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Interleukin 16 (IL16) is a proinflammatory cytokine and a chemoattractant factor for CD4+ T cells. IL16 has been detected at higher concentrations in rheumatoid arthritis (RA) synovial fluid than in osteoarthritis(OA) specimens.1 IL16 is expressed in inflammatory infiltrates and in CD68− synovial lining cells of patients with RA as detected by in situ hybridisation.1–3
In this study we compared the modulation of IL16 steady state mRNA in synovial fibroblasts (SF) from six patients with RA and from three patients with OA. SF were prepared, expanded, and characterised as described previously.4 To examine the IL16 encoding transcript amounts, SF were incubated in complete medium for 24 or 48 hours in the presence of one of the following chemicals: 1 ng/ml phorbol-12-myristate-13-acetate (PMA), an activator of protein kinase C (PKC); 200 ng/ml ionomycin (Iono), a calcium ionophor; 10 μM of adenosine-3′,5′-cyclic monophosphate (cAMP), which stimulates protein kinase A (PKA); 10 nM okadaic acid (Oka), a phosphatase inhibitor; 10 μM MAS-7, which activates G-proteins; 100 μM H-7 dihydrochloride (H-7), an inhibitor of protein kinases; and 10 nM staurosporine (Stauro), a protein kinase inhibitor (all from Calbiochem or Biomol). Differences of IL16 encoding steady state mRNA amounts in activated cells compared with controls were detected after 33 cycles of reverse transcriptase-polymerase chain reaction (RT-PCR) amplification (Taq DNA polymerase, Roche Biochemicals).1 Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RT-PCR served as a control for RNA content. The PCR amplification plateau was reached after 35 cycles. This suggested that the IL16-specific RT-PCR was suitable for detecting different levels of IL16 encoding transcripts as the PCR was stopped before reaching the amplification plateau. Still, the limitations of this method are evident and we therefore consider our data as a semiquantitative enumeration of transcripts encoding IL16.
Both, early passage RA SF and OA SF spontaneously transcribed IL16 encoding mRNA. Addition of protein kinase inhibitor staurosporine enhanced the IL16 RT-PCR signals in all samples of OA SF, whereas specific protein kinase C activator PMA reduced the IL16 encoding RT-PCR signals in OA SF (fig 1). Ionomycin, cAMP, and MAS-7 had minor and variable effects in OA SF (fig 1). Addition of protein kinase inhibitor staurosporine also enhanced the IL16 encoding signal in RA SF (fig 2). Incubation of the cells with PMA and ionomycin reduced the IL16 encoding RT-PCR signal intensity in these cells (fig 2). Again, cAMP and MAS-7 produced minor and variable effects in the different samples analysed (fig 2). Application of okadaic acid or H-7 dihydrochloride reduced the IL16 RT-PCR signals. As okadaic acid and H-7 reduced the cell viability prominently, the decreased IL16 signals probably result from the induction of cell death. In contrast, incubation of the cells with PMA, ionomycin, cAMP, MAS-7, or staurosporine did not reduce the viability.
Protein kinase inhibitor staurosporine has been reported to induce apoptosis in some cells.5 Enumeration of dead cells and observation of morphological changes by microscopy upon staurosporine treatment did not give any indication of reduced cell viability at concentrations 10- to 100-fold above the concentrations used in our experiments. RA SF are resistant to induction apoptosis by overexpression of sentrin, Bcl-2, and mutant forms of p53.6–9 Therefore the RA SF, especially, may be able to respond to a staurosporine induced pathway with enhanced IL16 transcript amounts. Because protein kinase C activator PMA reduced IL16 transcripts in SF, the data suggest that in SF the transcription of IL16 might be regulated through protein kinase C dependent pathways.
We thank A Hack for excellent technical service. This study was supported by grants to WKA (fortüne 411, in part by DFG Ai 16/10–1) and by institutional funding.
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