736-W.
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Chemokine Regulation by HIV-1 and Pro-Inflammatory Cytokines in Human Astrocytes and Neurons
D. M. Lawrence*, L. C. Durham, T. W. Sanchez, and E. O. Major
NINDS, NIH, Bethesda, MD
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Background: In the HIV-1 infected brain, production of chemokines such as MCP-1 is believed to be an important factor in neuropathogenesis. Human astrocytes produce high levels of MCP-1, and can harbor latent HIV-1 infection that may be reactivated in the context of neuroinflammation. Using the immortalized human fetal glial cell line SVG, progenitor-like cells which can be differentiated into an astrocytic or neuronal phenotype depending on culture conditions, we examined the regulation of MCP-1 by both HIV-1 infection and exposure to pro-inflammatory cytokines.
Methods: Cells were grown as astrocytic (SVG) or neuronal cells (SVG-N), then treated with either TNF-alpha, IFN-gamma, or transfected with the HIV-1 clone pNL4-3. In some studies, cells were transfected with plasmids containing the full or partial MCP-1 promoter + CAT reporter gene to identify critical promoter regions. Supernatants were collected at various times and tested by ELISA for levels of MCP-1 and p24 viral antigen; lysates were tested for CAT reporter activity.
Results: Unstimulated SVG cells produced moderate levels of MCP-1 protein (5 ng/mL); treatment with TNF-alpha and IFN-gamma increased MCP-1 levels 5- to 10-fold. As expected, MCP-1 promoter activation by TNF-alpha required both the distal and proximal promoter regions, whereas IFN-gamma stimulation required only the proximal region (-213 bp). The SVG-N cells produced much less basal MCP-1 (<500 pg/mL), and only TNF-alpha stimulated MCP-1 production; IFN-gamma had no effect. After transfection, HIV-1 transcription occurred in both phenotypes; peak p24 production was 3 days post-transfection. Maximal p24 production was 2-fold higher in glial cells compared to neuronal cells. At all time points tested during the productive phase of HIV-1 infection, MCP-1 production was increased 10-25% in the astrocytic cells, but reduced 30% in the neuronal cells, compared to uninfected cells.
Conclusions: Using the immortalized SVG cell line grown as either astrocytic or neuronal cells, we have shown that astrocytes have a greater capacity than the neuronal phenotype to produce MCP-1 in response to both HIV-1 infection and pro-inflammatory cytokines. This cell line will be useful to understand the molecular regulation of MCP-1 production and HIV replication in brain-derived cells.
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