Methamphetamine enhances HIV infection of macrophages

Abstract

Epidemiological studies have demonstrated that the use of methamphetamine (meth), a sympathomimetic stimulant, is particularly common among patients infected with HIV. However, there is a lack of direct evidence that meth promotes HIV infection of target cells. This study examined whether meth is able to enhance HIV infection of macrophages, the primary target site for the virus. Meth treatment resulted in a significant and dose-dependent increase of HIV reverse transcriptase activity in human blood monocyte-derived macrophages. Dopamine D1 receptor antagonists (SCH23390 and SKF83566) blocked this meth-mediated increase in the HIV infectivity of macrophages. Investigation of the underlying mechanisms of meth action showed that meth up-regulated the expression of the HIV entry co-receptor CCR5 on macrophages. Additionally, meth inhibited the expression of endogenous interferon-alpha and signal transducer and activator of transcription-1 in macrophages. These findings provide direct in vitro evidence to support the possibility that meth may function as a cofactor in the immunopathogenesis of HIV infection and may lead to the future development of innate immunity-based intervention for meth users with HIV infection.

Figure 1

Dose-dependent (A) and time-course (B) effect of meth on HIV Bal replication in macrophages. A: Seven-day-cultured macrophages were incubated with or without meth at indicated concentrations for 24 hours and then incubated with HIV Bal strain for 2 hours in the presence or absence of meth. Day-8 culture supernatants were collected for HIV RT assay. B: Seven-day-cultured macrophages were incubated with or without 100 μmol/L meth for 24 hours before infection with HIV Bal strain for 2 hours and then cultured for 12 days. HIV RT activity was determined in cultured supernatants at indicated time points after infection. Data are expressed as HIV RT activity in meth-treated cells (percentage of control) compared with those in untreated cells, which are defined as 100%. The results represent the mean ± SD of three independent experiments using macrophages from three different donors. Statistical analysis was performed by one-way analysis of variance (A) or Student’s t-test (B), and significance is shown with *P < 0.05 and **P < 0.01 (meth versus control).

Figure 2

Dopamine 1 receptor (D1R) expression in macrophages. A: Total mRNA was extracted from 7-day-cultured macrophages and subjected to the real time RT-PCR for D1R. Sizes are estimated from DNA ladder (100-bp fragments) co-electrophoresed as markers. Lane 1, markers; lanes 2 to 4, macrophages from three different donors, respectively; lane 5, human neuronal cells (NT2-N) as a positive control (+); lane 6, negative control (−; the same cells as used in the positive control but processed without reverse transcriptase). B: The macrophages plated on coverslips were fixed, permeabilized, and stained with (top panel) or without (bottom panel) antibody to D1R (green) and with nuclear staining dye (blue). The specimens were examined by a fluorescence microscopy with magnification of ×200. One representative result of three independent experiments is shown.

Figure 3

Effect of the D1R antagonists on meth-mediated up-regulation of HIV Bal infection. Seven-day-cultured macrophages were incubated with or without the D1R antagonist, 10 μmol/L SCH23390, or 10 μmol/L SKF83566 for 1 hour before treatment with or without 100 μmol/L meth for 24 hours and then infected with HIV Bal strain for 2 hours in the presence or absence of meth and/or SCH23390 or SKF83566. Culture supernatants were collected at day 8 after infection for HIV RT assay. Data are expressed as HIV RT activity in meth-treated cells (percentage of control) to those in untreated cells, which is defined as 100%. The results represent the mean ± SD of three independent experiments. Statistical analysis was performed using one-way analysis of variance, and significance is shown with *P < 0.05 and **P < 0.01 (METH + SCH or METH + SKF versus METH).

Figure 4

Effect of meth on the expression of CD4, CD14, and CCR5 receptors. Seven-day-cultured macrophages were treated with or without 100 μmol/L meth for 24 hours. Expression of CD4, CD14, and CCR5 on macrophages was determined by flow cytometry assay. The data shown are the percentage of positive cells for the indicated receptors and represent the mean ± SD of three independent experiments. Statistical analysis was performed using Student’s t-test, and significance is shown with *P < 0.05 (meth versus control).

Figure 5

Effect of meth on endogenous IFN-α mRNA (A) and protein (B) expression in macrophages. A: Seven-day-cultured macrophages were treated with or without 100 μmol/L meth for the indicated time points, and then cellular RNA were subjected to the real-time RT-PCR for IFN-α mRNA. B: Cell lysates from seven-day-cultured macrophages treated with or without 100 μmol/L METH for 24 hours were subjected to enzyme-linked immunosorbent assay for IFN-α protein. Data are expressed as IFN-α mRNA (A) or protein (B) levels in meth-treated cells (percentage of control) to those in untreated cells, which are defined as 100%. The results represent the mean ± SD of three independent experiments. Statistical analysis was performed by Student’s t-test and significance is shown with *P < 0.05 and **P < 0.01 (meth versus control).

Figure 6

Effect of the D1R antagonists on meth-mediated down-regulation of IFN-α. Seven-day-cultured macrophages were incubated with or without the D1R antagonist, 10 μmol/L SCH23390, or 10 μmol/L SKF83566 for 1 hour before treatment with or without 100 μmol/L meth for 3 hours. Cellular RNA was subjected to the real-time RT-PCR for IFN-α mRNA. Data are expressed as IFN-α mRNA levels in meth-treated cells (percentage of control) to those in untreated cells, which are defined as 100%. The results represent the mean ± SD of three independent experiments. Statistical analysis was performed using one-way analysis of variance, and significance is shown with *P < 0.05 and **P < 0.01 (METH + SCH or METH + SKF versus METH).

Figure 7

Effect of meth on STAT1 expression. Seven-day-cultured macrophages were incubated with or without 100 μmol/L meth for 24 hours. Equal amount of proteins extracted from the cells was subjected to the Western blot assay using rabbit antibodies against STAT1 and actin. The numbers in the right panel are the signal intensities expressed as densitometry scanning units (DSU) of protein bands of Western blot shown in the left panel. One representative experiment is shown.