Urinary B-cell-activating factor of the tumour necrosis factor family (BAFF) in systemic lupus erythematosus.

INTRODUCTION: We examined the clinical relevance of urinary concentrations of B-cell-activating factor of the tumour necrosis factor family (BAFF) and a proliferation-inducing ligand (APRIL) in systemic lupus erythematosus (SLE). METHODS: We quantified urinary BAFF (uBAFF) by enzyme-linked immunosorbent assay in 85 SLE, 28 primary Sjögren syndrome (pSS), 40 immunoglobulin A nephropathy (IgAN) patients and 36 healthy controls (HCs). Urinary APRIL (uAPRIL) and monocyte chemoattractant protein 1 (uMCP-1) were also quantified. Overall and renal SLE disease activity were assessed using the Systemic Lupus Erythematosus Disease Activity Index 2000. RESULTS: uBAFF was detected in 12% (10/85) of SLE patients, but was undetectable in HCs, IgAN and pSS patients. uBAFF was detectable in 28% (5/18) of SLE patients with active nephritis vs 5/67 (7%) of those without ( p = 0.03), and uBAFF was significantly higher in active renal patients ( p = 0.02) and more likely to be detected in patients with persistently active renal disease. In comparison, uAPRIL and uMCP-1 were detected in 32% (25/77) and 46% (22/48) of SLE patients, respectively. While no difference in proportion of samples with detectable uAPRIL was observed between SLE, HCs and IgAN patients, both uAPRIL and uMCP-1 were significantly detectable in higher proportions of patients with active renal disease. CONCLUSIONS: uBAFF was detectable in a small but a significant proportion of SLE patients but not in other groups tested, and was higher in SLE patients with active renal disease.

Glucocorticoid-induced leucine zipper governs the therapeutic potential of mesenchymal stem cells by inducing a switch from pathogenic to regulatory Th17 cells in a mouse model of collagen-induced arthritis.

OBJECTIVE: Mesenchymal stem cells (MSCs) are potent immunosuppressive cells that have shown promise in the treatment of rheumatoid arthritis (RA). Deciphering the intrinsic characteristics of MSCs that correlate with their biologic activity will facilitate their clinical use. Recently, the role of glucocorticoid-induced leucine zipper (GILZ) in the development of RA has been documented. The aim of this study was to evaluate whether GILZ expression by MSCs may contribute to their therapeutic effect. METHODS: MSCs were isolated from GILZ-deficient (GILZ(-/-) ) mice and wild-type mice. MSCs (1 × 10(6) cells) were injected twice via the tail vein into mice with collagen-induced arthritis (CIA). RESULTS: In vitro, we showed that GILZ is a key factor involved in the immunosuppressive potential of MSCs. MSCs derived from GILZ(-/-) mice did not suppress the proliferation of CD4+ T cells and were less efficient than MSCs derived from WT mice in altering Th17 cell polarization. Thus, we investigated the role of GILZ in an experimental model of arthritis and demonstrated that although WT MSCs significantly reduced paw swelling in arthritic mice, GILZ(-/-) MSCs did not. Moreover, the magnitude of the effects of GILZ(-/-) MSCs on Th17 cell frequency was significantly lower than that of WT MSCs. The therapeutic effect of MSCs correlated with the generation of Treg cells bearing the CD4 + RORγt+IL-17(low) IL-10+ signature, and Th17 cell polarization was GILZ dependent. CONCLUSION: This study demonstrates that GILZ has an essential role in the therapeutic effectiveness of MSCs in arthritis by favoring Th17 cell polarization toward a regulatory phenotype. Therefore, potentiation of GILZ expression in MSCs could represent a means to enhance their therapeutic effect in autoimmune diseases.

Association of serum B cell activating factor from the tumour necrosis factor family (BAFF) and a proliferation-inducing ligand (APRIL) with central nervous system and renal disease in systemic lupus erythematosus.

