OBJECTIVES:To examine the potential role of the angiogenic growth factor angiopoietin-1 (Ang-1) in inflammatory arthritis. METHODS:Eighteen synovial tissue samples were obtained from 17 patients with a clinical diagnosis of rheumatoid arthritis (RA) and compared with six synovial tissue samples from six patients with osteoarthritis (OA). Ang-1 expression in synovial tissues was determined by immunohistochemistry and in situ hybridisation. Ang-1 mRNA and protein expression were also examined by northern blot analysis and enzyme linked immunosorbent assay (ELISA) in cultured synovial fibroblasts and human umbilical vein endothelial cells (HUVECs) before and after treatment with tumour necrosis factor (TNF)alpha. RESULTS:Ang-1 protein expression was detected by immunohistochemistry in 16/18 RA synovial tissue samples. Ang-1 protein was frequently observed in the synovial lining layer and in cells within the sublining synovial tissue, in both perivascular areas and in areas remote from vessels. In contrast, Ang-1 was only weakly detected in these sites in OA samples. Ang-1 mRNA and protein were also expressed in cultured synovial fibroblasts derived from patients with RA. In addition, induction of Ang-1 mRNA and protein was observed by northern blot analysis and ELISA after stimulation of RA synovial fibroblasts, but not HUVECs, with the proinflammatory cytokine TNF alpha. CONCLUSIONS:Ang-1 mRNA and protein are expressed in the synovium of patients with RA, and are up regulated in synovial fibroblasts by TNF alpha. Ang-1 may therefore be an important regulator of angiogenesis in inflammatory arthritis.

Receptor activator of NF-kB ligand (RANKL) is an essential factor for osteoclast differentiation, and plays a major role in physiologic bone remodeling and in pathologic bone loss in conditions such as inflammatory arthritis and cancer. There is limited understanding of the pathways regulating transcription of the human RANKL gene. Therefore, we have performed analyses to identify transcription factors responsible for RANKL gene regulation in T cells, an important source of RANKL in several diseases associated with bone loss. Jurkat T cells, a human T lymphoma cell line, constitutively express low RANKL mRNA levels and are a useful cell system for investigating RANKL gene regulation. In these cells, RANKL mRNA was induced by stimulation with phorbol 12-myristate 13-acetate and ionomycin (P/I) or by plate-bound anti-CD3/CD28. We cloned a 1902 base pair fragment of the RANKL putative promoter, which contains a nuclear factor of activated T cells (NFAT) and an NFAT/activator protein (AP)-1 consensus element, into a luciferase reporter vector (pXP2-R1902). pXP2-R1902 luciferase activity was assessed in transient transfections of resting and activated Jurkat T cells. Inhibition of the activiation of NFATs activation was achieved by blockade with cyclosporine A (CsA), which inhibits calcineurin activation and subsequently NFAT nuclear translocation. In Jurkat T cells transfected with pXP2-R1902 we observed up to a 40-fold increase in luciferase activity above basic vector. An additional 6- to 8-fold induction of luciferase activity was seen when transfected Jurkat cells were stimulated with P/I, correlating with our mRNA observations. P/I induction of RANKL mRNA and pXP2-R1902 luciferase activity were inhibited by CsA blockade, implicating NFAT transcription factors as regulators of RANKL mRNA induction. We also examined regulation of RANKL mRNA expression by GeneChip analysis in P/I stimulated murine CD4+ T cells isolated from wild type (WT) or NFATc1 and c2 double-deficient (-/-) mice. GeneChip analysis demonstrated that RANKL mRNA expression in stimulated NFATc1-/-/c2-/- CD4+ T cells was 3.5-fold less than expression in stimulated WT CD4+ T cells. Similarly, induction of RANKL protein expression, as assessed by flow cytometry, was impaired in NFATc2-/- CD4+ T cells activated with anti-CD3/CD28 compared with activated WT CD4+ T cells. These data support the hypothesis that the NFAT family of transcription factors may be important transcriptional regulators of the RANKL gene in T cells and may provide novel therapeutic targets for inhibiting RANKL expression.

