David L. Boyle

c-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis

Mitogen-activated protein kinase (MAPK) cascades are involved in inflammation and tissue destruction in rheumatoid arthritis (RA). In particular, c-Jun N-terminal kinase (JNK) is highly activated in RA fibroblast-like synoviocytes and synovium. However, defining the precise function of this kinase has been difficult because a selective JNK inhibitor has not been available. We now report the use of a novel selective JNK inhibitor and JNK knockout mice to determine the function of JNK in synoviocyte biology and inflammatory arthritis. The novel JNK inhibitor SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one) completely blocked IL-1--induced accumulation of phospho-Jun and induction of c-Jun transcription in synoviocytes. Furthermore, AP-1 binding and collagenase mRNA accumulation were completely suppressed by SP600125. In contrast, complete inhibition of p38 had no effect, and ERK inhibition had only a modest effect. The essential role of JNK was confirmed in cultured synoviocytes from JNK1 knockout mice and JNK2 knockout mice, each of which had a partial defect in IL-1--induced AP-1 activation and collagenase-3 expression. Administration of SP600125 modestly decreased the rat paw swelling in rat adjuvant-induced arthritis. More striking was the near-complete inhibition of radiographic damage that was associated with decreased AP-1 activity and collagenase-3 gene expression. Therefore, JNK is a critical MAPK pathway for IL-1--induced collagenase gene expression in synoviocytes and in joint arthritis, indicating that JNK is an important therapeutic target for RA.

Prevention of cold-associated acute inflammation in familial cold autoinflammatory syndrome by interleukin-1 receptor antagonist

BACKGROUND Familial cold autoinflammatory syndrome (FCAS) is an autosomal dominant disorder characterised by recurrent episodes of rash, arthralgia, and fever after cold exposure. The genetic basis of this disease has been elucidated. Cryopyrin, the protein that is altered in FCAS, is one of the adaptor proteins that activate caspase 1, resulting in release of interleukin 1. METHODS An experimental cold challenge protocol was developed to study the acute inflammatory mechanisms occurring after a general cold exposure in FCAS patients and to investigate the effects of pretreatment with an antagonist of interleukin 1 receptor (IL-1Ra). ELISA, real-time PCR, and immunohistochemistry were used to measure cytokine responses. FINDINGS After cold challenge, untreated patients with FCAS developed rash, fever, and arthralgias within 1-4 h. Significant increases in serum concentrations of interleukin 6 and white-blood-cell counts were seen 4-8 h after cold challenge. Serum concentrations of interleukin 1 and cytokine mRNA in peripheral-blood leucocytes were not raised, but amounts of interleukin 1 protein and mRNA were high in affected skin. IL-1Ra administered before cold challenge blocked symptoms and increases in white-blood-cell counts and serum interleukin 6. INTERPRETATION The ability of IL-1Ra to prevent the clinical features and haematological and biochemical changes in patients with FCAS indicates a central role for interleukin 1beta in this disorder. Involvement of cryopyrin in activation of caspase 1 and NF-kappaB signalling suggests that it might have a role in many chronic inflammatory diseases. RELEVANCE TO PRACTICE These findings support a new therapy for a disorder with no previously known acceptable treatment. They also offer insights into the role of interleukin 1beta in more common inflammatory diseases.

Inhibitor of nuclear factor κB kinase β is a key regulator of synovial inflammation

