Deleting Suppressor of Cytokine Signaling-3 in chondrocytes reduces bone growth by disrupting mitogen-activated protein kinase signaling.

OBJECTIVE: To investigate the impact of deleting Suppressor of Cytokine Signaling (SOCS)-3 (SOCS3) in chondrocytes during murine skeletal development. METHOD: Mice with a conditional Socs3 allele (Socs3fl/fl) were crossed with a transgenic mouse expressing Cre recombinase under the control of the type II collagen promoter (Col2a1) to generate Socs3Δ/Δcol2 mice. Skeletal growth was analyzed over the lifespan of Socs3Δ/Δcol2 mice and controls by detailed histomorphology. Bone size and cortical bone development was evaluated by micro-computed tomography (micro-CT). Growth plate (GP) zone width, chondrocyte proliferation and apoptosis were assessed by immunofluorescence staining for Ki67 and TUNEL. Fibroblast growth factor receptor-3 (FGFR3) signaling in the GP was assessed by immunohistochemistry, while the effect of SOCS3 overexpression on FGFR3-driven pMAPK signaling in HEK293T cells was evaluated by Western blot. RESULTS: Socs3Δ/Δcol2 mice of both sexes were consistently smaller compared to littermate controls throughout life. This phenotype was due to reduced long bone size, poor cortical bone development, reduced Ki67+ proliferative chondrocytes and decreased proliferative zone (PZ) width in the GP. Expression of pMAPK, but not pSTAT3, was increased in the GPs of Socs3Δ/Δcol2 mice relative to littermate controls. Overexpression of FGFR3 in HEK293T cells increased Fibroblast Growth Factor 18 (FGF18)-dependent Mitogen-activated protein kinase (MAPK) phosphorylation, while concomitant expression of SOCS3 reduced FGFR3 expression and abrogated MAPK signaling. CONCLUSION: Our results suggest a potential role for SOCS3 in GP chondrocyte proliferation by regulating FGFR3-dependent MAPK signaling in response to FGF18.

Preclinical characterisation of the GM-CSF receptor as a therapeutic target in rheumatoid arthritis.

OBJECTIVE: Previous work has suggested that the granulocyte macrophage colony stimulating factor (GM-CSF)-GM-CSF receptor α axis (GM-CSFRα) may provide a new therapeutic target for the treatment of rheumatoid arthritis (RA). Therefore, we investigated the cellular expression of GM-CSFRα in RA synovial tissue and investigated the effects of anti-GM-CSFRα antibody treatment in vitro and in vivo in a preclinical model of RA. METHODS: We compared GM-CSFRα expression on macrophages positive for CD68 or CD163 on synovial biopsy samples from patients with RA or psoriatic arthritis (PsA) to disease controls. In addition, we studied the effects of CAM-3003, an anti-GM-CSFR antibody in a collagen induced arthritis model of RA in DBA/1 mice. The pharmacokinetic profile of CAM-3003 was studied in naïve CD1(ICR) mice (see online supplement) and used to interpret the results of the pharmacodynamic studies in BALB/c mice. RESULTS: GM-CSFRα was expressed by CD68 positive and CD163 positive macrophages in the synovium, and there was a significant increase in GM-CSFRα positive cells in patients in patients with RA as well as patients with PsA compared with patients with osteoarthritis and healthy controls. In the collagen induced arthritis model there was a dose dependent reduction of clinical arthritis scores and the number of F4/80 positive macrophages in the inflamed synovium after CAM-3003 treatment. In BALB/c mice CAM-3003 inhibited recombinant GM-CSF mediated margination of peripheral blood monocytes and neutrophils. CONCLUSIONS: The findings support the ongoing development of therapies aimed at interfering with GM-CSF or its receptor in various forms of arthritis, such as RA and PsA.

Distinctive pro-inflammatory gene signatures induced in articular chondrocytes by oncostatin M and IL-6 are regulated by Suppressor of Cytokine Signaling-3.

