In order to elucidate the pathogenesis of AIDS encephalitis and myelopathy, we have infected primary cultures derived from cortex of patients with intractable epilepsy or malignant astrocytoma with different isolates of HIV-1 and HIV-2-. Viral infection-was measured by reverse transcriptase (RT) activity and HIV-1 core antigen (p24) in cell free supernatant. The three isolates which showed signs of replication were derived from either macrophages (HIV-1-AD87(M) and HIV-1 BaL) or brain (HIV-l JR-FL), whereas T-cell adapted and HIV-2 strains did not replicate. Peak levels of RT activity (1-2xl05 cpm/ml) and p24 antigen (50-1 00 ng/ml in productivity infected cultures were generally seen at 9-15 days post infection. The antigenic phenotype of infected cells was characterized by a triple label assay using antibodies against HIV p24 or p17 gag proteins, glial fibrillary acidic protein (GFAP) antibody to identify astrocytes and Dil-labelled low density lipoprotein (Dil-LDL), which binds to microglia, a cell of the macrophage lineage residing in the brain. In infected cultures, Dil-LDL labelled microglia often stained positively for gag but GFAP, astrocytes did not. Viral production was further confirmed by electron microscopy which showed budding of HIV particles from the plasma membrane of microglial cells. After 4-5 days post infection progressive syncitia formation began among infected microglia and after 3 weeks up to 25 times more Dil-LDL + cells had died as compared to controls. This progressive clustering and fusion of microglial cells in vitro mimics the two pathological hallmarks of HIV infection in the brain: microglial nodules and multi-nucleated giant cells. When cells were incubated prior to infection with large amounts of anti-CD4 antibody which recognizes the gp120 binding site (25 pg/ml Leu 3a), both viral replication and cytopathic effects were reduced to control levels. In addition, microglia isolated from control cultures were found to express MRNA for CD4 by polymerase chain reaction (PCR). This demonstrates that HIV can enter microglia via the CD4 receptor as it does in T-cells and monocytes. We propose that the death of microglial cells which normally serve immune functions in the central nervous system may be a key factor in the pathogenesis of AIDS encephalitis and/or myelopathy.
