The hypothesis of this proposal is that significant biological responses occur to HIV-1 envelope (Env) proteins, in the absence of infection, in glial cells in brains and spinal cords of AIDS patients which account for the neuropathology and clinical changes. We wish to determine what the characteristics are of the induced toxicities in glia using cells cultured from uninfected human brain. We believe that the induction of cytokines by HIV-1 Env leads to nitric oxide (NO) production and NO inhibits the glutamate transporter leading to accumulation of extracellular glutamate. We shall map the Env epitopes which induce the proinflammatory cytokines Interleukin 1 (IL 1) and tumor necrosis factor alpha (TNFalpha) and nitric oxide and inhibit uptake of glutamate and examine the interrelatedness of these three biological responses.
In Specific Aim #1, we will map HIV-1 Env protein epitopes involved in binding to or cytokine induction in glial cells cultured from uninfected human fetal, neonatal, or adult brain. This will be accomplished by stimulating glia with high and low cytokine inducer wild type HIV-1 strains (JR-FL, NL 4-3), hybrid Env recombinants between these two strains, and truncation deletion Env mutants of the high inducer strain, all of which have been engineered into a vaccinia virus vector for expression. Bioassays will be used to measure the production of IL 1 and TNFalpha. To map Env binding epitopes, these recombinant Env proteins can be expressed on the surface of smaller lymphocytes which will be rosetted with the larger glia.
In Specific Aim #2, we will, using the cytokine inducers found in Specific Aim #1, measure Env induction of inducible nitric oxide synthase (iNOS) by (a) determinations of enzyme activity (NO production), (b) immunohistochemical identification, and (c) Northern blot analysis for mRNA. Radiolabeled glutamate uptake will be measured to assess inhibition of the transporter by Env proteins. We will determine whether Env induction of iNOS and NO and inhibition of glutamate uptake is due to cytokine induction by blocking with anti-cytokine antibodies. The role of NO in inhibition of glutamate uptake will be examined using NO antagonists. If the Env epitopes inducing cytokines do not cause NO production or inhibit glutamate uptake, alternative Env epitopes will be mapped using the strategies identified for mapping cytokine induction (Specific Aim #1).
Specific Aim #3 will assess which anti-inflammatory agents, such as pentoxifylline, Interleukins 4 and 10, transforming growth factor beta, can inhibit Env-induced cytokine and NO production, and inhibition of glutamate uptake. These inhibitory agents will be used singly and in combination. We shall assess whether inhibition of IL 1, TNFalpha, and iNOS occurs at the protein and/or mRNA level and whether their inhibition leads to a normalization of glutamate uptake.
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