Sera from 735 patients with systemic sclerosis were classified according to antinuclear antibody (ANA) pattern as follows: centromere (25%), homogeneous (26%), fine speckled (21%), fine speckled with nucleolar (14%), coarse speckled (7%), nucleolar only (3%) and cytoplasmic only (3%). Immunoprecipitations using 35S-labelled HeLa cell antigen extract were performed using sera from 374 of these patients to detect the systemic sclerosis-specific antibodies to RNA polymerases I and III. The sera were selected to represent each ANA group, but focused on those giving fine speckled nucleoplasmic staining (with or without nucleolar staining) where all 86 sera positive for these antibodies were concentrated. Immunoprecipitates from a further 93 sera from patients with ANA-positive autoimmune diseases other than systemic sclerosis did not precipitate RNA polymerases. In addition, all sera were tested for antibodies to the extractable nuclear antigens topoisomerase I, nRNP, Ro, La and PM-Scl. Sera positive for antibodies to these antigens gave clear correlations with ANA patterns but, of the examples tested, none contained antibodies precipitating RNA polymerase I or III. Thus, sera containing antibodies to RNA polymerases I and III were exclusive of both anticentromere and anti-topoisomerase I, and formed a major serological subgroup (11.7%). Clinically, 77% were patients with diffuse cutaneous disease reflected by higher skin scores and a significantly higher incidence of renal involvement (33%) than patients with antibodies to topoisomerase I (3%).
Microvascular damage occurs in systemic sclerosis (SSc) and is associated with increased expression of endothelial adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin. Elevated levels of the soluble circulating forms of these molecules have recently been reported in SSc. We have extended this observation by collecting serial serum samples from 12 patients with systemic sclerosis, at intervals between 4 and 12 months, through the course of their disease (mean period of observation 44 months). Circulating ICAM-1, VCAM-1 and E-selectin were measured by ELISA, and changes in these levels were compared with alterations in disease activity as assessed by skin sclerosis score, serum creatinine, erythrocyte sedimentation rate and pulmonary function tests coincident with each serum sample. The mean levels were ICAM-1 627 ng/ml, VCAM-1 959 ng/ml and E-selectin 81 ng/ml. In 8/12 patients, there was a substantial change in at least one disease parameter during the assessment period. In seven (88%) of these patients, changes in circulating VCAM-1 or E-selectin were associated with disease severity, falling with improvement in renal function or skin score, and rising with deterioration in pulmonary function tests. The maximum recorded level of VCAM-1 (3550 ng/ml) shortly preceded an acute renal SSc crisis. In two cases (25%), the correlation was statistically significant (P < or = 0.01). The ICAM-1 level did not reflect clinical changes in any patients. These results provide further evidence for endothelial cell dysfunction in SSc, and suggest that serial measurements of VCAM-1 and E-selectin may have potential value as surrogate markers for clinical progression or remission in this disease.
k d t h Cardiovascular events PAPPA c0.01 mlU/ml PAPPA >or = 0.01 (n42) mlU/ml (N=62) 9 (21%) 17 (27%) 10 (24%) I I (18%) 4(10%) Stroke 2 (5%) 0th 4 (10%)
BackgroundInflammation, vasculopathy and tissue fibrosis are key features of scleroderma (SSc). While healthy forearm skin which has a Young's modulus of 4–12kPa, SSc fibrotic skin measures 50–80kPa with its increased mechanical stiffness1. We have shown that mechano-sensing properties of fibroblasts in SSc are mediated by myocardin-related transcription factor A (MRTF-A)3. Monocytes/macrophages are likely to be involved in SSc pathogenesis but the effect of mechanical stress on these cells remain to be elucidated3,4.ObjectivesTo investigate if a mechanically-stressed microenvironment like that in SSc tissue, promotes macrophages towards a pathogenic phenotype, and whether MRTF-A is involved in this process.MethodsControl and scleroderma skin sections were immunostained with anti-CD68 and anti-MRTF-A antibodies (n=3). Human PBMC-derived macrophages were cultured in RPMI/M-CSF on 4kPa and 50kPa collagen-fibronectin-coated plates to mimic “soft”/healthy and “stiff”/fibrotic skin, and activated with LPS (10ng/ml) or IL-10 (10ng/ml) for macrophages designated M(LPS) and M(IL-10) (n=4). MRTF-A expression was assessed by qPCR and conditioned media profiled by Luminex array for inflammatory cytokines. Mouse bone marrow-derived macrophages (BMDMs) of wildtype and MRTF-A-null mice were maintained in RPMI/M-CSF on soft and stiff substrates. The data were analysed by two-way ANOVA and Tukey test (p<0.05, CI 95%).ResultsWe observed a greater number of CD68+ macrophages in diffuse SSc skin compared to control skin, mainly around perivascular regions. These macrophages also expressed MRTF-A. Human macrophages expressed MRTF-A mRNA and showed differential cytokine expression when cultured on soft and stiff substrates. M(LPS) on soft matrix expressed IFN-γ, which was undetectable with M(LPS) on stiff substrate (mean difference 0.2075±0.1576pg/ml, p<0.01). LPS- and IL-10 activation on soft substrate increased MCP-3 expression compared to controls (mean difference 68.51±49.19pg/ml, p<0.01, 92.88±49.22pg/ml, p<0.0001, respectively). M(LPS) on stiff compared to soft substrate showed lower MCP-3 expression (mean difference 57.01±49.22pg/ml, p<0.05). M(IL-10) on soft substrate showed higher MCP-1 expression compared to controls (mean difference 2448±2232pg/ml, p<0.05). M(IL-10) on stiff substrate decreased expression of MCP-1 (mean difference 2590±2233pg/ml, p<0.05) and increased fractalkine levels compared to soft substrate (mean difference 51.22±36.28pg/ml, p<0.01). Wildtype BMDMs on stiff compared to soft substrate displayed a more elongated morphology. MRTF-A-null BMDMs remained rounded on stiff substrate.ConclusionsMRTF-A is a mechanical stress-responsive factor which co-activates transcription of cytoskeletal and extracellular matrix-modifying genes. MRTF-A may couple mechanical stress to macrophage activation in SSc, where stiff matrix promotes macrophage secretion of cytokines and growth factors that exacerbate fibrosis.ReferencesSacksen et al., Arthritis Rheum 2013.Shiwen et al., PLoS One 2015.Stifano et al., Curr Rheumatol Re...
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