INTRODUCTION: The objective of this study is to determine whether serum concentrations of B cell activating factor from the tumour necrosis factor family (BAFF) and/or a proliferation-inducing ligand (APRIL) are associated with clinical manifestations of systemic lupus erythematosus (SLE). METHODS: BAFF and APRIL concentrations were quantified using a commercial ELISA in serum samples obtained at the time of clinical assessment in 98 patients, and on 245 samples from 75 of these patients followed prospectively. RESULTS: Serum BAFF was significantly increased, and APRIL decreased, in patients with either renal or central nervous system (CNS) lupus. In contrast, in cross-sectional analysis, there was no correlation between disease activity (SLEDAI-2k) and serum BAFF or APRIL. In longitudinal follow-up, there was no association between changes in serum BAFF or APRIL and changes in SLEDAI-2k, or between baseline serum BAFF or APRIL and subsequent changes in SLEDAI-2k. However, between-visit changes in BAFF were significantly different in patients with increases in SLEDAI-2k ≥ 4, compared to patients whose SLEDAI-2k did not change. CONCLUSIONS: Although neither serum BAFF nor APRIL correlated with disease activity in the overall population, elevated serum BAFF and reduced APRIL may be markers of renal and CNS disease in SLE patients.

Identification of a novel cell type-specific intronic enhancer of macrophage migration inhibitory factor (MIF) and its regulation by mithramycin.

The aim of this study was to determine the genetic regulation of macrophage migration inhibitory factor (MIF). DNase I hypersensitivity was used to identify potential hypersensitive sites (HS) across the MIF gene locus. Reporter gene assays were performed in different human cell lines with constructs containing the native or mutated HS element. Following phylogenetic and transcription factor binding profiling, electrophoretic mobility shift assay (EMSA) and RNA interference were performed and the effects of incubation with mithramycin, an antibiotic that binds GC boxes, were also studied. An HS centred on the first intron of MIF was identified. The HS acted as an enhancer in human T lymphoblasts (CEMC7A), human embryonic kidney cells (HEK293T) and human monocytic cells (THP-1), but not in a fibroblast-like synoviocyte (FLS) cell line (SW982) or cultured FLS derived from rheumatoid arthritis (RA) patients. Two cis-elements within the first intron were found to be responsible for the enhancer activity. Mutation of the consensus Sp1 GC box on each cis-element abrogated enhancer activity and EMSA indicated Sp1 binding to one of the cis-elements contained in the intron. SiRNA knock-down of Sp1 alone or Sp1 and Sp3 together was incomplete and did not alter the enhancer activity. Mithramycin inhibited expression of MIF in CEMC7A cells. This effect was specific to the intronic enhancer and was not seen on the MIF promoter. These results identify a novel, cell type-specific enhancer of MIF. The enhancer appears to be driven by Sp1 or related Sp family members and is highly sensitive to inhibition via mithramycin.

Endogenous estrogen regulation of inflammatory arthritis and cytokine expression in male mice, predominantly via estrogen receptor alpha.

OBJECTIVE: A number of experimental observations have associated elevated estrogen levels with amelioration of inflammation. The involvement of estrogen and estrogen receptor (ER) isotypes in the regulation of inflammation in males is not well understood. In this study, we used specific ERalpha and ERbeta agonists in male mice deficient in estrogen because of a deletion of aromatase (aromatase-knockout [ArKO] mice) to investigate ER isotype utilization in estrogen regulation of inflammation. METHODS: Lipopolysaccharide (LPS)-induced cytokine expression and antigen-induced arthritis (AIA) were investigated in male ArKO and WT littermate mice, as well as in response to selective agonists of ERalpha (16alpha-LE2) and ERbeta (8beta-VE2). The therapeutic effect of selective ER agonists was also examined in mice with collagen-induced arthritis (CIA). RESULTS: Estrogen deficiency in ArKO mice was associated with significant increases in LPS-induced serum interleukin-6 (IL-6), tumor necrosis factor, monocyte chemotactic protein 1, and interferon-gamma levels, which were significantly abrogated by administration of 16alpha-LE2, but not 8beta-VE2. In contrast, both 16alpha-LE2 and 8beta-VE2 significantly increased LPS-induced IL-10 levels. Estrogen deficiency was also associated with significant exacerbation of AIA and antigen-specific T cell proliferation, which was reversed by administration of either 16alpha-LE2 or 8beta-VE2. ArKO mice showed increased antigen-specific T cell proliferation in response to immunization with type II collagen (CII). Administration of 16alpha-LE2, but not 8beta-VE2, significantly reduced the severity of CIA, which was associated with inhibition of anti-CII-specific IgG. CONCLUSION: These data indicate that endogenous estrogen plays an essential inhibitory role in inflammation in male mice and that ERalpha is the dominant receptor that mediates these effects.

