11th International AIDS Conference Vancouver, British Columbia — July 7-12, 1996

Int Conf AIDS 1996 Jul 7-12; 11:5 (abstract no. Mo.A.143)
Rimaniol AC, Boussin FD, Dormont D, Bach JF, Zavala F; Hopital Necker, Paris, France. Fax: 33-1-43-06-23-88.

OBJECTIVE: To investigate the cellular mechanisms involved in the profound remodeling of the TNF receptor expression induced by HIV-1 LAI infection of human blood monocytes.

METHODS: Adherent monocytes isolated from blood of HIV-1 seronegative donors were cultured for various periods in RPMI 1640 medium supplemented with 5% heat-inactivated FCS, penicillin and streptomycin, and 5 micrograms/ml polymyxin B, in the presence of a live or heat-inactivated HIV-1-LAI preparation (0.1-6 ng/ml of p24 core Ag titer) obtained from culture supernatants of infected human umbilical cord blood mononuclear cells. TNF-alpha, TNF-sR75 and TNF-sR55 produced in the supernatants were measured by specific ELISA from Medgenix Diagnostics (Fleurus, Belgium). Total cellular RNA was extracted with TRIzol and Northern blot analysis performed with cDNA probes for TNF-R75 and TNF-R55, kindly provided by Dr. W. Lesslauer (Hoffman-La Roche LTD, Basel, Switzerland). Prior to TNF-receptor binding assay, cell-associated TNF was removed by incubation in HCl-glycine buffer for 10 min at 4 degrees C. Cells were washed twice with PBS and incubated with 0.5 nM 125I-TNF (400-800 Ci / mmol), in the presence or absence of 50 nM unlabeled TNF for 2 h at 4 degrees C. After 4 washes with PBS, cells were lysed with 0.1% triton X100 for 30 min. Cell-bound radioactivity was then determined with a gamma counter. Specific TNF binding was defined as total binding minus non specific binding observed in the presence of 50 nM unlabeled TNF.

RESULTS: Release of sTNF-R75 was triggered at an early step of interaction of the virus particles with the monocyte, involving the envelope glycoprotein gp120, but independent of productive infection. HIV-1 LAI induced an upregulation of TNF-R75 mRNA, whereas TNF-R55 mRNA was not detectable. sTNF-R75 release required exocytosis (inhibited by monensin), proteolytic cleavage by serine protease(s), but was independent of prior endocytosis of the receptor. Activation with HIV-1 LAI for 60 min induced an almost total (80%) but transient disappearance of the membrane TNF receptor P75, the major form of TNF-R expressed on the surface of adherent monocytes (as determined in TNF-binding assay with blocking IgG raised against the recombinant extracellular domains of TNF-R55 and TNF-R75). Only a small fraction (15-20%) of surface TNF-R was internalized, while sTNF-R75 was already detectable in supernatants suggesting that early shedding of TNF-R75 accounted for the down-modulation of the cell-surface receptors. Endogenous TNF-alpha had no role in the disappearance of its own receptor. Complete and stable restoration of TNF-R expression at the cell membrane, dependent on de novo protein synthesis, occurred after 5h, followed by massive sTNF-R75 release.

CONCLUSIONS: These results demonstrate that infection of human monocytes with HIV-1 LAI triggers at an early stage a cascade of cellular events that lead to profound remodeling of the cell TNF-R pool. Understanding the mechanisms of these receptor movements could be of importance to document the central role of the TNF system in HIV infection.

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MoA143

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