Variety of endosomal TLRs and Interferons (IFN-α, IFN-ß, IFN-γ) expression profiles in patients with SLE, SSc and MCTD.

We investigated Toll-like receptor (TLR)-3/-7/-8/-9 and interferon (IFN)-α/ß/γ mRNA expression in whole blood and serum IFN-α/ß/γ levels in patients with mixed connective tissue disease (MCTD), systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) and in healthy subjects to assess the association between the TLR-IFN expression and severity of and susceptibility to diseases, and identify potential biomarkers. Expression of the IFN-γ, TLR-3 and TLR-8 was detected only in SLE patients. TLR-7, IFN-α and IFN-ß expression was highest in SLE, while TLR-9 expression was highest in SSc patients. In SLE and MCTD patients a strong correlation was observed between TLR-7 and IFN-α expression and IFN-ß and IFN-α expression. In MCTD patients, negative correlation between IFN-α and TLR-9 and TLR-7 and TLR-9 was revealed. TLR-9 expression in anti-U1-70k-negative, anti-C negative and anti-SmB-negative MCTD patients was higher than in MCTD-positive patients. We observed negative correlations between serum IFN-α levels and TLR-7 expression and C3 and C4 levels in SLE patients. In SLE patients we observed that with increased IFN-γ, TLR-3 and TLR-8 expression increased the value of C3 and C4. Our results confirmed that the endosomal TLR-IFN pathway seems to be more important in SLE than in MCTD or SSc, and that IFN-α and IFN-ß may be possible biomarkers for SLE.

The diagnosis and classification of undifferentiated connective tissue diseases.

The term undifferentiated connective tissue disease (UCTD) refers to unclassifiable systemic autoimmune diseases which share clinical and serological manifestations with definite connective tissue diseases (CTDs) but not fulfilling any of the existing classification criteria. In this review we will go through the more recent evidence on UCTD and we will discuss in what extent the availability of new criteria for the CTDs could interfere with the "UCTD concept". The development of criteria able to identify early phases of defined CTD, may help in the differentiation of stable UCTD form their early stages and may offer a valuable guide to the treating physician to set up appropriate follow up schedules as well as therapeutic protocols. This simplified subset of CTD could offer a model to study clinic pathological correlations as well as the role of possible environmental factors in the development of autoimmunity.

T-regs in autoimmune hepatitis-systemic lupus erythematosus/mixed connective tissue disease overlap syndrome are functionally defective and display a Th1 cytokine profile.

Autoimmune hepatitis (AIH), a severe hepatopathy characterized by hypergammaglobulinaemia, autoantibodies and interface hepatitis, is occasionally associated with systemic autoimmune manifestations [systemic lupus erythematosus (SLE); mixed connective tissue disease (MCTD)]. In both AIH and SLE/MCTD numerical and/or functional impairment of regulatory T-cells (T-regs) is believed to favour autoimmunity. To investigate whether immune-tolerance breakdown profiles differ in patients with AIH and SLE/MCTD, isolated AIH or systemic autoimmunity, we studied phenotypic and functional features of T-regs in 10 patients with AIH-SLE/MCTD, 22 with AIH, 12 with SLE and 20 healthy subjects. Compared to health, CD4(pos)CD25(pos) cells were decreased in number and expressed high levels of the CD127 activation marker in all three disease groups; in AIH-SLE/MCTD and in SLE they displayed low levels of FOXP3. In AIH-SLE/MCTD, they also contained a high proportion of IFNγ positive cells, indicating a Th1 profile. Similarly, in AIH-SLE/MCTD, CD4(pos)CD25(pos)CD25(high) T-regs were reduced in number and contained an increased proportion of activated CD127(pos) and IFNγ(pos) cells. Such skewing towards a Th1 profile was also present at effector level, as a high frequency of IFNγ-producing cells was observed within AIH-SLE/MCTD CD4(pos)CD25(neg) responder cells. Impairment in suppressor function both of CD4(pos)CD25(pos) cells and CD4(pos)CD25(pos)CD127(neg) T-regs was observed in all three autoimmune conditions, but while addition of CD4(pos)CD25(pos)CD127(neg) T-regs decreased CD4(pos)CD25(neg) responder cell proliferation in healthy subjects and partially in AIH patients, it had no effect in AIH-SLE/MCTD and SLE patients. In conclusion, in AIH-SLE/MCTD T-regs display a distinctive phenotypic and functional signature, characterized by marked activation, elevated IFNγ production and by a profound impairment of suppressive function, suggesting that multiple autoimmune manifestations may derive from a complex defect of immune-regulation.

Defective expression and function of the leukocyte associated Ig-like receptor 1 in B lymphocytes from systemic lupus erythematosus patients.

Systemic lupus erythematosus (SLE) is characterized by the production of a wide array of autoantibodies and dysregulation of B cell function. The leukocyte associated Immunoglobulin (Ig)-like receptor (LAIR)1 is a transmembrane molecule belonging to Ig superfamily which binds to different types of collagen. Herein, we have determined the expression and function of LAIR1 on B lymphocyte from SLE patients. LAIR1 expression in peripheral blood B lymphocytes from 54 SLE, 24 mixed connective tissue disease (MCTD), 20 systemic sclerosis (SSc) patients, 14 rheumatoid arthritis (RA) and 40 sex and age matched healthy donors (HD) have been analyzed by immunofluorescence. The effect of LAIR1 ligation by specific monoclonal antibodies, collagen or collagen producing mesenchymal stromal cells from reactive lymph nodes or bone marrow on Ig production by pokeweed mitogen and B cell receptor (BCR)-mediated NF-kB activation was assessed by ELISA and TransAM assay. The percentage of CD20(+) B lymphocytes lacking or showing reduced expression of LAIR1 was markedly increased in SLE and MCTD but not in SSc or RA patients compared to HD. The downregulation of LAIR1 expression was not dependent on corticosteroid therapy. Interestingly, LAIR1 engagement by collagen or collagen-producing mesenchymal stromal cells in SLE patients with low LAIR1 expression on B cells delivered a lower inhibiting signal on Ig production. In addition, NF-kB p65 subunit activation upon BCR and LAIR1 co-engagement was less inhibited in SLE patients than in HD. Our findings indicate defective LAIR1 expression and function in SLE B lymphocytes, possible contributing to an altered control of B lymphocytes behavior.