The tumour suppressor gene p53 modulates the severity of antigen-induced arthritis and the systemic immune response.

p53 is a transcription factor with a well-described role in the induction of apoptosis and cell cycle arrest as part of a protective response to a variety of stressful stimuli. Expansion of inflamed tissue in rheumatoid arthritis has been related to the loss of functioning p53, and the severity of collagen-induced arthritis is increased in p53-/- mice. Our objective was to assess the role of p53 in a model of adaptive immunity, antigen-induced arthritis (AIA). AIA was induced in p53-/- and wild-type mice by priming with methylated bovine serum albumin followed by intra-articular challenge. Severity of arthritis was assessed using a standardized scoring system and synovial apoptosis was detected by TdT-mediated biotin-dUTP nick-end labelling. Splenocyte proliferation was measured by [H(3)] incorporation and interferon (IFN)-gamma release. Splenocyte viability was assessed using Titreglow. Splenic T cell activation status was assessed by flow cytometry. Serum cytokines were measured using enzyme-linked immunosorbent assay. Increased severity of AIA in p53-/- mice was associated with decreased synovial apoptosis and with increased delayed-type hypersensitivity response, increased mitogen and antigen-induced splenocyte proliferation and increased IFN-gamma release in p53-/- mice compared with wild-type mice. Antigen-specific immunoglobulin responses were equivalent in both groups. Splenocyte viability was increased in p53-/- mice but T cell apoptosis was equivalent. T cell activation markers were increased in p53-/- mice compared with wild-type mice. Lipopolysaccharide-induced tumour necrosis factor release was increased in p53-/- mice with a trend to increased interleukin-6 in p53-/- mice compared with littermates. p53 is involved in the modulation of adaptive and innate immune responses relevant to arthritis models and is also involved in the modulation of severity of AIA by both cell-cycle dependent and cell-cycle-independent mechanisms.

New therapeutic target in inflammatory disease: macrophage migration inhibitory factor.

The cytokine macrophage migration inhibitory factor (MIF) participates in fundamental events in innate and adaptive immunity. The profile of activities of MIF in vivo and in vitro is strongly suggestive of a role for MIF in the pathogenesis of many inflammatory diseases, including rheumatoid arthritis (RA), and hence antagonism of MIF is suggested as a potential therapeutic strategy in inflammatory disease. The best developed case for therapeutic antagonism of MIF is in RA. In RA, MIF is abundantly expressed in serum and synovial tissue. MIF induces synovial expression of key pro-inflammatory genes, regulates the function of endothelial cells and leucocytes, and is implicated in the control of synoviocyte proliferation and apoptosis via direct effects on the expression of the tumour suppressor protein p53. In animal models of RA, anti-MIF antibodies or genetic MIF deficiency are associated with significant inhibition of disease. A similar case has been made, for example using MIF-deficient mice, in models of atheroma, colitis, multiple sclerosis and other inflammatory diseases. The relationship with p53 also means MIF may be important in the link between inflammatory disease and cancer, such as is seen in RA or colitis. MIF also has a unique relationship with glucocorticoids, in that despite antagonizing their effects, the expression of MIF is in fact induced by glucocorticoids. Thus, MIF functions as a physiological counter-regulator of the anti-inflammatory effects of glucocorticoids. This may be entrained by selective activation of mitogen-activated protein kinases rather than nuclear factor kappa B. Therapeutic MIF antagonism may therefore provide a specific means of 'steroid sparing'. Exploitation of antibody, soluble receptor or small molecule technologies may soon lead to the ability to test in the clinic the importance of MIF in human inflammatory diseases.

Macrophage migration inhibitory factor in rheumatoid arthritis: clinical correlations.