Objective Inhibitor of nuclear factor κB kinase β (IκB kinase β, or IKKβ) has emerged as a key regulator of the transcription factor nuclear factor κB (NF-κB). Since IKKβ could have both pro- and antiinflammatory activity, we examined whether its constitutive activation was sufficient to cause a chronic inflammatory disease such as rheumatoid arthritis. Methods Normal Lewis rats were evaluated for paw swelling by plethysmometry and histologic assessment after intraarticular injection of an adenoviral construct encoding the IKKβ wild-type gene (Ad.IKKβ-wt); controls received an adenoviral construct encoding green fluorescent protein (Ad.GFP). The rats were killed after 7 days. Additionally, rats were killed 48 hours after intraarticular injection of Ad.IKKβ-wt or Ad.GFP for studies of IKK activity and NF-κB binding. For studies of the effects of inhibition of IKKβ activity, Lewis rats were immunized with Mycobacterium tuberculosis in mineral oil. The ankle joints were injected on day 12 with an adenoviral construct encoding IKKβ KM (dominant negative, IKKβ-dn) or Ad.GFP. We evaluated paw swelling and NF-κB expression on day 25. Results Intraarticular gene transfer of IKKβ-wt into the joints of normal rats resulted in significant paw swelling and histologic evidence of synovial inflammation. Increased IKK activity was detectable in the IKKβ-wt–injected ankle joints, coincident with enhanced NF-κB DNA binding activity. Intraarticular gene transfer of IKKβ-dn significantly ameliorated the severity of adjuvant arthritis, accompanied by a significant decrease in NF-κB DNA expression in the joints of Ad.IKKβ-dn–treated animals. Conclusion IKKβ plays a key role in rodent synovial inflammation. Intraarticular gene therapy to inhibit IKKβ activity represents an attractive strategy for the treatment of chronic arthritis.

Inflammasome-mediated disease animal models reveal roles for innate but not adaptive immunity.

NLRP3 nucleates the inflammasome, a protein complex responsible for cleavage of prointerleukin-1beta (IL-1beta) to its active form. Mutations in the NLRP3 gene cause the autoinflammatory disease spectrum cryopyrin-associated periodic syndromes (CAPS). The central role of IL-1beta in CAPS is supported by the response to IL-1-targeted therapy. We developed two Nlrp3 mutant knockin mouse strains to model CAPS to examine the role of other inflammatory mediators and adaptive immune responses in an innate immune-driven disease. These mice had systemic inflammation and poor growth, similar to some human CAPS patients, and demonstrated early mortality, primarily mediated by myeloid cells. Mating these mutant mice to various gene mutant backgrounds showed that the mouse disease phenotype required an intact inflammasome, was only partially dependent on IL-1beta, and was independent of T cells. These data suggest that CAPS are true inflammasome-mediated diseases and provide insight for more common inflammatory disorders.

DNA methylome signature in rheumatoid arthritis

Objectives Epigenetics can influence disease susceptibility and severity. While DNA methylation of individual genes has been explored in autoimmunity, no unbiased systematic analyses have been reported. Therefore, a genome-wide evaluation of DNA methylation loci in fibroblast-like synoviocytes (FLS) isolated from the site of disease in rheumatoid arthritis (RA) was performed. Methods Genomic DNA was isolated from six RA and five osteoarthritis (OA) FLS lines and evaluated using the Illumina HumanMethylation450 chip. Cluster analysis of data was performed and corrected using Benjamini–Hochberg adjustment for multiple comparisons. Methylation was confirmed by pyrosequencing and gene expression was determined by qPCR. Pathway analysis was performed using the Kyoto Encyclopedia of Genes and Genomes. Results RA and control FLS segregated based on DNA methylation, with 1859 differentially methylated loci. Hypomethylated loci were identified in key genes relevant to RA, such as CHI3L1, CASP1, STAT3, MAP3K5, MEFV and WISP3. Hypermethylation was also observed, including TGFBR2 and FOXO1. Hypomethylation of individual genes was associated with increased gene expression. Grouped analysis identified 207 hypermethylated or hypomethylated genes with multiple differentially methylated loci, including COL1A1, MEFV and TNF. Hypomethylation was increased in multiple pathways related to cell migration, including focal adhesion, cell adhesion, transendothelial migration and extracellular matrix interactions. Confirmatory studies with OA and normal FLS also demonstrated segregation of RA from control FLS based on methylation pattern. Conclusions Differentially methylated genes could alter FLS gene expression and contribute to the pathogenesis of RA. DNA methylation of critical genes suggests that RA FLS are imprinted and implicate epigenetic contributions to inflammatory arthritis.

Fibroblast-like synoviocytes of mesenchymal origin express functional B cell-activating factor of the TNF family in response to proinflammatory cytokines.

