41   Substance P Antagonist (CP96,345) Inhibits HIV Replication in Human Mononuclear Phagocytes.

J. P. Lai*, W. Z. Ho, G. X. Zhan, Y. Yi, R. G. Collman, and S .D. Douglas. The Children's Hosp. of Philadelphia and Univ. of Pennsylvania Med. Sch., Philadelphia.

Background:Substance P (SP) is a potent modulator of neuroimmunoregulation. SP affects macrophage function in an autocrine manner. SP—NK-1R interaction is an important trigger of intracellular events and may be involved in modulation of HIV infection of human MDM. We investigated the hypothesis that interruption of the SP autocrine loop affects HIV infectivity of human MDM.

Methods:MDM were treated with or without the SP antagonist CP96,345 or its inactive enantiomer, CP96,344, or with SP and/or anti-SP antibody for 2 h. The MDM were then infected with HIV overnight. Untreated and HIV-infected MDM were used as controls. Culture supernatants were harvested for measurement of HIV RT activity, and cellular RNA was extracted for determination of HIV gag mRNA using real time RT-PCR. MDM were examined for CCR5, CD4 and CD14 expression by flow cytometry.

Results:SP enhanced HIV replication in MDM, while CP96,345 inhibited HIV infectivity of MDM in a concentration-dependent manner by 83% (10^-5M), 54% (10^-6M), 43% (10^-7M), or 1% (10^-8M). CP96,345 prevented the formation of typical giant syncytia induced by HIV Bal in the MDM. CP96,344 failed to inhibit HIV Bal replication. Both CP96,345 and anti-SP antibody inhibited SP-enhanced HIV replication in MDM. Among HIV strains tested, only R5 strains, both prototype and primary isolates (Bal, ADA, BL-6 and CSF-6), were significantly inhibited by CP96,345, while an R4 strain (UG024) was not affected. Furthermore, ADA- pseudotyped HIV infection of MDM was markedly inhibited by CP96,345, while MLV-pseudotyped HIV was not affected, indicating that the effect of CP96,345 is regulated by env-determined early events. In addition, CCR5 expression on MDM was down-regulated by CP96,345, while expression of CD4, CXCR4 and CD14 on MDM was not affected. In addition, SP significantly increased HIV LTR-driven CAT activity, which was abrogated by CP96,345, indicating that SP activates the HIV LTR through the NK-1R on the cell membrane.

Conclusions:CP96,345 inhibits HIV replication in human MDM by interrupting the SP autocrine loop, resulting in down-regulation of CCR5 expression and antagonism of the SP effect on activation of the HIV LTR promoter.