OBJECTIVE: To describe gene expression in murine chondrocytes stimulated with IL-6 family cytokines and the impact of deleting Suppressor of Cytokine Signaling-3 (SOCS-3) in this cell type. METHOD: Primary chondrocytes were isolated from wild type and SOCS-3-deficient (Socs3(Δ/Δcol2)) mice and stimulated with oncostatin M (OSM), IL-6 plus the soluble IL-6 receptor (IL-6/sIL-6R), IL-11 or leukemia inhibitory factor (LIF) for 4 h. Total RNA was extracted and gene expression was evaluated by microarray analysis. Validation of the microarray results was performed using Taqman probes on RNA derived from chondrocytes stimulated for 1, 2, 4 or 8 h. Gene ontology was characterized using DAVID (database for annotation, visualization and integrated discovery). RESULTS: Multiple genes, including Bcl3, Junb, Tgm1, Angptl4 and Lrg1, were upregulated in chondrocytes stimulated with each gp130 cytokine. The gene transcription profile in response to OSM stimulation was pro-inflammatory and was highly correlated to IL-6/sIL-6R, rather than IL-11 or LIF. In the absence of SOCS-3, OSM and IL-6/sIL-6R stimulation induced an interferon (IFN)-like gene signature, including expression of IL-31ra and S100a9. CONCLUSION: While each gp130 cytokine induced a transcriptional response in chondrocytes, OSM- and IL-6/sIL-6R were the most potent members of this cytokine family. SOCS-3 plays an important regulatory role in this cell type, as it does in hematopoietic cells. Our results provide new insights into a hierarchy of gp130-induced transcriptional responses in chondrocytes that is normally restrained by SOCS-3 and suggest therapeutic inhibition of OSM may have benefit over and above antagonism of IL-6 during inflammatory arthritis.

Arterial stiffness is increased in systemic sclerosis: a cross-sectional comparison with matched controls.

OBJECTIVES: Increased arterial stiffness is a predictor of cardiovascular and all-cause mortality. Atherosclerosis may be increased in systemic sclerosis (SSc). Our aims were to determine if arterial stiffness is elevated and to evaluate correlates of arterial stiffness in SSc. METHODS: We carried out two studies: 1. a comparison of arterial stiffness in 40 SSc patients free from cardiovascular disease or significant vascular manifestations of SSc and 40 healthy controls (HC), and 2. an analysis of determinants of arterial stiffness in 80 SSc patients free from cardiovascular disease. RESULTS: In Study 1, the groups were well-matched for age (52.2 vs. 50.0 years, p=0.432) and sex (80% female in both). SSc patients had higher augmentation index (AIx) than HC (31.0% [IQR 25.7-38.7] vs. 23.8% [IQR 13.5- 30.1], p<0.001). Pulse wave velocity (PWV) was also higher, however this did not reach statistical significance (6.9 m/s [IQR 6.0-8.3] vs. 6.5 m/s [IQR 6.1-7.4], p=0.275). In Study 2, age (p<0.001) and calcium channel blocker (CCB) therapy (p=0.016) were independently associated with higher AIx; and age (p<0.001), disease duration (p=0.042) and systolic blood pressure (p=0.001) with higher PWV. CONCLUSIONS: SSc patients had higher AIx than HC. The paradoxical association between CCB therapy and higher AIx could reflect generalised vasculopathy rather than atherosclerotic disease. Prospective studies in larger cohorts are warranted to clarify this point and elucidate other determinants of arterial stiffness in SSc.

Defective gp130-mediated signal transducer and activator of transcription (STAT) signaling results in degenerative joint disease, gastrointestinal ulceration, and failure of uterine implantation.