Immunohistochemical analysis revealed that the intimal lining cells of synovial tissue of inflamed joints of patients with rheumatoid arthritis differed from that of normal joints or of diseased joints in osteoarthritis in that they stained with mAb specific for the B cell-activating factor of the TNF family (BAFF; also called BLyS). We generated fibroblast-like synoviocytes (FLS) cell lines that were bereft of myelomonocytic cells to examine whether mesenchymal-derived FLS could express this critical B cell survival factor. We found that FLS expressed low amounts of BAFF mRNA relative to that of myelomonocytic cells. However, when various cytokines/factors were added to such FLS cell lines, we found that IFN-γ or TNF-α were unique in that they could induce significant increases in BAFF mRNA and protein. Even minute amounts of IFN-γ primed FLS for TNF-α, allowing the latter to stimulate significantly higher levels of BAFF mRNA and protein than could TNF-α alone. Consistent with this, B cells cocultured with IFN-γ and/or TNF-α-treated FLS had a significantly greater viability than B cells cocultured with nontreated FLS. The enhanced protection of B cells afforded by IFN-γ/TNF-α-treated FLS was inhibited by the addition of BAFF-R:Fc fusion protein. We conclude that the proinflammatory cytokines IFN-γ and TNF-α can induce mesenchymal-derived FLS to express functional BAFF in vitro. The induced expression of BAFF on FLS by proinflammatory cytokines may enhance the capacity of such cells to protect B cells from apoptosis in inflammatory microenvironments in vivo.

Regional analysis of p53 mutations in rheumatoid arthritis synovium.

The p53 tumor suppressor protein plays a central role in cell cycle regulation, DNA repair, and apoptosis. Recent studies indicate that DNA damage and somatic mutations in the p53 gene can occur because of genotoxic stress in many tissues, including the skin, colon, and synovium. Although somatic mutations in the p53 gene have been demonstrated in rheumatoid arthritis (RA) synovial tissue and synoviocytes, no information is available on the location or extent of p53 mutations. Using microdissected RA synovial tissue sections, we observed abundant p53 transition mutations, which are characteristic DNA damage caused by oxidative stress. p53 mutations, as well as p53 mRNA expression, were located mainly in the synovial intimal lining rather than the sublining (P < 0.01). Clusters of p53 mutant subclones were observed in some microdissected regions, suggesting oligoclonal expansion. Because IL-6 gene expression is regulated by wild-type p53, IL-6 mRNA expression in microdissected tissues was quantified by using real-time PCR. The regions with high rates of p53 mutations contained significantly greater amounts of IL-6 mRNA compared with the low mutation samples (P < 0.02). The microdissection findings suggest that p53 mutations are induced in RA synovial tissues by inflammatory oxidative stress. This process, as in sun-exposed skin and inflamed colonic epithelium, provides some of the mutant clones with a selective growth advantage. A relatively low percentage of cells containing p53 mutations can potentially affect neighboring cells and enhance inflammation through the elaboration of proinflammatory cytokines.

Expression and Regulation of Aggrecanase in Arthritis: The Role of TGF-β

Aggrecanases are key matrix-degrading enzymes that act by cleaving aggrecan at the Glu373-Ala374 site. While these fragments have been detected in osteoarthritis (OA) and rheumatoid arthritis (RA) cartilage and synovial fluid, no information is available on the regulation or expression of the two key aggrecanases (aggrecanase-1 and aggrecanase-2) in synovial tissue (ST) or fibroblast-like synoviocytes (FLS). The aggrecanase-1 gene was constitutively expressed by both RA and OA FLS. Real-time PCR demonstrated that TGF-β significantly increased aggrecanase-1 gene expression in FLS. Aggrecanase-1 induction peaked after 24 h of TGF-β stimulation. The expression of aggrecanase-1 mRNA was significantly greater in RA ST than in OA or nonarthritis ST. Aggrecanase-2 mRNA and protein were constitutively produced by nonarthritis, OA, and RA FLS but were not increased by IL-1, TNF-α, or TGF-β. Furthermore, OA, RA, and nonarthritis ST contained similar amounts of immunoreactive aggrecanase-2. The major form of the aggrecanase-2 enzyme was 70 kDa in nonarthritis ST, whereas a processed 53-kDa form was abundant in RA ST. Therefore, aggrecanase-1 and -2 are differentially regulated in FLS. Both are constitutively expressed, but aggrecanase-1 is induced by cytokines, especially TGF-β. In contrast, aggrecanase-2 protein may be regulated by a post-translational mechanism in OA and RA ST. Synovial and FLS production of aggrecanase can contribute to cartilage degradation in RA and OA.