OBJECTIVETo investigate the expression of the novel Ets transcription factor ESE-1 in rheumatoid synovium and in cells derived from joint tissues, and to analyze the role of nuclear factor kappaB (NF-kappaB) as one of the central downstream targets in mediating the induction of ESE-1 by proinflammatory cytokines.METHODSESE-1 protein expression was analyzed by immunohistochemistry using antibodies in synovial tissues from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). ESE-1 messenger RNA (mRNA) levels were analyzed by reverse transcriptase-polymerase chain reaction or Northern blotting in human chondrocytes, synovial fibroblasts, osteoblasts, and macrophages, before and after exposure to interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), or lipopolysaccharide (LPS) with or without prior infection with an adenovirus encoding the inhibitor of nuclear factor kappaB (IkappaB). The wild-type ESE-1 promoter and the ESE-1 promoter mutated in the NF-kappaB site were cloned into a luciferase reporter vector and analyzed in transient transfections. Electrophoretic mobility shift assays (EMSAs) and supershift assays with antibodies against members of the NF-kappaB family were conducted using the NF-kappaB site from the ESE-1 promoter as a probe.RESULTSImmunohistochemical analysis showed specific expression of ESE-1 in cells of the synovial lining layer and in some mononuclear and endothelial cells in RA and OA synovial tissues. ESE-1 mRNA expression could be induced by IL-1beta and TNFalpha in cells such as synovial fibroblasts, chondrocytes, osteoblasts, and monocytes. Transient transfection experiments and EMSAs showed that induction of ESE-1 gene expression by IL-1beta requires activation of NF-kappaB and binding of p50 and p65 family members to the NF-kappaB site in the ESE-1 promoter. Overexpression of IkappaB using an adenoviral vector blocked IL-1beta-induced ESE-1 mRNA expression. Chromatin immunoprecipitation further confirmed that NF-kappaB binds to the ESE-1 promoter in vivo.CONCLUSIONESE-1 is expressed in synovial tissues in RA and, to a variable extent, in OA, and is specifically induced in synovial fibroblasts, chondrocytes, osteoblasts, and monocyte/macrophages by IL-1beta, TNFalpha, or LPS. This induction relies on the translocation of the NF-kappaB family members p50 and p65 to the nucleus and transactivation of the ESE-1 promoter via a high-affinity NF-kappaB binding site. ESE-1 may play a role in mediating some effects of proinflammatory stimuli in cells at sites of inflammation.

OBJECTIVE:Osteoclast differentiation factor (ODF; also known as osteoprotegerin ligand, receptor activator of nuclear factor kappaB ligand, and tumor necrosis factor-related activation-induced cytokine) is a recently described cytokine known to be critical in inducing the differentiation of cells of the monocyte/macrophage lineage into osteoclasts. The role of osteoclasts in bone erosion in rheumatoid arthritis (RA) has been demonstrated, but the exact mechanisms involved in the formation and activation of osteoclasts in RA are not known. These studies address the potential role of ODF and the bone and marrow microenvironment in the pathogenesis of osteoclast-mediated bone erosion in RA.METHODS:Tissue sections from the bone-pannus interface at sites of bone erosion were examined for the presence of osteoclast precursors by the colocalization of messenger RNA (mRNA) for tartrate-resistant acid phosphatase (TRAP) and cathepsin K in mononuclear cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to identify mRNA for ODF in synovial tissues, adherent synovial fibroblasts, and activated T lymphocytes derived from patients with RA.RESULTS:Multinucleated cells expressing both TRAP and cathepsin K mRNA were identified in bone resorption lacunae in areas of pannus invasion into bone in RA patients. In addition, mononuclear cells expressing both TRAP and cathepsin K mRNA (preosteoclasts) were identified in bone marrow in and adjacent to areas of pannus invasion in RA erosions. ODF mRNA was detected by RT-PCR in whole synovial tissues from patients with RA but not in normal synovial tissues. In addition, ODF mRNA was detected in cultured adherent synovial fibroblasts and in activated T lymphocytes derived from RA synovial tissue, which were expanded by exposure to anti-CD3.CONCLUSION:TRAP-positive, cathepsin K-positive osteoclast precursor cells are identified in areas of pannus invasion into bone in RA. ODF is expressed by both synovial fibroblasts and by activated T lymphocytes derived from synovial tissues from patients with RA. These synovial cells may contribute directly to the expansion of osteoclast precursors and to the formation and activation of osteoclasts at sites of bone erosion in RA.