Morphine enhances Tat-induced activation in murine microglia

Abstract

There is increasing evidence that opiates accelerate the pathogenesis and progression of acquired immunodeficiency syndrome (AIDS), as well as the incidence of human immunodeficiency virus (HIV) encephalitis (HIVE), a condition characterized by inflammation, leukocyte infiltration, and microglial activation. The mechanisms, by which the HIV-1 transactivating protein Tat and opioids exacerbate microglial activation, however, are not fully understood. In the current study, we explored the effects of morphine and HIV-1 Tat(1-72) on the activation of mouse BV-2 microglial cells and primary mouse microglia. Both morphine and Tat exposure caused up-regulation of the chemokine receptor CCR5, an effect blocked by the opioid receptor antagonist naltrexone. Morphine in combination with Tat also induced morphological changes in the BV-2 microglia from a quiescent to an activated morphology, with a dramatic increase in the expression of the microglial activation marker CD11b, as compared with cells exposed to either agent alone. In addition, the mRNA expression of inducible nitric oxide synthase (iNOS), CD40 ligand, Interferon-gamma-inducible protein 10 (IP-10), and the proinflammatory cytokines tumor necrosis factor alpha (TNFalpha), interleukin (IL)-1beta, and IL-6, which were elevated with Tat alone, were dramatically enhanced with Tat in the presence of morphine. In summary, these findings shed light on the cooperative effects of morphine and HIV-1 Tat on both microglial activation and HIV coreceptor up-regulation, effects that could result in exacerbated neuropathogenesis.

Figure 1

Morphine up-regulates CCR5 through the μ-opioid receptor in BV-2 mouse microglia. Cells were treated with 10⁻⁷ M morphine (10⁻¹³ to 10⁻⁵ M for dose response) for 6 h (24-h treatment for immunocytochemistry). Cells were pretreated with 10⁻⁶ M naltrexone for 1 h to block opioid receptor. RNA was isolated and subjected to reverse transcription and real-time PCR analysis using primers specific for CCR5 or MOR (μ-opioid receptor) and HPRT as an internal control. Data were plotted as fold increase compared to the untreated control. (A) Dose response effect of morphine on the mRNA expression of CCR5 chemokine receptor. **P<.01 (B) Immunocytochemical analysis using antibody specific to the CCR5 chemokine receptor. (C) Fold increase in the MOR mRNA.

Figure 2

Morphine plus Tat enhance the expression of CCR5 in BV-2 microglia. Cells were pretreated with morphine (10⁻⁷ M) for 1 h and treated with 20 nM of Tat_1–72 for 24 h and stained for the CCR5 receptor. (A and B) FACS analysis of mouse microglial cells using antibody specific to the CCR5 chemokine receptor. Samples were run on the BD LSRII cytometer. Data were expressed as averages of the mean fluorescence intensities (MFIs), whereas the graphical data were obtained by converting the MFIs into percentage increase/decrease. **P<.01; *P<.05. (C) Western blot analysis of mouse microglial cells using antibody specific to the CCR5 chemokine receptor. Total proteins were isolated, denatured, separated by SDS-PAGE, and analyzed by Western blotting.

Figure 3

(A) Morphine and Tat effect changes in microglial morphology from quiescent to an activated macrophage-like. Cells were treated with morphine (10⁻⁷ M) for 6 h and treated with 20 nM of Tat_1–72 for 24 h and stained for the CCR5 receptor. Morphine enhances the effect of Tat on microglial activation. (B) Immunocytochemical analysis of mouse microglial cells pretreated with morphine (10⁻⁷ M) for 1 h and treated with 20 nM of Tat_1–72 for 24 h. Cells were stained for the activation marker CD11b. Data were expressed as averages of the mean fluorescence intensities (MFIs). ***P<.001.

Figure 4

Morphine enhances the effect of Tat on microglial activation. FACS analysis of mouse microglial cells using antibodies specific to CD11b microglial activation marker. Cells were pretreated with morphine (10⁻⁷ M) for 6 h, followed by treatment with 20 nM of Tat_1–72 for 24 h. Samples were run on the BD LSRII cytometer. Data were expressed as average of mean fluorescence intensities (MFIs) expressed as percentages. *P<.05.

Figure 5

Morphine potentiates the mRNA expression of inflammatory cytokines in BV-2 mouse microglia. Cells were pretreated with morphine (10⁻⁷ M) for 1 h and treated with Tat_1–72 for 6 h. RNA was isolated and subjected to reverse transcription and real-time PCR analysis using primers specific for the selected cytokines and HPRT as internal control. Data were plotted as fold increase compared to the untreated control.

Figure 6

Morphine enhances the mRNA expression of inflammatory cytokines in primary mouse microglia. (A) Immunofluorescence staining for CD11b (green) showing the purity of primary mouse microglial cultures. (B) Quantitative PCR of cDNA from primary mouse microglia. Cells were pretreated with morphine (10⁻⁷ M) for 1 h and treated with Tat_1–72 for 6 h. RNA was isolated and subjected to reverse transcription and real-time PCR analysis using primers specific for the selected cytokines and HPRT as internal control. Data were plotted as fold increase compared to the untreated control.

Figure 7

Morphine increases the protein expression of IL-6 and TNFα in mouse microglia. Cells were pretreated with morphine (10⁻⁷ M) for 6 h and treated with Tat_1–72 for 24 h. Cell culture supernatants were analyzed for the expression of various cytokines using the Milliplex mouse cytokine panel.