Heterogeneous requirement of IkappaB kinase 2 for inflammatory cytokine and matrix metalloproteinase production in rheumatoid arthritis: implications for therapy

OBJECTIVE: To investigate the potential role of IkappaB kinase 1 (IKK-1) and IKK-2 in the regulation of nuclear factor kappaB (NF-kappaB) activation and the expression of tumor necrosis factor alpha (TNFalpha), as well as interleukin-1beta (IL-1beta), IL-6, IL-8, vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs), in rheumatoid arthritis (RA). METHODS: Recombinant adenoviruses expressing beta-galactosidase, dominant-negative IKK-1 and IKK-2, or IkappaBalpha were used to infect ex vivo RA synovial membrane cultures and synovial fibroblasts obtained from patients with RA undergoing joint replacement surgery, or human dermal fibroblasts, human umbilical vein endothelial cells (HUVECs), and monocyte-derived macrophages from healthy volunteers. Then, their effect on the spontaneous or stimulus-induced release of inflammatory cytokines, VEGF, and MMPs from RA synovial membrane cells was examined. RESULTS: IKK-2 was not required for lipopolysaccharide (LPS)-induced NF-kappaB activation or TNFalpha, IL-6, or IL-8 production in macrophages, but was essential for this process in response to CD40 ligand, TNFalpha, and IL-1. In synovial fibroblasts, dermal fibroblasts, and HUVECs, IKK-2 was also required for LPS-induced NF-kappaB activation and IL-6 or IL-8 production. In RA synovial membrane cells, IKK-2 inhibition had no effect on spontaneous TNFalpha production but significantly reduced IL-1beta, IL-6, IL-8, VEGF, and MMPs 1, 2, 3, and 13. CONCLUSION: Our study demonstrates that IKK-2 is not essential for TNFalpha production in RA. However, because IKK-2 regulates the expression of other inflammatory cytokines (IL-1beta, IL-6, and IL-8), VEGF, and MMPs 1, 2, 3, and 13, which are involved in the inflammatory, angiogenic, and destructive processes in the RA joint, it may still be a good therapeutic target.     

Interleukin-18 enhances monocyte tumor necrosis factor alpha and interleukin-1beta production induced by direct contact with T lymphocytes: implications in rheumatoid arthritis

OBJECTIVE: At sites of inflammation, T cells exert pathologic effects through direct contact with monocyte/macrophages, inducing massive up-regulation of interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFalpha). We examined the regulatory effects of IL-18 on monocyte activation by direct contact with T lymphocytes in rheumatoid arthritis (RA). METHODS: Activated T cells were isolated from RA synovial fluid. Resting T cells and monocytes were isolated from peripheral blood mononuclear cells. RA synovial T cells or phytohemagglutinin (PHA)-stimulated T cells were fixed by paraformaldehyde and then cocultured with monocytes at a ratio of 4:1. Levels of TNFalpha, IL-1beta, IL-10, and IL-18 were measured by enzyme-linked immunosorbent assay. Expression of adhesion molecules, IL-18 receptor, and TNF receptors was analyzed by flow cytometry. Expression of NF-kappaB p65, phosphorylated IkappaBalpha, and phosphatidylinositol 3-kinase (PI 3-kinase) p110 was analyzed by Western blotting. RESULTS: IL-18 dose-dependently enhanced the production of IL-1beta and TNFalpha, but not IL-10, by monocytes following contact with RA synovial T cells or PHA-prestimulated T cells. NF-kappaB inhibitors N-acetyl-L-cysteine and Bay 11-7085 and PI 3-kinase inhibitor LY294002 inhibited the enhancing effects of IL-18, but MAPK p38 inhibitor SB203580, ERK inhibitor PD98059, and JNK inhibitor SP600125 did not. Increased levels of NF-kappaB in the nucleus, phosphorylated IkappaB, and PI 3-kinase were confirmed in monocytes cocultured with PHA-prestimulated T cells, and the levels were further increased by stimulation with IL-18. Neutralizing antibody to IL-18 inhibited monocyte activation induced by direct contact with PHA-prestimulated T cells. Via cell-cell contact, PHA-prestimulated T cells increased autocrine production of IL-18 by monocytes, which was mediated by activation of the NF-kappaB and PI 3-kinase pathways, and up-regulated the expression of the IL-18 receptor in monocytes. IL-18 up-regulated the expression of the TNF receptors vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) on monocytes. Blocking the binding of the TNF receptors VCAM-1 or ICAM-1 on monocytes to their ligands on stimulated T cells suppressed the IL-18-enhanced production of TNFalpha and IL-1beta in monocytes induced by contact with PHA-prestimulated T cells. CONCLUSION: IL-18 augments monocyte activation induced by contact with activated T cells in RA synovitis, which is dependent on activation of the NF-kappaB and PI 3-kinase pathways. IL-18 up-regulates the expression of the TNF receptors VCAM-1 and ICAM-1 on monocytes, which mediate the enhancing effects of IL-18 on T cell-monocyte contact.     