The receptor subunit gp130 transduces multiple cell type-specific activities of the leukemia inhibitory factor (LIF)/interleukin (IL)-6 family of cytokines through the signal transducer and activator of transcription (STAT) and src homology 2 domain-bearing protein tyrosine phosphatase (SHP)-2/ras/Erk pathways. To define STAT-dependent physiological responses, we generated mice with a COOH-terminal gp130(DeltaSTAT) "knock-in" mutation which deleted all STAT-binding sites. gp130(DeltaSTAT) mice phenocopyed mice deficient for IL-6 (impaired humoral and mucosal immune and hepatic acute phase responses) and LIF (failure of blastocyst implantation). However, unlike mice with null mutations in any of the components in the gp130 signaling pathway, gp130(DeltaSTAT) mice also displayed gastrointestinal ulceration and a severe joint disease with features of chronic synovitis, cartilaginous metaplasia, and degradation of the articular cartilage. Mitogenic hyperresponsiveness of synovial cells to the LIF/IL-6 family of cyto-kines was caused by sustained gp130-mediated SHP-2/ras/Erk activation due to impaired STAT-mediated induction of suppressor of cytokine signaling (SOCS) proteins which normally limits gp130 signaling. Therefore, the joint pathology in gp130(DeltaSTAT) mice is likely to arise from the disturbance of the otherwise balanced activation of the SHP-2/ras/Erk and STAT signaling cascades emanating from gp130.

Severe inflammatory arthritis and lymphadenopathy in the absence of TNF.

It has been postulated that TNF has a pivotal role in a cytokine cascade that results in joint inflammation and destruction in rheumatoid arthritis (RA). To evaluate this, we examined the response of TNF-deficient (Tnf(-/-)) mice in two models of RA. Collagen-induced arthritis (CIA) was induced by injection of chick type II collagen (CII) in CFA. Tnf(-/-) mice had some reduction in the clinical parameters of CIA and, on histology, significantly more normal joints. However, severe disease was evident in 54% of arthritic Tnf(-/-) joints. Tnf(-/-) mice had impaired Ig class switching, but preserved T cell proliferative responses to CII and enhanced IFN-gamma production. Interestingly, CII-immunized Tnf(-/-) mice developed lymphadenopathy and splenomegaly associated with increased memory CD4(+) T cells and activated lymph node B cells. Acute inflammatory arthritis was also reduced in Tnf(-/-) mice, although again some mice exhibited severe disease. We conclude that TNF is important but not essential for inflammatory arthritis; in each model, severe arthritis could proceed even in the complete absence of TNF. These results call into doubt the concept that TNF is obligatory for chronic autoimmune and acute inflammatory arthritis and provide a rationale for further studies into TNF-independent cytokine pathways in arthritis.

Distinct roles for the NF-kappaB1 (p50) and c-Rel transcription factors in inflammatory arthritis.

Rheumatoid arthritis (RA) is a complex disease, with contributions from systemic autoimmunity and local inflammation. Persistent synovial joint inflammation and invasive synovial pannus tissue lead to joint destruction. RA is characterized by the production of inflammatory mediators, many of which are regulated by the Rel/NF-kappaB transcription factors. Although an attractive target for therapeutic intervention in inflammatory diseases, Rel/NF-kappaB is involved in normal physiology, thus global inhibition could be harmful. An alternate approach is to identify and target the Rel/NF-kappaB subunits critical for components of disease. To assess this, mice with null mutations in c-rel or nfkb1 were used to examine directly the roles of c-Rel and p50 in models of acute and chronic inflammatory arthritis. We found c-Rel-deficient mice were resistant to collagen-induced arthritis but had a normal response in an acute, destructive arthritis model (methylated BSA/IL-1 induced arthritis) suggesting c-Rel is required for systemic but not local joint disease. In contrast, p50-deficient mice were refractory to induction of both the chronic and acute arthritis models, showing this subunit is essential for local joint inflammation and destruction. Our data suggest Rel/NF-kappaB subunits play distinct roles in the pathogenesis of inflammatory arthritis and may provide a rationale for more specific therapeutic blockade of Rel/NF-kappaB in RA.

Bacterial lipopolysaccharide copurifies with plasmid DNA: implications for animal models and human gene therapy.