OBJECTIVE: Cytokines play an important role in the pathology of rheumatoid arthritis (RA). Macrophage migration inhibitory factor (MIF) is a cytokine with a broad spectrum of actions, including induction of monocyte tumour necrosis factor alpha (TNF-alpha). Evidence of the expression and proinflammatory activity of MIF has recently been demonstrated in RA synovium and in animal models of RA. We wished to assess the relationship between MIF expression in synovium and clinical disease. METHODS: Computer-assisted analysis of the cytokine content of arthroscopically obtained biopsies of RA synovium, using paired samples from eight patients with active and inactive/treated disease, was compared with documented clinical parameters. RESULTS: Synovial MIF immunostaining correlated strongly with disease activity as measured by CRP concentration. Reductions in clinical disease parameters, including CRP, tender and swollen joint counts, were accompanied by significant reductions in synovial MIF. Synovial TNF-alpha, transforming growth factor beta (TGF-beta) and interleukin (IL) 10 also showed a significant reduction in association with reduced disease activity, while IL-1 beta and IL-1 receptor agonist did not. CONCLUSION: The correlation of synovial MIF with disease activity corroborates existing evidence of the role of this cytokine in RA. The demonstration that only MIF and TNF-alpha show significant variation in synovial cytokine content with clinical remission suggests that MIF is an important member of the cytokine hierarchy in RA.

Regulation of synoviocyte phospholipase A2 and cyclooxygenase 2 by macrophage migration inhibitory factor.

OBJECTIVE: Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with known actions in macrophage and T cell activation. MIF also has the unique capacity to reverse the inhibitory effects of glucocorticoids on these cells. We have recently demonstrated MIF expression in human rheumatoid arthritis (RA) synovium and cultured fibroblast-like synoviocytes (FLS), as well as the ability of FLS-derived MIF to induce monocyte release of tumor necrosis factor alpha. We investigated the effects of MIF on aspects of RA FLS activation, including the induction of phospholipase A2 (PLA2) and cyclooxygenase (COX). METHODS: PLA2 activity was measured by 3H-arachidonic acid released from treated FLS supernatants. COX activity was measured by prostaglandin E2 enzyme-linked immunosorbent assay. Cytosolic PLA2 (cPLA2) and COX-2 messenger RNA (mRNA) were determined using semiquantitative reverse transcriptase-polymerase chain reaction. RESULTS: Constitutive PLA2 activity was detected in RA FLS. Recombinant human MIF up-regulated PLA2 activity (P < 0.01) and cPLA2 mRNA expression, but had no effect on secretory PLA2. Recombinant human MIF up-regulated COX activity (P < 0.05) and COX-2 mRNA, but had no observable effect on COX-1. Interleukin-1beta (IL-1beta) significantly up-regulated PLA2 activity (P < 0.005) and cPLA2 mRNA expression while anti-MIF monoclonal antibody (mAb) significantly inhibited this IL-1beta-induced PLA2 activity (P < 0.02). Anti-MIF mAb significantly reduced IL-1beta-induced COX activity (P < 0.05) and COX-2 mRNA expression. CONCLUSION: MIF exerts a proinflammatory effect on key aspects of RA FLS activation. That anti-MIF mAb inhibited IL-1beta up-regulation of FLS indicates an additional cofactor role for MIF in IL-1beta-induced FLS activation. These data suggest that MIF antagonism has important therapeutic potential in RA.

Annexin I surface binding sites and their regulation on human fibroblast-like synoviocytes.

OBJECTIVE: Annexin I is a glucocorticoid-inducible protein whose expression in rheumatoid synovium and inhibitory actions in animal models of arthritis suggests its involvement in human arthritis. The present study explored the potential for annexin I to mediate its antiinflammatory actions via specific cell-surface binding sites on human fibroblast-like synoviocytes (FLS). METHODS: Annexin I binding sites on cultured FLS from patients with osteoarthritis (OA) and rheumatoid arthritis (RA) were determined by ligand-binding flow cytometry. Phospholipase A2 (PLA2) activity was determined by arachidonic acid release. RESULTS: FLS exhibited saturable, concentration-dependent cell-surface annexin I binding, with >99% of the OA FLS exhibiting binding at an annexin I concentration of 10 microM. Annexin I binding of RA FLS was significantly lower than that of OA FLS. FLS annexin I binding sites were not affected by elastase or a specific elastase inhibitor, and elastase release did not differ between RA and OA cells. In contrast, collagenase significantly increased annexin I binding sites on OA FLS and approached a significant effect on RA FLS. Tumor necrosis factor alpha increased annexin I binding sites on OA and RA FLS. Similarly, interleukin-1beta significantly increased annexin I binding on OA FLS; but the increased binding on RA FLS was not significant. Dexamethasone exerted no significant effect on OA or RA FLS annexin I binding sites. Treatment of RA FLS with an annexin I N-terminal peptide significantly inhibited RA FLS PLA2 activity. CONCLUSION: This is the first description of the expression, regulation, and function of cell surface annexin I binding sites on FLS. Reduced annexin I binding sites in RA FLS may impair the sensitivity of certain proinflammatory events to glucocorticoids.