Galectin 3 and its binding protein in rheumatoid arthritis

OBJECTIVE: To characterize the expression pattern and role of galectin 3 and galectin 3 binding protein (G3BP) in rheumatoid arthritis (RA), in comparison with galectin 1, and to explore whether soluble galectin 3 and G3BP, investigated in serum, synovial fluid, or cell culture supernatant, are associated with disease. METHODS: Synovial tissues from patients with RA or osteoarthritis (OA), as well as from healthy controls, were analyzed for galectins 1 and 3 and G3BP by in situ hybridization and immunohistochemistry. Levels of galectin 3 and G3BP in serum and synovial fluid from patients with RA and OA and controls, as well as in cell culture supernatants, were determined by enzyme-linked immunosorbent assay (ELISA). In vitro, the intracellular expression of galectin 3 in RA and OA synovial fibroblasts after modulation with tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), and anti-CD40 monoclonal antibodies was measured by flow cytometry. RESULTS: In RA, galectin 3 messenger RNA and protein stained throughout the synovial membrane, whereas G3BP was particularly expressed at sites of bone destruction. In contrast, the expression of galectin 1 was not uniform in different RA specimens, and was never found at sites of invasion. In OA and normal synovial tissues, only a small number of cells were positive for galectins and/or G3BP. Galectin 3 was elevated in RA sera and synovial fluids, whereas G3BP was increased in RA synovial fluids only. In RA, serum galectin 3 correlated with C-reactive protein levels, whereas G3BP was associated with joint destruction and/or synovial cell activation as measured by the levels of cartilage oligomeric matrix protein. In vitro, RA synovial fibroblasts showed an increased release of galectin 3 into culture medium, as measured by ELISA, but decreased secretion of G3BP. In RA synovial fibroblasts with low basal expression of galectin 3, TNFalpha increased its intracellular level in a dose-dependent manner. In contrast, IL-1beta or anti-CD40 monoclonal antibodies showed no effect. CONCLUSION: Our data indicate that galectin 3 and G3BP are not only involved in inflammation, but also contribute to the activation of synovial fibroblasts. The intracellular accumulation of galectin 3 can be enhanced by TNFalpha. Thus, galectin 3 and G3BP represent novel markers of disease activity in RA.     

NF-kappaB-regulated expression of cellular FLIP protects rheumatoid arthritis synovial fibroblasts from tumor necrosis factor alpha-mediated apoptosis

OBJECTIVE: Little apoptosis has been observed in rheumatoid arthritis (RA) synovial tissues. Tumor necrosis factor alpha (TNFalpha) is expressed in the joints of patients with RA, yet RA synovial fibroblasts are relatively resistant to apoptosis induced by TNFalpha. Recently, we demonstrated that FLIP is highly expressed in the RA joint. These studies were performed to determine if TNFalpha-induced NF-kappaB controls the expression of FLIP long (FLIP(L)) and FLIP short (FLIP(S)) in RA synovial fibroblasts and to determine the role of FLIP in the control of TNFalpha-induced apoptosis. METHODS: RA synovial fibroblasts were isolated from RA synovial tissues and used between passages 3 and 9. RA synovial or control fibroblasts were sham infected or infected with a control adenovirus vector or one expressing the super-repressor IkappaBalpha (srIkappaBalpha). The cells were stimulated with TNFalpha or a control vehicle, and expression of FLIP(L) and FLIP(S) was determined by isoform-specific real-time polymerase chain reaction and Western blot analysis. Cell viability was determined by XTT cleavage, and apoptosis was determined by annexin V staining, DNA fragmentation, and activation of caspases 8 and 3. RESULTS: TNFalpha induced the expression of both isoforms of FLIP messenger RNA (mRNA) in RA synovial fibroblasts; however, FLIP(L) was the dominant isoform detected by Western blot analysis. In control fibroblasts, TNFalpha induced the expression of FLIP(L) and FLIP(S) mRNA and protein. The TNFalpha-induced, but not the basal, expression of FLIP was regulated by NF-kappaB. When NF-kappaB activation was suppressed by the expression of srIkappaBalpha, TNFalpha-mediated apoptosis was induced. TNFalpha-induced apoptotic cell death was mediated by caspase 8 activation and was prevented by the ectopic expression of FLIP(L) or the caspase 8 inhibitor CrmA. CONCLUSION: The TNFalpha-induced, but not the basal, expression of FLIP is regulated by NF-kappaB in RA synovial fibroblasts. The resistance of RA synovial fibroblasts to TNFalpha-induced apoptosis is mediated by the NF-kappaB-regulated expression of FLIP. These observations support the role of NF-kappaB and FLIP as attractive therapeutic targets in RA.     