During the course of gene therapy experiments in rodents, using intramuscular injections of plasmid DNA derived from Escherichia coli, we noted dose-related toxicity. This observation prompted a search for possible contaminants of DNA samples. We used the highly specific and sensitive limulus amoebocyte lysate assay (LAL), to monitor endotoxin bioactivity in DNA samples, and found plasmid DNA derived from standard E. coli bacterial strains, using traditional DNA isolation protocols, to be heavily contaminated with endotoxin, or lipopolysaccharide (LPA). Standard DNA isolation procedures resulted in the copurification of up to 500 micrograms/ml of LPS. LPS is a potent inducer of cytokines and other inflammatory mediators, and may complicate the use of naked DNA in gene therapy. The copurification of endotoxin with plasmid DNA also has important implications for in vitro transfection studies and microinjection of DNA into embryos. A simple and efficient protocol to reduce LPS contamination of plasmid DNA was developed. The conversion of intact bacteria to spheroplasts prior to the isolation of plasmid DNA, incubation with lysozyme, treatment with the detergent n-octyl-beta-D-thioglucopyranoside (OSPG) and polymyxin-B (PMB) chromatography, allowed the isolation of plasmid DNA containing less than 50 ng/ml LPS. This represents a 10,000-fold reduction in LPS contamination, compared to conventional methods of plasmid DNA purification, avoids potentially toxic reagents such as ethidium bromide, and produces a higher yield of plasmid DNA.

The effect of cytokines on the expression of MHC antigens and ICAM-1 by normal and transformed synoviocytes.

We report the expression on synovial cells of cell surface molecules known to be involved in T cell activation by antigen presenting cells. Normal human synovial fibroblasts and a human synovial cell line transformed with the SV40 large T antigen were used for in vitro stimulation studies with recombinant cytokines. We demonstrate an increase in MHC-A, B, C expression in normal synovial cells in response to recombinant interferon gamma (r gamma IFN), tumour necrosis factor alpha and beta (rTNF alpha and beta) and interleukin-1 (rIL-1 alpha). Intercellular adhesion molecular-1 (ICAM-1) expression was increased in parallel with MHC Class I. The combination of r gamma IFN and rTNF alpha was additive in its effect on ICAM-1 expression. Northern blot analysis suggests that ICAM-1 expression in synovial cells is controlled at the level of transcription. In contrast, MHC Class II (HLA-DR) was only significantly induced by r gamma IFN. Other stimuli including interleukin-4 (IL-4), interleukin 6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF) and prostaglandin E2 (PGE2) did not affect the expression of ICAM-1 or MHC Class I and II. Leucocyte function antigen 3 (LFA-3) expression was not affected by any of the stimuli tested. Immunoperoxidase staining of rheumatoid synovial tissue confirmed enhanced in vivo expression of ICAM-1 in rheumatoid arthritis. These changes are discussed in the context of T cell activation in inflammatory arthritis.

An unusual cause of back pain.

This case describes what may become an increasingly common clinical problem in Australia as the proportion of our population originally derived from South East Asia, ages. Our patient was of Chinese origin and presented with back pain which was eventually found to be due to metastatic disease from an otherwise silent hepatoma, in association with unrecognised chronic hepatitis B infection.

Tumour necrosis factor antagonists improve disease activity but not arterial stiffness in rheumatoid arthritis.

OBJECTIVES: Systemic inflammation may play an important role in the accelerated atherosclerosis and increased cardiovascular mortality of rheumatoid arthritis (RA). Atorvastatin reduced arterial stiffness in RA patients after only 6 weeks, an effect that may be partially mediated by the immunomodulatory effects of this drug. Suppression of inflammation with tumour necrosis factor (TNF) antagonists may therefore also improve vascular function in RA; however, TNF antagonists have also been shown to cause or exacerbate congestive heart failure in patients with RA and heart failure. The aim of the present study was to examine the effect of treatment with TNF antagonists on arterial stiffness in RA patients with active disease. METHODS: Fourteen RA patients (age 55.1 +/- 3.8 yr; disease duration 7.9 +/- 1.3 yr) with high disease activity [disease activity score (DAS28) 7.1 +/- 0.3] commencing treatment with TNF antagonists for the first time were studied. Clinical status and arterial stiffness were measured before and after 6 weeks of TNF antagonist therapy (etanercept, adalimumab or infliximab). RESULTS: Arterial stiffness did not change during the study period (the mean augmentation index was 29.1 +/- 2.2% at baseline vs 30.1 +/- 1.8% at week 6; P = 0.504). The DAS28 improved significantly from 7.1 +/- 0.3 to 4.3 +/- 0.4 (P < 0.0001). The erythrocyte sedimentation rate and C-reactive protein [median (range)] were reduced from 44 (12-85) to 15 (3-82) mm/h (P = 0.02) and from 34 (3-95) to 10 (2-61) mg/l (P = 0.007), respectively. CONCLUSIONS: Despite significant reductions in synovitis and inflammatory markers in these RA patients, arterial stiffness was not improved by 6 weeks of treatment with TNF antagonists. This result is of relevance given recent reports of potential adverse cardiovascular effects of TNF antagonists in some RA patients.