Regulation of macrophage migration inhibitory factor by endogenous glucocorticoids in rat adjuvant-induced arthritis.

OBJECTIVE: To explore the regulation of macrophage migration inhibitory factor (MIF) by endogenous glucocorticoids in adjuvant-induced arthritis (AIA). METHODS: Adrenalectomy or sham operation was performed 2 days prior to adjuvant arthritis induction. Synovial explant supernatant levels of MIF and tumor necrosis factor alpha (TNFalpha) were measured by enzyme-linked immunosorbent assay (ELISA). Synovial MIF immunostaining was detected by 3-layer immunohistochemistry. Serum MIF levels were measured by Western blotting. Pituitary MIF release was measured by ELISA. Anti-MIF monoclonal antibody (mAb) or isotype-matched control antibody was administered to adrenalectomized (ADX) animals throughout AIA development. RESULTS: Compared with sham operation, adrenalectomy was associated with significant exacerbation of clinical disease parameters (P < 0.05). Adrenalectomy was associated with significantly reduced levels of synovial MIF, but not TNFalpha. In contrast, adrenalectomy was associated with increased serum MIF levels. Concomitant increased pituitary MIF levels were observed in ADX rats, consistent with the pituitary being the principal source of this increase. The administration of specific anti-MIF mAb conferred 100% protection from lethality during arthritis development and decreased arthritis disease expression. CONCLUSION: These findings provide the first in vivo confirmation of the observation that endogenous glucocorticoids are involved in the regulation of MIF in a site of inflammation, and that local and systemic MIF production are differentially regulated in this setting. The reversal of disease in ADX rats by anti-MIF mAb suggests that balance between glucocorticoids and MIF may influence the expression of inflammatory disease.

Endogenous glucocorticoids modulate experimental anti-glomerular basement membrane glomerulonephritis.

The influence of endogenous glucocorticoids (GC) on glomerular injury was studied in a rat model of heterologous anti-glomerular basement membrane (GBM) glomerulonephritis (GN). Sprague-Dawley rats underwent adrenalectomy (ADX) or sham-operation 3 days prior to i.v. administration of both nephritogenic (100 microgram/g) and subnephritogenic (50 microgram/g) doses of sheep anti-rat GBM globulin. Administration of a subnephritogenic dose of anti-GBM globulin resulted in GN in adrenalectomized animals only. Similarly, ADX performed prior to administration of anti-GBM in the nephritogenic dose range resulted in exacerbation of GN compared with sham-operated animals (24 h protein excretion: 190.8 +/- 32.8 versus 42.5 +/- 2.6 mg/24 h; P < 0.005). In ADX animals receiving subnephritogenic doses of anti-GBM injury was manifested by abnormal proteinuria (62.7 +/- 5.8 mg/24 h), accumulation of neutrophils which peaked at 6 h (7.2 +/- 1.37 neutrophils per glomerular cross-section (neut/gcs)) and macrophage accumulation in glomeruli at 24 h (6.8 +/- 1.2 macrophages/gcs). Sham-adrenalectomized animals given the same dose of anti-GBM globulin developed minimal or no glomerular injury: urinary protein excretion (8.7 +/- 1.5 mg/24 h, P < 0.001); neutrophils (0.2 +/- 0.04 neutrophils/gcs, P < 0.001); macrophages (1.2 +/- 0.5 macrophages/gcs, P < 0.001). The increased cellular recruitment to glomeruli in adrenalectomized animals was associated with glomerular endothelial P-selectin expression. P-selectin expression was not detected in sham-operated rats after anti-GBM injection. Complement deposition in glomeruli was minimal in both groups. Physiologic GC replacement of ADX rats receiving subnephritogenic-dose anti-GBM reversed the observed susceptibility to GN development, with urinary protein excretion (7.8 +/- 1.12, P < 0.005) and no detectable P-selectin expression or leucocyte accumulation in glomeruli. These results suggest that endogenous GC modulate heterologous anti-GBM nephritis in rats and that this may be attributable, in part, to regulation of P-selectin expression.

