Substance P antagonist (CP-96,345) inhibits HIV-1 replication in human mononuclear phagocytes

Abstract

Substance P (SP) is a potent modulator of neuroimmunoregulation. We recently reported that human immune cells express SP and its receptor. We have now investigated the possible role that SP and its receptor plays in HIV infection of human mononuclear phagocytes. SP enhanced HIV replication in human blood-isolated mononuclear phagocytes, whereas the nonpeptide SP antagonist (CP-96,345) potently inhibited HIV infectivity of these cells in a concentration-dependent fashion. CP-96,345 prevented the formation of typical giant syncytia induced by HIV Bal strain replication in these cells. This inhibitory effect of CP-96,345 was because of the antagonism of neurokinin-1 receptor, a primary SP receptor. Both CP-96,345 and anti-SP antibody inhibited SP-enhanced HIV replication in monocyte-derived macrophages (MDM). Among HIV strains tested (both prototype and primary isolates), only the R5 strains (Bal, ADA, BL-6, and CSF-6) that use the CCR5 coreceptor for entry into MDM were significantly inhibited by CP-96,345; in contrast, the X4 strain (UG024), which uses CXCR4 as its coreceptor, was not inhibited. In addition, the M-tropic ADA (CCR5-dependent)-pseudotyped HIV infection of MDM was markedly inhibited by CP-96,345, whereas murine leukemia virus-pseudotyped HIV was not affected, indicating that the major effect of CP-96,345 is regulated by Env-determined early events in HIV infection of MDM. CP-96,345 significantly down-regulated CCR5 expression in MDM at both protein and mRNA levels. Thus, SP-neurokinin-1 receptor interaction may play an important role in the regulation of CCR5 expression in MDM, affecting the R5 HIV strain infection of MDM.

Figures

Figure 1

(A) SP up-regulated HIV Bal replication in MDM. MDM were incubated with different concentrations of SP as indicated and infected with HIV Bal strain. Untreated and HIV-infected MDM were used as an infection control (HIV only). HIV RT activity was determined in triplicates in the culture supernatants 12 days after infection. The data shown are presented as the mean ± SD of triplicate cultures and are representative of three experiments (**, P < 0.01, SP 10−6 M vs. untreated). (B) Effect of CP-96,345 on HIV infection of human MDM. The HIV Bal strain was used to infect MDM in the presence or absence of CP-96,345 (10−8 to 10−6 M). HIV RT activity was measured in the supernatants 8 and 12 days after infection. HIV RT activity in CP-96,345-treated and infected MDM culture supernatants were expressed as percentage of that of untreated and HIV-infected MDM controls.

Figure 2

Effect of CP-96,345 on HIV-induced syncytium formation in MDM. The morphology of untreated and HIV-infected (A), CP-96,345-treated (10−7 M) and infected (B), and untreated and uninfected MDM (C) was observed and photographed under a light microscopy (×400) 12 days after infection.

Figure 3

Effect of CP-96,345 on the levels of HIV gag mRNA. The HIV Bal strain was used to infect MDM in the presence or absence of CP-96,345 (10−11 to 10−7 M). HIV gag mRNA levels were determined by RT-PCR and real-time RT-PCR by using RNA extracted from the MDM 12 days after infection. β-actin was used as the control to monitor the amount and integrity of RNA in each sample. (A) RT-PCR: −, negative control; +, DNA isolated from ACH-2 cells was used as a PCR positive control (100 copies). HIV only: HIV Bal infected MDM as an infection control; MDM were infected with HIV Bal strain in the presence of CP-96,345 (10−11 M to 10−7 M) as indicated. M: 100-bp DNA ladder coelectrophoresed as markers. (B) Real-time RT-PCR: HIV gag mRNA levels were quantified by real-time RT-PCR by using the same RNA samples as indicated in A, and the data are expressed as HIV gag mRNA copy numbers per PCR. The data shown are presented as the mean ± SD of triplicate cultures and are representative of three experiments (*, P < 0.05; **, P < 0.01; CP-96,345 vs. HIV only).

Figure 4

Effect of CP-96,345 on HIV replication by antagonism of NK-1R on MDM. HIV Bal strain was used to infect MDM in the presence or absence of CP-96,345 (10−7 M) or CP-96,344 (10−7 M). HIV RT activity was determined in the culture supernatants collected 8 days after infection. RT activity in HIV-infected MDM was used as a positive control (HIV only), whereas that in HIV-infected MDM in the presence of CP-96,344 was used as a specificity control. The data shown are presented as the mean ± SD of triplicate cultures and are representative of three experiments (*, P < 0.05; CP-96,345 vs. HIV only).