Chondrosarcoma of the calcaneum and massive soft tissue calcification in a patient with hereditary and acquired connective tissue diseases.

We describe the first case of the coexistence of the hereditary connective tissue disorder multiple exostoses (HME) and an acquired connective tissue disorder manifest by the overlap of dermatomyositis (DM), scleroderma (PSS), high titre speckled pattern antinuclear antibodies, and increased antibodies to double stranded deoxyribonucleic acid (DNA). Furthermore this patient developed chondrosarcoma of the calcaneum (an unusual site for this malignancy) and massive soft tissue calcification (an unusual feature of PSS, adult DM, and systemic lupus erythematosus (SLE)).

Collagen-induced arthritis in C57BL/6 (H-2b) mice: new insights into an important disease model of rheumatoid arthritis.

Collagen-induced arthritis (CIA) is a widely used model of rheumatoid arthritis (RA) and has been important for understanding autoimmunity. CIA is purportedly restricted to mice bearing the MHC class II H-2q or H-2r haplotypes. In this study, we re-examined established concepts regarding susceptibility to CIA. We found mice derived from the C57BU6 (B6) (H-2b) background can develop CIA with high incidence (60-70%), and sustained severity by using an immunization procedure modified for optimum response in DBA/1 (D1) (H-2q) mice. Clinically and histologically the B6 disease resembles that of D1 mice and is dependent on immunization with type II collagen, as well as on B and CD4+ T cells. In contrast, 129/Sv mice, which share H-2b, are resistant to CIA. We conclude that susceptibility to CIA may reflect immunization conditions and/or important contributions from non-MHC genes, revealed by different immunization protocols. A practical outcome is that CIA can be directly applied to gene knockout mice generated from B6 embryonic stem cells without need for backcross onto the D1 background. This model may lead to improved understanding of autoimmunity in CIA and RA and may provide a platform for analysis of the contribution of non-MHC genes to CIA.

Australian mortality statistics for rheumatoid arthritis 1950-81: analysis of death certificate data.

An analysis of mortality related to rheumatoid arthritis (RA) in Australia for the period 1950 to 1981 was undertaken based on information recorded in death certificates. These data include every death over a 32 year period where RA was considered to be the underlying cause. Death from RA was commonly reported (0.17% of all deaths). The mean age at death from RA in both sexes exceeded that of the general population for most of the period. There was little difference between patients dying of RA and the general population for age at death in the over 50 years' age group. There was a significant decrease in mortality for women dying of RA over the age of 75. RA accounted for more deaths in women than in men (in a ratio of 2.2:1). Men tended to die at a younger age from RA than did women. The impact of RA remained relatively constant in relation both to the total causes of death and to deaths due to other musculoskeletal diseases. There was a significant decline, however, in female RA deaths as a percentage of deaths due to all musculoskeletal diseases. Cohort analysis does not indicate any marked effect from extrinsic factors on mortality due to RA.

Atorvastatin reduces arterial stiffness in patients with rheumatoid arthritis.