Macrophage migration inhibitory factor in rheumatoid arthritis: evidence of proinflammatory function and regulation by glucocorticoids.

OBJECTIVE: Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine whose involvement in tumor necrosis factor alpha (TNFalpha) synthesis and T cell activation suggests a role in the pathogenesis of rheumatoid arthritis (RA). Antagonism of MIF is associated with marked inhibition of animal models of RA. Uniquely, MIF is inducible by low concentrations of glucocorticoids. We sought to investigate the expression of MIF in RA synovial tissue. METHODS: MIF was demonstrated in human RA synovium by immunohistochemistry, flow cytometry, enzyme-linked immunosorbent assay (ELISA), and reverse transcription-polymerase chain reaction (RT-PCR). Regulation of MIF expression was investigated by treatment of cultured fibroblast-like synoviocytes (FLS) with interleukin-1beta (IL-1beta), TNFalpha, or interferon-gamma (IFNgamma), and dexamethasone (DEX). Mononuclear cell TNFalpha release after exposure to FLS-conditioned medium was measured by ELISA. RESULTS: MIF was present in RA synovial lining CD14+ macrophages and FLS. Constitutive MIF messenger RNA (mRNA) expression was demonstrated by RT-PCR of RNA from unstimulated cultured RA FLS, which also released abundant MIF. Serum, synovial fluid, and FLS intracellular MIF were significantly higher in RA patients than in controls. Synoviocyte MIF was not increased by IL-1beta, TNFalpha, or IFNgamma. In contrast, DEX 10(-7)M significantly reduced synoviocyte MIF, while DEX 10(-10)-10(-12)M induced a significant increase in MIF and MIF mRNA. Peripheral blood mononuclear cell TNFalpha release was induced by culture in RA FLS-conditioned medium, and this induction was significantly abrogated by monoclonal anti-MIF antibody, suggesting that MIF is an upstream regulator of TNFalpha release. CONCLUSION: These data represent the first demonstration of the cytokine MIF in human autoimmune disease and suggest MIF as a potential therapeutic target in RA.

Inhibitory effect of annexin I on synovial inflammation in rat adjuvant arthritis.

OBJECTIVE: Annexin I is an endogenous antiinflammatory mediator, expressed in rheumatoid arthritis (RA) synovium, the contribution of which to autoregulation of the synovial inflammatory response has not been examined in models of RA. We investigated the antiinflammatory role of annexin I in rat adjuvant arthritis. METHODS: Rats with adjuvant-induced arthritis (AIA) were treated with a specific anti-annexin I monoclonal antibody (mAb), isotype control IgG, and/or dexamethasone. Clinical outcomes and synovial synthesis of tumor necrosis factor alpha (TNFalpha), prostaglandin E2 (PGE2), and nitric oxide were examined, and annexin I expression was assessed by flow cytometry and reverse transcription-polymerase chain reaction. RESULTS: Anti-annexin I mAb reversed the effects of dexamethasone on the clinical features of AIA and exacerbated AIA in the absence of exogenous glucocorticoid. Clinical exacerbation of AIA by anti-annexin I mAb was accompanied by significantly increased synovial TNFalpha and PGE2, suggesting that annexin I tonically inhibits the production of these mediators. Anti-annexin I mAb treatment was associated with significantly reduced leukocyte intracellular annexin I, despite increased annexin I messenger RNA expression, consistent with a depletion effect of extracellular mAb via the cell surface. CONCLUSION: Annexin I is a key endogenous inhibitory mediator of arthritis via mechanisms that include inhibition of cytokine and effector molecule production. Moreover, a synthesis-independent depletion of intracellular annexin I by extracellular antibody supports the hypothesis that externalization of annexin I is involved in its mode of action.

Three year follow-up of body composition changes in pre-menopausal women with systemic lupus erythematosus.