Importance of NF-κB in rheumatoid synovial tissues: in situ NF-κB expression and in vitro study using cultured synovial cells

OBJECTIVES: To examine whether inhibition of NF-kappaB induces apoptosis of human synovial cells stimulated by tumour necrosis factor alpha (TNFalpha), interleukin 1beta (IL1beta), and anti-Fas monoclonal antibody (mAb). METHODS: The expression of proliferating cell nuclear antigen (PCNA), NF-kappaB, and the presence of apoptotic synovial cells were determined in synovial tissues. Apoptosis of cultured synovial cells was induced by inhibition of NF-kappaB nuclear translocation by Z-Leu-Leu-Leu-aldehyde (LLL-CHO). The activation of caspase-3 and expression of XIAP and cIAP2 in synovial cells in LLL-CHO induced apoptosis was also examined. RESULTS: Abundant PCNA+ synovial cells were found in rheumatoid arthritis (RA) synovial tissue, though a few apoptotic synovial cells were also detected in the RA synovial tissues. Nuclear NF-kappaB was expressed in RA synovial cells. Electrophoretic mobility shift assay showed that treatment of cells with TNFalpha or IL1beta significantly stimulated nuclear NF-kappaB activity. A small number of apoptotic synovial cells expressing intracellular active caspase-3 were found after treatment of cells with LLL-CHO. Although treatment of RA synovial cells with TNFalpha or IL1beta alone did not induce apoptosis, apoptosis induced by LLL-CHO and caspase-3 activation were clearly enhanced in TNFalpha or IL1beta stimulated synovial cells compared with unstimulated synovial cells. Furthermore, induction of apoptosis of synovial cells with caspase-3 activation by anti-Fas mAb was clearly increased by LLL-CHO. The expression of cIAP2 and XIAP in synovial cells may not directly influence the sensitivity of synovial cells to apoptosis induced by LLL-CHO. CONCLUSION: The results suggest that NF-kappaB inhibition may be a potentially important therapeutic approach for RA by correcting the imbalance between apoptosis and proliferation of synovial cells in RA synovial tissue.     

Expression of interleukin-21 receptor, but not interleukin-21, in synovial fibroblasts and synovial macrophages of patients with rheumatoid arthritis

OBJECTIVE: To determine the role and expression of the cytokine/receptor pair interleukin-21 (IL-21)/IL-21 receptor (IL-21R) in rheumatoid arthritis (RA). METHODS: The expression of IL-21R and IL-21 was analyzed by TaqMan real-time polymerase chain reaction (PCR) and in situ hybridization of synovial biopsy samples from patients with RA and osteoarthritis (OA). Double labeling by immunohistochemistry after in situ hybridization was performed with anti-CD68 antibodies. The expression of IL-21R at the protein level was confirmed by Western blotting. Stimulation experiments were performed with recombinant IL-1beta, tumor necrosis factor alpha (TNFalpha), platelet-derived growth factor (PDGF), and transforming growth factor beta (TGFbeta). The role of IL-21R in cartilage destruction was analyzed in the SCID mouse coimplantation model of RA. RESULTS: IL-21R was found in total RNA extracts and in synovial biopsy samples from RA patients, whereas no expression or only minimal expression was seen in samples from OA patients. Double labeling indicated that both synovial macrophages and synovial fibroblasts expressed IL-21R. Western blotting with anti-IL-21R antibodies confirmed the expression of IL-21R protein in RA synovial fibroblasts (RASFs). Of note, IL-21 was not detectable by real-time PCR and in situ hybridization in the same samples in vivo as in vitro. The level of expression of IL-21R messenger RNA (mRNA) was not altered by stimulation with IL-1beta, TNFalpha, PDGF, or TGFbeta. Interestingly, in the SCID mouse coimplantation model, RASFs did not maintain their expression of IL-21R at sites of invasion into the cartilage. Similarly, IL-21R mRNA was not expressed at sites of invasion into cartilage and bone in RA synovium. CONCLUSION: Our data demonstrate that IL-21R is expressed in RA synovium by RASFs and synovial macrophages. IL-21R is associated with the activated phenotype of RASFs independently of the major proinflammatory cytokines IL-1beta and TNFalpha, but correlates negatively with the destruction of articular cartilage and bone.     

