E. be biologically active. Binding to LM-111 in the presence of TGF-1 activates SF for expression of IL-6 and IL-8 and thus may contribute to synovial inflammation and to infiltration of leukocytes. Keywords:arthritis, inflammation, IL-6, IL-8, TGF- synovial fibroblast == INTRODUCTION == In rheumatoid arthritis (RA), inflammation and infiltration of mononuclear cells contribute to synovial hyperplasia (1). The key regulatory factors for inflammation in RA are TNF- and IL-1, but IL-6 or IL-8 contribute to the RA pathology as well (25). IL-6 is a key factor for activation of B- and T-cells and for the mobilization of neutrophils (6,7). IL-8 promotes chemotaxis of neutrophils (8), lymphocytes (9) and mononuclear cells (10). Laminin-111, previously called laminin-1 (LN-1) or EHS laminin, was detected in the pericellular matrix of articular cartilage (11). Using an anti-EHS-laminin AZ1 antibody, high expression of laminins has been detected in the lining layer of the synovial membrane of RA patients (12). Activation of synovial fibroblasts (SF) by binding to LM-111 in the presence of TGF-1 activated the expression of MMP-3, MMP-10 and IL-16 (13,14). This activation of SF by TGF-1 and LM-111 works in the absence of TNF- and IL-1 does not depend on NFB signalling, and thus may contribute to tissue destruction in the joints of RA patients, even after administration of pharmaceuticals controlling bioactive TNF- or IL-1. High expression of IL-6, IL-8 and IL-16 were measured in RA synovial fluid (15,16), and the activation of SF by LM-111 in the presence of TGF-1 resulted in a significant IL-16 expression (14). Therefore, we extended our recent studies and investigated whether LM-111 and TGF-1 cooperate in regulation of additional RA-associated interleukins in SF, specifically of IL-6 and IL-8. == METHODS == == Cell culture == SF were isolated from synovial tissue of 17 OA and 16 RA patients as described recently (Table1) (14). The cells were expanded in DMEM medium (Life Technologies) enriched with ITS (Life Technologies), 10% FCS (Biochrome) and antibiotics (Sigma). This study was approved by the local ethics committee. == Table 1. == Clinical data of patients included in the study RA, rheumatoid arthritis; OA, osteoarthritis; F, female; M, male; CRP, C-reactive protein; BSR, blood sedimentation rate; DMARDs, disease modifying anti-rheumatic drugs; NSAR, non-steroidal anti-inflammatory drugs. For activation, SF were incubated in DMEM complete medium in the presence of rhTGF-1 (10 ng/mL, 24 h, Calbiochem). Cells in medium without TGF-1 served as controls. In other experiments, cells were AZ1 incubated for 24 h in flasks coated with LM-111 (BD Biosciences). Uncoated flasks and flasks coated with LM-511/521 (Chemicon) served as controls. AZ1 == Transcript analysis == RNA was extracted (RNeasy, Qiagen) and cDNA was generated by oligo-(dT) priming and AMV-reverse transcriptase (Clontech) as described (14). Transcripts were quantified by qRT-PCR utilizing commercially available primers (SearchLC) and normalized to GAPDH and serial dilutions of a recombinant standard (Roche) in each run (17). The results are presented as mean transcript induction index of activated fibroblasts normalized to the mock-treated cells and the recombinant EZH2 standards. == Protein analysis == Production of cytokines was detected in SF supernatants by a multiplexed cytokine array technique (Luminex). Cells were activated for 24 h by the addition of 10 ng/mL rhTGF-1, by binding to coated LM-111 or by binding to LM-111 in the presence of TGF-1. Controls were incubated in normal tissue culture flasks without stimuli. Supernatants were harvested and pre-cleared by centrifugation (12000 x g, 4C, 5 min), and aliquots were mixed with dye-loaded microbeads coated with antibodies reactive with the cytokines followed by biotinylated detection antibodies and fluorochrome-labelled detection reagent for quantification (Invitrogen). The cytokine array data were confirmed by ELISA (scan Bio-Tek) and commercially available kits (GE Health Care). Biological activity of IL-6 was confirmed by the B9 B-cell hybridoma proliferation assay (a generous gift of Prof. Kolodziej, Berlin). Briefly, supernatants of activated SF (n=8) were added to B9 cells and IL-6-dependent cell proliferation was determined utilizing.