Direct role of plasma membrane-expressed gp120/41 in toxicity to human astrocytes induced by HIV-1-infected macrophages

Objective: To compare astrocyte toxicity induced by plasma membrane-expressed gp120/41 and soluble gp120. Design: Analysis of morphological alterations and lactate dehydrogenase (LDH) release from astrocytes in culture with monocytes infected with HIV-1, microglia expressing Env of a macrophage-tropic HIV-1 isolate or soluble Env. Methods: Primary human embryonic astrocytes were cultured with: monocytes infected with two M-tropic HIV-1 isolates (HIV-1(9533), HIV-1(BX08)); human microglia infected with the defective Semliki Forest virus (SFV) vector coding for the env gene of HIV-1(BX08) isolate (SFVenvBX08); and soluble gp140 purified from baby hamster kidney cells transfected with the env gene of HIV-1(BX08) lacking the intracytoplasmic region of gp41 (SFV Delta envBX08). Gp120 mRNA levels were assessed by quantitative reverse transcriptase-polymerase chain reaction and the protein was detected by immunofluorescence in infected monocytes or microglia. Results: Contact of HIV-infected monocytes induced morphological changes in astrocytes and a 137% increase in LDH release at day 2 of co-culture compared with controls (uninfected monocytes). Gp120/41 (BX08)-expressing microglia induced a 170% increase in LDH release (relative to SFV LacZ-infected microglia). Pretreatment of cocultures with an anti-gp120 monoclonal antibody (mAb; NEA-9305) directed against the V3 loop inhibited LDH release. Soluble purified gp140 from BX08 isolate induced only a weak LDH release (104%). Finally, cytotoxicity was not blocked by treatment of the co-culture with Bordetella pertussis toxin, an inhibitor of G(i)alpha protein-dependent receptors. Conclusion: HIV envelope glycoprotein expressed at the plasma membrane induced astrocyte damage more efficiently than its soluble counterpart. The V3 loop was involved in toxicity induction through a pathway independent of the G(i)alpha protein-coupled receptor. (C) 2000 Lippincott Williams & Wilkins.