Prostaglandins increase proMMP-1 and proMMP-3 secretion by human ciliary smooth muscle cells

PURPOSE The mechanism by which prostaglandin(PG)F2 alpha increases uveoscleral outflow and lowers intraocular pressure in primates is not known. In cultured human ciliary muscle cells, PGF2 alpha induces the expression of the protooncogene c-fos which is known to induce the transcription of genes such as matrix metalloproteinase-1 (MMP-1) and MMP-3 in other cell systems. As these enzymes are initially secreted as proenzymes, the present study was undertaken to determine if PG treatment induces ciliary muscle cells to secrete either proMMP-1 or proMMP-3. METHODS Human ciliary smooth muscle cells were grown to confluence in monolayer cell cultures and then treated with PGF2 alpha, 17-phenyltrinor-PGF2 alpha, or 11-deoxy-PGE1. Medium harvested at various times after treatment was assayed for proMMP-1 and proMMP-3 content using sandwich ELISAs. RESULTS Three days after adding 10 nM PGF2 alpha, proMMP-1 and proMMP-3, concentrations in the culture medium were increased by 254 +/- 33% (mean +/- SE) and 128 +/- 13%, respectively. Compared with vehicle controls, 24 h treatment with 200 nM PGF2 alpha, 17-phenyltrinor-PGF2 alpha, or PGE1, increased proMMP-1 by 116 +/- 29%, 169 +/- 26%, and 273 +/- 16%, respectively. In parallel experiments, proMMP-3 was increased by 99 +/- 18%, 82 +/- 24%, and 214 +/- 16%, respectively. CONCLUSIONS These results suggest that induction of MMPs in situ following topical PG treatment may degrade ciliary muscle extracellular matrix and possibly contribute to increased uveoscleral outflow, as well.

A Novel Spleen Tyrosine Kinase Inhibitor Blocks c-Jun N-Terminal Kinase-Mediated Gene Expression in Synoviocytes

Spleen tyrosine kinase (Syk) is a key regulator of cell signaling induced by cytokines or Fc receptor engagement. However, the role of Syk in rheumatoid arthritis (RA) is not known yet. We investigated the pathways activated by Syk in tumor necrosis factor-α (TNFα)-stimulated fibroblast-like synoviocytes (FLS) using the novel Syk inhibitor N4-(2,2-dimethyl-3-oxo-4H-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine (R406). Using immunohistochemistry, Syk was detected in RA synovial tissue (ST), primarily in the synovial intimal lining. Western blot analysis demonstrated significantly greater amounts of phospho-Syk expression in RA ST compared with osteoarthritis ST. The kinase was expressed and functionally activated by TNFα in FLS and was blocked by R406. Western blot analysis demonstrated that Syk inhibition by R406 markedly suppressed TNFα-induced c-Jun N-terminal kinase (JNK) phosphorylation in FLS, with a modest decrease in extracellular signal-regulated kinase phosphorylation. Surprisingly, p38 activation was not affected by R406. The Syk inhibitor also decreased TNFα-induced mitogen-activated protein kinase kinase (MKK) 4 phosphorylation but not MKK3 and MKK6 phosphorylation, which is consistent with its selective sparing of p38. The connection between Syk and JNK was confirmed by demonstrating decreased phospho-c-Jun protein expression and complete inhibition of JNK function in R406-treated cells. R406 also suppressed downstream actions of JNK, as determined by activator protein 1 binding, as well as matrix metalloproteinase 3 gene expression. These data demonstrate that Syk activation plays an essential role in TNFα-induced cytokine and matrix metalloproteinase production in RA FLS, especially by suppressing activation of the JNK pathway.