title: Cytokine mRNA and protein expression in primary-culture and repeated-passage synovial fibroblasts from patients with rheumatoid arthritis.
abstract: Constitutive mRNA expression and secretion of proinflammatory and anti-inflammatory cytokines was comparatively analyzed in rheumatoid arthritis (RA) synovial fibroblasts (SFB), isolated from primary culture or derived by repeated passage; normal-skin fibroblasts were used as controls. First-passage RA-SFB (n = 3) secreted large amounts of IL-6 (15,800 +/- 2,110 pg/ml; mean +/- SEM), but only limited amounts of tumor necrosis factor (TNF)-alpha (22.1 +/- 1.1 pg/ml) or IL-10 (35.7 +/- 34.2 pg/ml; only one of three samples was positive). IL-1beta, IL-15, and IL-18 were not detectable at the protein level and showed very low mRNA levels by semiquantitative RT-PCR. In repeated-passage RA-SFB (tenth passage), protein secretion was significantly lower for IL-6 (one-twentieth of the initial level) and TNF-alpha (two-thirds), and markedly reduced for IL-10 (one-quarter, with only one of three samples positive). While the decrease of IL-10 protein from first to tenth passage was paralleled by a corresponding decrease of mRNA, the relative mRNA levels for IL-6 and TNF-alpha were actually increased (20-fold and 300-fold, respectively), indicating post-transcriptional and/or post-translational regulation of these cytokines. Due to highly variable levels among individual patients, however, no significant differences were observed for any cytokine mRNA between primary-culture and repeated-passage RA-SFB (ninth passage). Likewise, no significant differences were detectable between RA-SFB and normal-skin fibroblasts (primary-culture and repeated-passage). By producing high amounts of IL-6 and limited amounts of TNF-alpha, RA-SFB may contribute to the (im)balance of proinflammatory and anti-inflammatory cytokines in the inflamed joint.
authors.affiliation: Institute of Clinical Immunology and Transfusion Medicine, University of Leipzig, Johannisallee 30, D-04103 Leipzig, Germany.
authors.forename: Astrid
authors.initials: A
authors.lastname: Hirth
authors.forename: Alla
authors.initials: A
authors.lastname: Skapenko
authors.forename: Raimund W
authors.initials: RW
authors.lastname: Kinne
authors.forename: Frank
authors.initials: F
authors.lastname: Emmrich
authors.forename: Hendrik
authors.initials: H
authors.lastname: Schulze-Koops
authors.forename: Ulrich
authors.initials: U
authors.lastname: Sack
mesh_terms: D001172:Arthritis, Rheumatoid / Q000378:metabolism* / Q000473:pathology; D002478:Cells, Cultured; D016207:Cytokines / Q000235:genetics* / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D007231:Infant, Newborn; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism*; D013583:Synovial Membrane / Q000473:pathology*
chemical_list: D016207:Cytokines; D012333:RNA, Messenger