OBJECTIVES: To measure the change in body composition in a pre-menopausal female systemic lupus erythematosus (SLE) population over 3 yr, and to identify predictors of change in body composition including the effects of disease-, corticosteroid (CS)- and patient-related variables. METHODS: All 55 pre-menopausal females with SLE who participated in a cross-sectional study of body composition in 1994 were invited to undergo interview, examination, medical record review, and body composition assessment by dual-energy X-ray absorptiometry (DXA). RESULTS: Twenty-eight subjects participated with a mean (S.E.M.) age of 34.4 (1.6) yr, duration of SLE of 6.8 (0.8) yr and mean (range) time to follow-up of 3.2 (2.9-3.4) yr. Seventeen subjects were exposed to CS during the study period with a mean (range) daily dose of prednisolone of 12.0 (2.8-22.9) mg. There was a significant increase in body mass index (BMI) (24.53+/-0.83 vs 25.37+/-1.04, P = 0.03) and fat-free mass (41.04+/-0.83 vs 41.53+/-0.92, P = 0.05) over the 3 yr period. Univariate analysis revealed that change in fat-free mass was significantly associated with change in total body bone mineral density (BMD) (P = 0.03). Stepwise multiple linear regression analysis revealed a significant independent association of disease activity with increases in both BMI (r2 = 0.41, P = 0.006) and fat mass (r2 = 0.39, P = 0.007), and of exercise and Modified Health Assessment Questionnaire with an increase in fat-free mass (r2 = 0.51, P = 0.007). Age at SLE diagnosis and smoking were significant independent predictors for loss of total body BMD, while CS duration was predictive of an increase in total body BMD (r2 = 0.80, P < 0.0001). CONCLUSION: In this SLE population, disease activity was predictive of deleterious changes in body composition, including increases in BMI and fat mass. Patient-related variables were also important predictors of body composition change with exercise independently predicting an increase in fat-free mass, and smoking predictive of loss of total body BMD. In contrast, CS-related variables were not found to have harmful effects on body composition. Change in fat-free mass, and not fat mass, was predictive of change in total body BMD.

Urinary excretion of the pyridinium cross-links of collagen in systemic lupus erythematosus.

The aim of this study was to measure the urinary excretion of the pyridinium cross-links of collagen and to determine their usefulness as markers of reduced bone mineral density in systemic lupus erythematosus (SLE). All female SLE patients managed at a single centre were invited to participate in a cross-sectional study of urinary pyridinium cross-links excretion (HPLC), bone mineral density (DXA), and SLE-related variables. Ninety-one women with SLE were studied, 35 of whom were postmenopausal. Pyridinoline/Creatinine (Pyd/Cr) and deoxypyridinoline/Cr (Dpd/Cr) levels in postmenopausal women were significantly increased compared with premenopausal values (p = 0.010 and p = 0.004, respectively). Univariate linear regression analysis revealed a significant association of Dpd/Cr with reduced femoral neck and lumbar spine BMD (p = 0.001, p<0.001), and of Pyd/Cr with reduced femoral neck BMD (p = 0.020). In addition, the association of Pyd/Cr with reduced lumbar spine BMD approached significance (p = 0.055). Stepwise multiple linear regression analysis adjusting for other variables confirmed a significant association of Dpd/Cr with reduced lumbar spine BMD (p = 0.006), and a significant association of both Pyd/Cr and Dpd/Cr with postmenopausal status (p = 0.003, p<0.001). It was concluded that in this SLE population, the urinary excretion of Dpd/Cr was a useful marker of reduced BMD at the lumbar spine. Menopausal status was a major predictor of cross-links excretion in SLE.

Involvement of macrophage migration inhibitory factor in the evolution of rat adjuvant arthritis.