BACKGROUND: Chronic systemic inflammation may contribute to accelerated atherosclerosis and increased arterial stiffness in patients with rheumatoid arthritis (RA). In addition to lowering cholesterol, statins have immunomodulatory effects which may be especially beneficial in patients with RA who have systemic immune activation. OBJECTIVE: To investigate the effect of atorvastatin on the augmentation index (AIx: a measure of arterial stiffness) and systemic inflammation in RA. METHODS: 29 patients with RA (mean (SD) age 55 (13) years) with moderately active disease of long duration were studied. AIx, lipid levels, serum inflammatory markers, and disease activity score were measured before and after 12 weeks of atorvastatin 20 mg daily. RESULTS: AIx improved significantly from 34.1 (11.6)% to 29.9 (11)% (p = 0.0002), with the greatest improvements in AIx occurring in those subjects with the highest disease activity scores (r = -0.5, p = 0.007). Total and LDL cholesterol were reduced from 5.5 (0.9) to 3.9 (0.7) mmol/l and 3.3 (0.8) to 1.9 (0.6) mmol/l, respectively (p = 0.0001). Serum inflammatory markers remained unchanged during the study. CONCLUSIONS: Atorvastatin significantly reduced arterial stiffness in patients with RA. The greatest improvements were seen in patients with more active disease, suggesting that, in addition to the beneficial effects of cholesterol reduction, immune modulation may contribute to the cardioprotective effect of statins.

Cushing's disease presenting with avascular necrosis of the femoral heads and complicated by pituitary apoplexy.

A case of Cushing's disease presenting with avascular necrosis of the femoral heads is described. Eighteen months after the onset of hip symptoms the patient developed pituitary apoplexy and presented to hospital as a medical emergency. Endogenous hypercortisolism is a rare and important cause of avascular necrosis of bone.

Contact allergy to gold after systemic administration of gold for rheumatoid arthritis.

A patient with seropositive, nodular rheumatoid arthritis (RA) developed contact allergy to gold jewellery following a severe skin rash which occurred after 13 weeks of treatment with sodium aurothiomalate. Patch testing confirmed that the contact allergy was due to gold. This is the first proved case of contact allergy to gold or any other compound initiated by systemic administration of the allergen.

Idiopathic retroperitoneal fibrosis presenting with back pain.

We describe 2 cases of idiopathic retroperitoneal fibrosis presenting with back pain. Additional features included weight loss, constitutional symptoms and elevated erythrocyte sedimentation rate (ESR). One patient had sacroiliitis and was HLA-B27 positive. A further finding was obstruction of the superior mesenteric artery which has not been previously described in retroperitoneal fibrosis. The importance of considering idiopathic retroperitoneal fibrosis in the differential diagnosis of back pain has not been emphasized in the rheumatological literature.

Molecular cloning of HEK, the gene encoding a receptor tyrosine kinase expressed by human lymphoid tumor cell lines.

We describe the molecular cloning of a receptor tyrosine kinase from a cell line (LK63) derived from a case of human pre-B-cell leukemia. We have previously shown that a monoclonal antibody (IIIA4) raised against LK63 recognized a glycosylated, cell-surface 135-kDa molecule (HEK), which displayed tyrosine kinase activity in vitro. The HEK protein was purified by using a IIIA4 antibody column and both N-terminal and internal amino acid sequences were obtained. A 51-mer degenerate oligonucleotide based on the internal amino acid sequence was used to screen an LK63-derived lambda gt10 cDNA library under low-stringency hybridization conditions. One clone of 2.5 kilobases (kb) was isolated and characterized and used to rescreen the library under more-stringent hybridization conditions. A 4.5-kb clone containing the entire HEK coding region was isolated and its complete DNA sequence was determined. The 4.5-kb insert was subcloned into the expression vector CDM8 and transfected into COS cells. COS cells transfected with the sense HEK/CDM8 construct stained specifically with the IIIA4 antibody, thereby confirming that the antigen recognized by the IIIA4 antibody and the expressed protein product of the HEK cDNA clone were identical. DNA sequence analysis revealed that HEK is a newly discovered member of the EPH/ELK family of receptor tyrosine kinases. Northern blot analysis of a number of cell lines demonstrated the expression of 5.5- to 6.0-kb HEK transcripts in LK63 and the T-cell lines JM and HSB-2. Southern blot analysis of DNA from LK63 suggested that the HEK gene was neither amplified nor rearranged in the LK63 tumor.