Background In previous studies we have shown that synovial fibroblasts from rheumatoid arthritis (RA) patients transcribe elevated levels of IL-16 encoding mRNA. In addition, in RA synovial fluid high concentrations of IL-16 were detected. The chemotactic activity to CD4+ cells was associated with IL-16. In osteoarthritis synovial fibroblasts, IL-16 was not found.
Objectives Therefore we initiated experiments to study the activation pathway regulating IL-16 transcription in synovial versus skin fibroblasts.
Methods OA synovial fibroblasts (n = 3), skin fibroblasts (n = 6) and neonatal preputial fibroblasts (n = 3) were expanded (less than 4 passages). Cells were incubated in complete medium (10% FCS) supplemented with MAS-7, ocadaic acid (ODA), ionomycin, cAMP, forskolin, staurosporin, phorbolester, H7 and an unrelated peptide as mock control. Untreated cells served as controls. After 6 to 48 h of incubation, mRNA was extracted from experimental samples and controls. The mRNA yield was determined by spectrophotometry and, cDNA was generated from 1 μg RNA using the oligo (dT) priming procedure. Steady state transcript amounts encoding IL-16 or IL-18 were determined by real time quantitative RT/PCR (LightCycler, Roche) using the SYBR green method. The PCR products were visualised by EtBr fluorescence after gel electrophoresis. Quantitative GAPDH RT/PCR served as internal standard.
Results mRNA encoding IL-16 and IL-18 was detected in all fibroblasts analysed. Incubation of the fibroblasts for 24 to 48 h with staurosporin or forskolin enhanced IL-16 steady state mRNA levels two to three fold. Incubation with ODA or H7 reduced IL-16 mRNA three-fold and ten-fold, respectively. PMA reduced IL-16 signals to about 70%. Interestingly, IL-18 signals were not reduced in ODA treated fibroblasts whereas H7 reduced IL-18 signals about ten-fold. Staurosporin and forskolin did not enhance IL-18 encoding signals but reduced the IL-18 signals two to three fold. IL-18 encoding mRNA amounts were elevated in ionomycin treated cells. All other components did not show considerable activation or reduction of the IL-16 or IL-18 RT/PCR signals. In addition, there was no remarkable difference in IL-16 or IL-18 transcriptional responses to all agents analysed so far.
Conclusion Staurosporin is a broad protein kinase inhibitor and activated IL-16 transcript levels whereas phorbolester PMA activates protein kinase C and reduced IL-16 mRNA levels. We conclude that IL-16 transcription is regulated by kinase/phosphatase signal transduction pathways. In addition, a cAMP dependent enhancement of IL-16 endocing steady state mRNA levels was observed. By contrast, IL-18 RT/PCR signals are reduced upon kinase inhibition but not activated after PMA incubation. Raising intracellular calcium by ionomycin induced IL-18 encoding mRNA. Therefore the IL-16 and IL-18 gene activities are regulated differently. Since in RA synovial fibroblasts IL-16 mRNA is elevated when compared to OA fibroblasts and blocking kinase activity activated IL-16 mRNA levels in OA synovial or skin fibrobasts, we hypothesise that a reduced kinase activity may be associated with enhanced levels of IL-16 message in RA synovial fibroblasts.
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