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author:("birth, Astrid")
1.  Cytokine mRNA and protein expression in primary-culture and repeated-passage synovial fibroblasts from patients with rheumatoid arthritis 
Arthritis Research  2001;4(2):117-125.
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)-α (22.1 ± 1.1 pg/ml) or IL-10 (35.7 ± 34.2 pg/ml; only one of three samples was positive). IL-1β, 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-α (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-α 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-α, RA-SFB may contribute to the (im)balance of proinflammatory and anti-inflammatory cytokines in the inflamed joint.
PMCID: PMC83845  PMID: 11879547
cytokines; inflammation; mRNA; rheumatoid arthritis; synovial fibroblasts
2.  Isolation and characterization of rheumatoid arthritis synovial fibroblasts from primary culture — primary culture cells markedly differ from fourth-passage cells 
Arthritis Research  2000;3(1):72-76.
To reduce culture artifacts by conventional repeated passaging and long-term culture in vitro, the isolation of synovial fibroblasts (SFB) was attempted from rheumatoid arthritis (RA) synovial membranes by trypsin/collagenase digest, short-term in vitro adherence (7 days), and negative isolation using magnetobead-coupled anti-CD14 monoclonal antibodies. This method yielded highly enriched SFB (85% prolyl-4-hydroxylase+/74% Thy-1/CD90+ cells; <2% contaminating macrophages; <1% leukocytes/endothelial cells) that, in comparison with conventional fourth-passage RA-SFB, showed a markedly different phenotype and significantly lower proliferation rates upon stimulation with platelet-derived growth factor and IL-1β. This isolation method is simple and reliable, and may yield cells with features closer to the in vivo configuration of RA-SFB by avoiding extended in vitro culture.
PMCID: PMC17827  PMID: 11178129
isolation; phenotype/function; primary culture; rheumatoid arthritis; synovial fibroblasts

Results 1-2 (2)