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Background: Quantifying cytokines on the protein level can be problematic because of low concentrations or degradation during sample handling. Aiming towards finding a simple method by which to quantify cytokines on the mRNA level, we combined existing and established molecular biology techniques. Based on the principle of quantitative competitive RT-PCR with a DNA-competitor, IL-1beta, IL-6, IL-12alpha and the housekeeping enzyme GAPDH are measured at levels down to 200 copies of mRNA.Methods: As a source of mRNA, the total RNA from 4 samples of 5 x 10(6) THP-1 cells stimulated with LPS (1 microg/ml for 24 h) was isolated. For competitors, we constructed sequences similar to the target sequences, but with deletion or insertion of 10-15% of the target length. For validating this method, we performed first strand synthesis on different days using different amounts of RNA (1-4 microg) isolated from the same pool of cells. Quantitative competitive PCR was accomplished using different amounts of cDNA (0.125-4 microL). Using IL-1beta as an example, the assay was validated for a dynamic range of 5-300 x 10(3) copies.Results: A linear correlation was found between output and amount of RNA for cDNA synthesis, signifying that the final result of the analysis was linearly related to the amount of RNA or cDNA when operating within the range 1-4 microg (RNA isolation).Conclusion: The quantitative, competitive RT-PCR produces highly reproducible results within a 60-fold dynamic range. [ABSTRACT FROM AUTHOR]

Summary We report that interleukin (IL)‐4 and IL‐10 can significantly up‐ or down‐regulate CXC chemokine receptor 4 (CXCR4) expression on CD4+ T lymphocytes, respectively. Stromal cell‐derived factor‐1α (SDF‐1α)‐induced CD4+ T‐lymphocyte chemotaxis was also correspondingly regulated by IL‐4 and IL‐10. IL‐4 and IL‐10 up‐ or down‐regulated CXCR4 mRNA expression in CD4+ T lymphocytes, respectively, as detected by real‐time quantitative reverse transcription–polymerase chain reaction (RT–PCR). Scatchard analysis revealed a type of CXCR4 with affinity (Kd ≈ 6·3 nm), and ≈ 70 000 SDF‐1α‐binding sites per cell, among freshly isolated CD4+ T lymphocytes, and two types of CXCR4 with different affinities (Kd1 ≈ 4·4 nm and Kd2 ≈ 14·6 nm), and a total of ≈ 130 000 SDF‐1α‐binding sites per cell, among IL‐4‐stimulated CD4+ T lymphocytes. The regulation of CXCR4 expression in CD4+ T lymphocytes by IL‐4 and IL‐10 could be blocked by a selective inhibitor of protein kinase (staurosporine) or by a selective inhibitor of cAMP‐ and cGMP‐dependent protein kinase (H‐8), indicating that these cytokines regulate CXCR4 on CD4+ T lymphocytes via both cAMP and cGMP signalling pathways. The fact that cyclosporin A or ionomycin were able to independently change the CXCR4 expression and block the effects of IL‐4 and IL‐10 on CXCR4 expression implied that the capacity of IL‐4 and IL‐10 to regulate CXCR4 on CD4+ T lymphocytes is not linked to calcium‐mobilization stimulation. These results indicate that the effects of IL‐4 and IL‐10 on the... [ABSTRACT FROM AUTHOR]