Quantitation of Simian Cytokine and β -Chemokine mRNAs,Using Real-Time Reverse Transcriptase-Polymerase Chain Reaction: Variations in Expression during Chronic Primate Lentivirus Infection

Abstract

Cytokines and β-chemokines are important mediators of the immune system and are expressed in many infectious diseases. To study cytokine and β-chemokine profiles during pathogenesis of lentiviral infection and progression to AIDS in rhesus macaques, we established new quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assays based on TaqMan chemistry. Using synthetic RNA standards, we quantified mRNAs of IL-2, IL-4, IL-6, IL-10, IL-12 p40, interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), RANTES, macrophage inflammatory protein 1α (MIP-1α), and MIP-1β in unstimulated peripheral blood mononuclear cells (PBMCs) and lymph nodes from macaques chronically infected with SIV or SHIV. Viremic monkeys with decreased CD4⁺ T cell counts (<500 cells/μl) had significantly higher IL-10 mRNA expression than uninfected controls, which parallels the findings in HIV-1-infected humans. In addition, MIP-1α, MIP-1β, and RANTES mRNA expression increased in viremic monkeys with decreased CD4⁺ T cell counts; gene expression was inversely correlated with CD4⁺ T cell counts, but not viral load. The newly established quantitative real-time RT-PCR assays will allow the determination of cytokine and β-chemokine patterns in rhesus macaques in studies of microbial pathogenesis or vaccine development.