylation, whereas gp120(IIIB) probably activated the NMDA receptor directly and phosphorylated JNK.

Fractalkine reduces N-methyl-d-aspartate-induced calcium flux and apoptosis in human neurons through extracellular signal-regulated kinase activation.

Our purpose was to investigate in human neurons the neuroprotective pathways induced by Fractalkine (FKN) against glutamate receptor-induced excitotoxicity. CX(3)CR1 and FKN are expressed constitutively in the tested human embryonic primary neurons and SK-N-SH, a human neuroblastoma cell line. Microfluorometry assay demonstrated that CX(3)CR1 was functional in 44% of primary neurons and in 70% of SK-N-SH. Fractalkine induced ERK1/2 phosphorylation within 1 min and Akt phosphorylation after 10 min, and both phosphorylation decreased after 20 min. No p38 and SAPK/JNK activation was observed after FKN treatment. Application of FKN triggered a 53% reduction of the NMDA-induced neuronal calcium influx, which was insensitive to pertussis toxin and LY294002 an inhibitor of Akt pathway, but abolished by PD98059, an ERK1/2 pathway inhibitor. Moreover, FKN significantly reduced neuronal NMDA-induced apoptosis, which was pertussis toxin insensitive and abolished in presence of PD98059 and LY294002. In conclusion, FKN protected human neurons from NMDA-mediated excitotoxicity in at least two ways with different kinetics: (i) an early ERK1/2 activation which reduced NMDA-mediated calcium flux; and (ii), a late Akt activation associated with the previously induced ERK1/2 activation.

Human cytomegalovirus infection reduces surface CCR5 expression in human microglial cells, astrocytes and monocyte-derived macrophages.

Within the brain, glial cells are target cells for human cytomegalovirus (HCMV) and HIV. We infected cultures of unstimulated human microglial cells and astrocytes of embryonic origin and of monocyte-derived macrophages (MDM) with HCMV strain AD169 and observed down-regulation of the plasma membrane expression of CCR5 in the three cell types, and of CXCR4 and CD4 in microglial cells only. Cells were then coinfected simultaneously or at a 24-h interval with both AD169 and two different HIV-1 monocytotropic strains. HCMV late antigens and HIV-1 tat protein colocalized in the cytoplasm of 5-10% of microglia and MDM. p24 antigen levels decreased 10- to 40-fold in supernatants of MDM and the reduction was greater when HCMV infection was performed 24 h before HIV-1 infection. These data suggest that HCMV-induced reduction in the cell-surface expression of the primary co-receptor of HIV-1 monocytotropic strains may impair the ability of HIV to infect these cells.