Objectives: To investigate the specificity of three anti-CD68 monoclonal antibodies (mAbs) for macrophages (Mw) in immunohistochemistry (IHC) and flow cytometry (FACS). Methods: IHC was performed on cryostat sections of rheumatoid arthritis (RA) and osteoarthritis (OA) synovial membranes using the anti-CD68 mAbs KP1, EBM11, and PGM1, and the fibroblast (FB) markers CD90 and prolyl 4-hydroxylase. Expression of CD68 was also analysed by FACS on the monocytic cell lines THP-1 and U937, as well as on synovial fibroblasts (SFB), skin FB, and gingival FB (both surface and intracellular staining). Results: In IHC, there was an overlap between CD68 (mAbs KP1 and EBM11) and the FB markers CD90/ prolyl 4-hydroxylase in the lining layer, diffuse infiltrates, and stroma of RA and OA synovial membranes. In FACS analysis of THP-1 and U937 cells, the percentage of cells positive for the anti-CD68 mAbs KP1 and EBM11 progressively increased from surface staining of unfixed cells, to surface staining of pre-fixed cells, to intracellular staining of the cells. Upon intracellular FACS of different FB, nearly all cells were positive for KP1 and EBM11, but only a small percentage for PGM1. In surface staining FACS, a small percentage of FB were positive for all three anti-CD68 mAbs. Conclusion: An overlap between CD68 (mAbs KP1 or EBM11) and the FB markers CD90 or prolyl 4-hydroxylase may prevent unequivocal identification of Mw in synovial tissue by IHC or in monocytic cells and FB upon intracellular FACS. This may be due to sharing of common markers by completely different cell lineages.

Objective: To examine the relative importance of tumour necrosis factor-receptor 1 (TNF-R1) and TNF-R2 and their signalling pathways for pro-inflammatory and pro-destructive features of early-passage synovial fibroblasts (SFB) from rheumatoid arthritis (RA) and osteoarthritis (OA). Methods: Cells were stimulated with tumour necrosis factor (TNF)a or agonistic anti-TNF-R1/TNF-R2 monoclonal antibodies. Phosphorylation of p38, ERK and JNK kinases was assessed by western blot; proliferation by bromodesoxyuridine incorporation; interleukin (IL)6, IL8, prostaglandin E 2 (PGE 2 ) and matrix metalloproteinase (MMP)-1 secretion by ELISA; and MMP-3 secretion by western blot. Functional assays were performed with or without inhibition of p38 (SB203580), ERK (U0126) or JNK (SP600125). Results: In RA-and OA-SFB, TNFa-induced phosphorylation of p38, ERK or JNK was exclusively mediated by TNF-R1. Reduction of proliferation and induction of IL6, IL8 and MMP-1 were solely mediated by TNF-R1, whereas PGE 2 and MMP-3 secretion was mediated by both TNF-Rs. In general, inhibition of ERK or JNK did not significantly alter the TNFa influence on these effector molecules. In contrast, inhibition of p38 reversed TNFa effects on proliferation and IL6/PGE 2 secretion (but not on IL8 and MMP-3 secretion). The above effects were comparable in RA-and OA-SFB, except that TNFa-induced MMP-1 secretion was reversed by p38 inhibition only in OA-SFB. Conclusion: In early-passage RA/OA-SFB, activation of MAPK cascades and pro-inflammatory/prodestructive features by TNFa is predominantly mediated by TNF-R1 and, for proliferation and IL6/PGE 2 secretion, exclusively regulated by p38. Strikingly, RA-SFB are insensitive to p38 inhibition of MMP-1 secretion. This indicates a resistance of RA-SFB to the inhibition of pro-destructive functions and suggests underlying structural/functional alterations of the p38 pathway, which may contribute to the pathogenesis or therapeutic sensitivity of RA, or both.

Chromosomal aberrations were comparatively assessed in nuclei extracted from synovial tissue, primary-culture (P-0) synovial cells, and early-passage synovial fibroblasts (SFB; 98% enrichment; P-1, P-4 [passage 1, passage 4]) from patients with rheumatoid arthritis (RA; n = 21), osteoarthritis (OA; n = 24), and other rheumatic diseases. Peripheral blood lymphocytes (PBL) and skin fibroblasts (FB) (P-1, P-4) from the same patients, as well as SFB from normal joints and patients with joint trauma (JT) (n = 4), were used as controls. Analyses proceeded by standard GTG-banding and interphase centromere fluorescence in situ hybridization. Structural chromosomal aberrations were observed in SFB (P-1 or P-4) from 4 of 21 RA patients (19%), with involvement of chromosome 1 [e.g. del(1)(q12)] in 3 of 4 cases. In 10 of the 21 RA cases (48%), polysomy 7 was observed in P-1 SFB. In addition, aneusomies of chromosomes 4, 6, 8, 9, 12, 18, and Y were present. The percentage of polysomies was increased in P-4. Similar chromosomal aberrations were detected in SFB of OA and spondylarthropathy patients. No aberrations were detected in i) PBL or skin FB from the same patients (except for one OA patient with a karyotype 45,X[10]/46,XX[17] in PBL and variable polysomies in long-term culture skin FB); or ii) synovial tissue and/or P-1 SFB of normal joints or of patients with joint trauma. In conclusion, qualitatively comparable chromosomal aberrations were observed in synovial tissue and early-passage SFB of patients with RA, OA, and other inflammatory joint diseases. Thus, although of possible functional relevance for the pathologic role of SFB in RA, these alterations probably reflect a common response to chronic inflammatory stress in rheumatic diseases. DMEM = Dulbecco's modified Eagle's medium; FB = fibroblasts; FCS = fetal calf serum; FISH = fluorescence in situ hybridization; HEPES = N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid; IL = interleukin; ISCN = International System for Human Cytogenetic Nomenclature; JT = joint trauma; OA = osteoarthritis; P-0 = primary culture; P-1 = passage 1; P-4 = passage 4; PBL = peripheral blood lymphocytes; PBS = phosphate-buffered saline; RA = rheumatoid arthritis; RPMI = Roswell Park Memorial Institute [medium]; SFB = synovial fibroblasts.

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