OBJECTIVE: Recent studies have established an essential role for macrophage migration inhibitory factor (MIF) in T cell and macrophage activation, both of which are characteristics of rat adjuvant arthritis. This study investigated the role of MIF in early adjuvant arthritis. METHODS: MIF was detected in rat synovium by immunohistochemistry and enzyme-linked immunosorbent assay using specific monoclonal antibodies (MAb). Anti-MIF MAb treatment was administered, and the effects on clinical aspects of adjuvant arthritis were assessed. RESULTS: MIF was absent from normal rat synovium prior to adjuvant injection, but was detectable on day 4 after injection (6 days before the onset of clinical disease) and was colocalized with ED-1+ macrophages throughout the evolution of the disease. Levels of MIF were increased in established adjuvant arthritis sera, and adjuvant arthritis synovial macrophages released MIF at a mean +/- SEM concentration of 607.9 +/- 201.5 pg/ml. Anti-MIF treatment led to profound, dose-dependent inhibition of the adjuvant arthritis clinical score, paw swelling, and synovial lavage leukocyte numbers (P < 0.001), and also resulted in reduced synovial macrophage and T cell accumulation. CONCLUSION: These findings demonstrate an important role for MIF in the evolution of rat adjuvant arthritis.

Glucocorticoid inhibition of adjuvant arthritis synovial macrophage nitric oxide production: role of lipocortin 1.

Nitric oxide (NO) is a mediator of inflammatory injury which is inhibited by glucocorticoids and is implicated in rheumatoid (RA) and adjuvant arthritis (AA). The glucocorticoid-induced anti-inflammatory molecule lipocortin 1 is expressed in RA synovium, but the effects of lipocortin 1 on synovial inflammation have been little studied. We investigated the effects of glucocorticoids and lipocortin 1 on inducible NO synthase (iNOS) and glucocorticoids on the induction of lipocortin 1 in AA synovial macrophages. NO production was measured by Griess assay in supernatants of day 14 AA rat synovial explants and of synovial macrophages purified from enzyme-digested synovium and treated with lipopolysaccharide (LPS) 1 microg/ml, dexamethasone (DEX) 10(-7) M, and anti-lipocortin 1 MoAb. iNOS and lipocortin 1 expression were detected by flow cytometry using specific MoAb. Cell surface lipocortin was determined by Western blot. NO was produced by all AA synovial explants and NO was released by cultured synovial macrophages (14.5 +/- 2.1 micromol/24 h). iNOS was detected in synovial macrophages (ED-1+) by permeabilization flow cytometry. LPS increased synovial macrophage NO release (P < 0.0001) and iNOS expression (P = 0.04). DEX inhibited constitutive (P = 0.002) and LPS-induced (P < 0.001) NO release and iNOS expression (P = 0.03). DEX inhibition of synovial macrophage NO was associated with induction of cell surface and intracellular lipocortin 1. Anti-lipocortin 1 MoAb treatment reduced the inhibition of NO release by DEX (P = 0.002), but had no effect on iNOS expression. These findings demonstrate a role for lipocortin I in the inhibition by glucocorticoids of AA synovial macrophage iNOS activity.

Suppression of adjuvant arthritis and synovial macrophage inducible nitric oxide by N-iminoethyl-L-ornithine, a nitric oxide synthase inhibitor.

Nitric oxide (NO.) is a pro-inflammatory effector molecule in certain inflammatory diseases, including arthritis. We investigated the production of NO. by adjuvant arthritis (AA) synovial macrophages, and studied the effects of a NO. synthase inhibitor. N-iminoethyl-L-ornithine (L-NIO). Compared to control rats, rats treated with L-NIO in vivo exhibited significantly lower articular index (p < 0.05), paw volume (p < 0.05), and synovial fluid cell count (p < 0.05). No effect on cutaneous delayed-type hypersensitivity to the disease-initiating antigen was observed. Inducible NO. synthase (iNOS) was detected in AA synovial macrophages, and cultured AA synovial macrophage iNOS levels were increased by a factor of 138 +/- 17% (p < 0.01) by 1 microgram/ml LPS in vitro. Constitutive NO. production by AA synovial macrophages (43 +/- 1 nmol/10(5) cells/24 h) was significantly inhibited by 10 nM L-NIO in vitro (32 +/- 0.5, p < 0.01). NO. production induced by 1 microgram/ml LPS (48 +/- 2) was also decreased by L-NIO (39 +/- 2, p < 0.05). In vivo L-NIO treatment also inhibited alveolar macrophage NO. production (p < 0.05). The ability of L-NIO to decrease iNOS-mediated synovial macrophage NO. production and inhibit the clinical parameters of AA implicate macrophage-derived NO. in the pathogenesis of this disease.