Figure 5

Effect of CP-96,345 and/or anti-SP antibody on SP-enhanced HIV replication. HIV Bal strain was used to infect MDM in the presence or absence of SP (10−6 M), CP-96,345 (10−6 M), or anti-SP antibody (1:1,000 dilution) as indicated. CP, CP-96,345. HIV only, untreated and HIV Bal strain-infected MDM was used as a control. HIV RT activity (A) in the culture supernatants was determined 12 days after infection, and HIV gag mRNA copy numbers (B) were quantified by using real-time RT-PCR 12 days after infection (B). The data shown are presented as the mean ± SD of triplicate cultures and are representative of three experiments (*, P < 0.05; **, P < 0.01; treatment vs. HIV only).

Figure 6

Effect of CP-96,345 on HIV infection of MDM. Different HIV strains were used to infect MDM in the presence or absence of CP-96,345 (10−7 M). HIV RT activity in the culture supernatants was determined 8 days after infection. HIV RT activity in the CP-96,345-treated and HIV-infected MDM were expressed as percentage of that of untreated and HIV (corresponding strains)-infected MDM controls, which were defined as 100%. The data shown are presented as the mean ± SD of triplicate cultures and are representative of three experiments (*, P < 0.05; **, P < 0.01; CP-96,345 vs. HIV only). R5, CCR5 tropic strains; X4, CXCR4 tropic strains; R5X4, dual tropic strains.

Figure 7

Effect of SP on activation of HIV LTR-driven CAT activity in U38 cells. U38 cells were incubated with SP at different concentrations (10−10 to 10−6 M) or with CP-96,345 (10−6 M) and SP (10−8 M) (SP + CP) or with CP-96,345 (10−6 M) alone (CP only) or untreated (U38 only) for 48 h. The untreated U38 cells were used as baseline control. The CAT activity (cpm) of the treated U38 cells was expressed as percentage of that of untreated baseline control cells. The data shown are presented as the mean ± SD of triplicate cultures and are representative of three experiments (*, P < 0.05; SP-treated U38 vs. U38 only).

Figure 8

Effect of CP-96,345 on macrophage receptor expression. MDM were incubated with or without CP-96,345 or CP-96,344 for 24 h, and the MDM receptor expression was determined by direct immunofluorescence. The results shown are the percentage of MDM positive for the receptors analyzed and are representative of three experiments. The data shown are presented as the mean ± SD of triplicate cultures and are representative of three experiments (*, P < 0.05; CP-96,345 treated vs. control).

Figure 9

Effect of CP-96,345 on CCR5 mRNA expression in MDM. −, PCR negative control, which represents lack of PCR products when RT was omitted from the RT reaction by using RNA extracted from untreated MDM. MDM only, untreated MDM was used as a control. MDM were incubated with CP-96,345 (10−8 to 10−6 M) as indicated for 3 h, and CCR5 mRNA was then amplified by using RT-PCR. M, 100-bp DNA ladder coelectrophoresed as markers. β-actin was used to monitor the amount and integrity of RNA in each sample.

Figure 10

Effect of CP-96,345 on pseudotyped HIV infection of MDM. Recombinant luciferase-encoding HIV reporter viruses pseudotyped with ADA Env or MLV Env were used to infect untreated MDM (ADA only or MLV only) and CP-96,345-pretreated MDM (10−8 and 10−7 M overnight) as indicated. The data are expressed as relative light units of CP-96,345-treated cells to that of untreated control (ADA only or MLV only), which is defined as 100%. The data shown are presented as the mean ± SD of triplicate cultures and are representative of three experiments (**, P < 0.01; CP-96,345 treated vs. ADA only).

Figure 11

Effect of CP-96,345 on cytokine production in MDM. MDM was incubated with or without CP-96,345 and/or LPS for 24 h. TNF-α and IL-6 levels in the culture supernatants were determined by ELISA. The treatment includes untreated MDM (MDM only), MDM incubated with LPS (1 ng/ml) only (LPS), and MDM incubated with LPS (1 ng/ml) plus CP-96,345 at the concentrations indicated. The data shown are presented as the mean ± SD of triplicate cultures and are representative of three experiments (*, P < 0.05; **, P < 0.01; LPS and CP-96,345 treated vs. LPS).