Transforming growth factor-beta inhibits interleukin-10 synthesis by human monocytic cells.

Transforming growth factor-beta (TGF-beta1) enhances interleukin-10 (IL-10) synthesis by mouse monocytes/macrophages, suggesting a potential role of IL-10 in mediating some of the anti-inflammatory properties of TGF-beta1. Since differences exist between the transcriptional regulation of human and mouse IL-10, the studies reported here examined whether TGF-beta1 up-regulated IL-10 production by human monocytes/macrophages as well. Exposure of PMA-differentiated U-937 promonocytic cells to TGF-beta1 resulted in an unexpected, dose-dependent decrease in IL-10 production as assessed by specific ELISA. TGF-beta1 was effective when added at the time of the PMA stimulus or 6 hours after. In addition, TGF-beta1 suppressed induction of IL-10 by three different stimuli other than PMA. TGF-beta1 inhibition of IL-10 protein release was associated with proportional changes in IL-10 mRNA accumulation as assessed by quantitative kinetic ELISA PCR. This would result from a decrease in IL-10 gene transcription as TGF-beta1 did not affect IL-10 mRNA stability, and TGF-beta1 limited the luciferase activity in cells transfected with reporter gene constructs containing 1,308 bp of the 5' non-coding sequence of human IL-10 gene. Blocking tumour necrosis factor-alpha (TNF-alpha) with neutralizing anti-TNF-alpha antibody did not modify the response to TGF-beta1, indicating the involvement of TNF-alpha-independent mechanisms in the overall process. Thus, the present study provides the first evidence that TGF-beta1 prevents IL-10 production by human monocytic cells at a transcriptional level.

Quantification of porcine cytokine gene expression using RT-PCR, a homologous internal control and chemiluminescence for microplate detection.

The polymerase chain reaction (PCR) has proved to be a sensitive and versatile method for the analysis of human and murine cytokine mRNA expression. This paper describes for the first time a reverse transcription-polymerase chain reaction (RT-PCR) at end-point for the quantification of five porcine cytokines: interferon (IFN)-gamma, interleukin (IL)-2, IL-4, IL-10 and IL-18. The main features of the methodology are: (1) a unique RT for all quantifications, (2) the addition of homologous DNA internal controls (IC) of equal length to the corresponding cytokine and consequently co-amplification of the target cytokine and the IC with equivalent efficacy, (3) PCR and detection of amplicons for all cytokines simultaneously, (4) cytokine quantification in relation to a housekeeping gene control (glyceraldehyde-3-phosphate dehydrogenase, GAPDH), (5) detection of the amplicons by enzyme linked immunosorbent assay (ELISA) using a chemiluminescent substrate with high sensitivity and wide dynamic range, (6) automation of the detection system for analysis of a large number of samples. This highly sensitive quantitative RT-PCR assay (able to detect 100-200 cytokines mRNA copies/75x10(3) cells) was validated on peripheral blood mononuclear cells (PBMC) from pigs infected or not with pseudorabies virus (PRV), re-stimulated in vitro by a mitogen or antigens.

Persistent alterations in T-cell repertoire, cytokine and chemokine receptor gene expression after 1 year of highly active antiretroviral therapy.

OBJECTIVES: To examine T-cell repertoire modifications, the evolution of T-helper (TH)1/TH2 cytokine imbalance and modifications in chemokine receptor expression when the viral load is decreased by 2-3 log10 copies/ml under highly active antiretroviral therapy (HAART). DESIGN: Sixteen patients previously treated with zidovudine and lamivudine, with CD4 cells below 300 x 10(6)/l and viraemia above 30000 copies/ml were treated by saquinavir and ritonavir together with both reverse transcriptase (RT) inhibitors (ANRS 069 trial). T-cell repertoire, chemokine receptor and lymphokine expression were studied from peripheral blood mononuclear cells sampled at weeks 0, 24 and 48. METHODS: T-cell repertoire study was carried out using the Immunoscope method. Interleukin (IL)-12 receptor beta2, CC-chemokine receptor (CCR)-3, CXC-chemokine receptor-4 and CCR-5 expression in CD4+ cells was measured by kinetic quantitative PCR and IL-2, IL-4, IL-10, IL-13, interferon (IFN)-gamma were measured using a quantitative RT-PCR assay with homologous internal standards. RESULTS: Repertoire alterations were more frequent in CD4- than in CD4+ cells and persisted despite undetectable viraemia. Increased CCR-3 expression and spontaneous IFN-gamma as well as mitogenic induced IL-13 were observed at baseline and decreased slightly under HAART. CONCLUSION: The CD8+ cell repertoire alterations were profound, whereas the CD4+ cell alterations were moderate and both persisted unchanged under HAART. The TH1/TH2 imbalance was more related to TH2 over-expression than to TH1 deficiency and persisted for at least 1 year under HAART.