Anti-interleukin-6 receptor antibody therapy reduces vascular endothelial growth factor production in rheumatoid arthritis

OBJECTIVE: To investigate whether interleukin-6 (IL-6) is a regulator of vascular endothelial growth factor (VEGF) in rheumatoid arthritis (RA). METHODS: Serum VEGF levels in RA patients were assayed before and after 8 weeks or 24 weeks of maintenance therapy with humanized anti-IL-6 receptor monoclonal antibody (anti-IL-6R mAb). VEGF secreted by RA synovial fibroblasts cultured in the presence of IL-6, IL-1beta, and/or tumor necrosis factor alpha (TNFalpha) was measured. The inhibitory effect of anti-IL-6R mAb, recombinant IL-1 receptor antagonist (IL-1Ra), and anti-TNFalpha mAb on VEGF production was also examined. RESULTS: Serum VEGF levels in RA patients before anti-IL-6R mAb therapy were significantly higher than those in healthy controls (P < 0.0005). Treatment of RA patients with anti-IL-6R mAb normalized serum VEGF levels. In the in vitro study, IL-6 and IL-1beta each induced a slight amount of VEGF production in synovial cells, but TNFalpha did not. Although VEGF-inducing activity of these cytokines was not remarkable when they were added alone, IL-6 acted synergistically with IL-1beta or TNFalpha to induce VEGF production. There was no synergistic effect between IL-1beta and TNFalpha. In the presence of all of these cytokines, anti-IL-6R mAb eliminated the synergistic effect of IL-6, IL-1beta, and TNFalpha, while IL-1Ra or anti-TNFalpha mAb did not. CONCLUSION: Anti-IL-6R mAb therapy reduced VEGF production in RA. IL-6 is the pivotal cytokine that induces VEGF production in synergy with IL-1beta or TNFalpha, and this may be the mechanism by which IL-6 blockade effectively suppresses VEGF production in synovial fibroblasts.     

Association of genotypes affecting the expression of interleukin-1beta or interleukin-1 receptor antagonist with osteoarthritis

OBJECTIVE: The majority of cytokines and growth factors known to be involved in cartilage metabolism are synthesized by the chondrocytes themselves. They are up-regulated in osteoarthritic (OA) cartilage, resulting in 2 opposite phenotypes, TNFalpha(high) and TNFalpha(low), that are characterized by an elevated number of tumor necrosis factor alpha (TNFalpha)-positive and interleukin-1beta (IL-1beta)-positive chondrocytes, respectively. To establish a hierarchy among the cytokines and growth factors expressed in articular chondrocytes, this study investigated cytokine genes for known polymorphisms that may contribute to the deregulated expression in OA cartilage. METHODS: Polymerase chain reaction techniques were performed either in a thermal cycler using standard methods or in a light cycler to analyze the frequencies of the TNFalpha (-308), IL-1 receptor antagonist (IL-1Ra) (intron 2), IL-1beta (exon 5), and IL-6 (-174) polymorphisms in 61 OA patients and 254 randomly chosen controls. RESULTS: For the TNFalpha(low) phenotype, a statistically significant association was found with the less frequent allele of IL-1beta, which carries a single-basepair substitution in exon 5 and may contribute to the characteristic increase in IL-beta-positive chondrocytes. In contrast, the TNFalpha(high) phenotype was significantly associated with the less frequent allele of IL-1Ra, which carries two 86-bp repeats in the second intron and is assumed to lead to an elevated expression of the antagonist. CONCLUSION: These results point to an association between the IL-1beta polymorphism and the TNFalpha(high) phenotype and between the IL-1Ra polymorphism and the TNFalpha(low) phenotype found in OA. Both associations suggest that IL-1beta may be more important than TNFalpha for the regulation of cytokine and growth factor expression in articular chondrocytes.