398-T.
|
Antagonism of the CCR5 Receptor by SCH-C Leads to Elevated beta-Chemokine Levels and Receptor Expression in Chronically Treated PBMC Cultures
S. Xu*, L. Wojcik, and J. Strizki
Schering-Plough Res. Inst., Kenilworth, NJ
|
Background: SCH-C, a CCR5 receptor antagonist, is a potent antiviral agent for R5-tropic HIV isolates and is being evaluated clinically. In this study we investigated the potential effects of CCR5 blockade on beta-chemokine production and receptor expression in vitro.
Methods: Human PBMCs were cultured in the presence or absence of SCH-C or an anti-CCR5 antibody, 2D7, and cultures tested for beta-chemokine levels by ELISA and CCR5 and CXCR4 expression by FACS. RT-PCR analysis was used to quantitate chemokine and CCR5 mRNA levels in the cultures. Susceptibility studies were done by pre-incubating PBMCs with compound prior to infection with serially diluted R5, X4, or R5/X4 viral isolates and measuring p24 production on day 4-6.
Results: Our results showed that cultures treated with SCH-C or 2D7 contained significantly higher levels of MIP-1alpha, MIP-1beta, and RANTES compared with untreated cultures. Elevated chemokine levels were sustained over the course of the cultures (15-20 days). CCR5 receptor expression was significantly increased on cells treated with SCH-C while no effect was seen on CXCR4 expression. Quantitative RT-PCR analysis of beta-chemokine and CCR5 mRNA levels revealed no significant difference between the control and treated groups for any of the genes tested. This suggests that SCH-C increases chemokine levels by inhibiting their binding and uptake by CCR5 and not by upregulating gene expression. Similarly, the increase in CCR5 surface expression observed in SCH-C cultures likely results from inhibition of receptor recycling by the compound. Since CCR5 receptor density can influence viral infectivity, we also tested the susceptibility of SCH-C treated cells to infection with R5, X4, and R5/X4 viruses. As expected, the CCR5 antagonists did not alter the susceptibility of cells to X4 or R5/X4 virus infection. Similarly, R5 virus infection was not affected by pretreatment with SCH-C, despite increased CCR5 expression on cells.
Conclusions: CCR5 antagonists and can effectively increase chemokine levels in long term PBMC cultures, likely due to inhibition of ligand uptake by the receptor. In addition CCR5 receptor expression is increased by SCH-C treatment, presumably resulting from inhibition of chemokine-induced receptor cycling.
|