Endothelial cell markers reflecting endothelial cell dysfunction in patients with mixed connective tissue disease.

INTRODUCTION: The aim of the present study was to investigate the association between cardiovascular risk factors and endothelial dysfunction in patients with mixed connective tissue disease (MCTD) and to determine which biomarkers are associated with atherosclerotic complications, such as cardiovascular disease. METHODS: Fifty MCTD patients and 38 healthy age-matched and sex-matched controls were enrolled in this study. In order to describe endothelial dysfunction, we assessed flow-mediated dilation (FMD), nitrate-mediated dilation (NMD) and carotid artery intima-media thickness (IMT). We investigated FMD of the brachial artery after reactive hyperemia and NMD after sublingual nitroglycerin administration, while the IMT of the common carotid artery was determined by ultrasound. Anti-U1 ribonucleoprotein (anti-U1RNP) antibodies, anti-cardiolipin (anti-CL) antibodies, anti-endothelial cell antibody (AECA) and endothelial cell markers, such as soluble thrombomodulin (TM) and von Willebrand factor antigen (vWFAg), were assessed. RESULTS: The endothelium-dependent vasodilation (FMD) was significantly impaired in patients with MCTD, as compared with controls (%FMD: 4.7+/-4.2% vs. 8.7+/-5.0%; P<0.001), while the percentage NMD did not differ (%NMD: 14.3+/-6.6% vs. 17.1+/-6.7%; P=0.073). Mean carotid IMT values were higher in patients than in controls (IMT: MCTD, 0.64+/-0.13 mm vs. controls, 0.53+/-0.14 mm; P<0.001). FMD negatively correlated with disease duration, the levels of apolipoprotein A1, the paraoxonase-1 activity, and systolic blood pressure in MCTD patients. The percentage FMD was significantly lower in MCTD patients with cardiovascular diseases (CVD), than in those without CVD (%FMD: 3.5+/-2.9 vs. 5.8+/-4.8, P<0.0002), while percentage NMD did not differ between patients with and without CVDs. Serum levels of autoantibodies (anti-U1RNP, AECA and anti-CL) were significantly higher in MCTD patients and differed between MCTD patients with and without CVD. Endothelial cell markers such as soluble TM (12.2+/-8.1 ng/ml vs. 3.2+/-1.3 ng/ml; P<0.001) and vWFAg (224.1+/-115% vs. 89.4+/-27.1%, P<0.001) were the highest in MCTD patients with CVD. CONCLUSIONS: FMD is a reliable sensitive marker of endothelial cell dysfunction in MCTD. Beside the traditional risk factors, anti-U1RNP, AECA and anti-CL antibodies may be important not only in the pathogenesis of MCTD but in the induction of endothelial cell activation, and may play crucial roles in the development of early atherosclerosis in MCTD.

Speckled antinuclear antibodies in keratinocytes--what does it mean?

OBJECTIVE: Epidermal in vivo nuclear staining is occasionally noted in the lupus band test (LBT) in patients with connective tissue diseases (CTD). The exact clinical significance of this finding remains unelucidated, especially in association with a positive LBT We have reviewed the clinical and serological characteristics of patients with in vivo-bound antinuclear antibodies (ANA) in keratinocytes. METHODS: Between 1990-1999 speckled IgG staining in keratinocyte nuclei was observed in 31 LBT specimens. We had detailed clinical and laboratory data for 22/31 patients. The present study comprises 22 patients with in vivo-bound ANA (8 cases with mixed CTD (MCTD), 10 with systemic lupus erythematosus (SLE), 2 with Sjögren's syndrome (SS), one with undefined CTD and one clinically healthy mother of a child with neonatal lupus erythematosus), and 22 consecutive CTD patients (2 MCTD, 15 SLE, 5 SS) without in vivo-bound ANA. Antinuclear, anti-dsDNA and anti-extractable nuclear antigens (ENA) antibodies were determined using indirect immunofluorescence and ELISA methods. RESULTS: A significant difference (p < 0.01) in anti-RNP antibodies between patients with and without in vivo-bound ANA was observed. Consequently, there was a strong association of in vivo-bound ANA and anti-RNP antibodies (p < 0.01). In SLE patients with in vivo-bound ANA the incidence of nephropathy was significantly lower (p < 0.01), regardless of LBT positivity. In SLE patients there were no differences in LBT positivity, anti-dsDNA antibodies and complement consumption between groups. CONCLUSION: Our study implies that the presence of speckled ANA in keratinocytes in LBT may be useful in the diagnosis of MCTD and in the prognosis of renal involvement, especially in SLE patients.

[The role of adrenal and gonadal hormones in the pathogenesis of autoimmune polyarthritis]. / Az adrenalis és a gonadális hormonok szerepe az autoimmun polyarthritisek patogenezisében.

A growing body of recently published results suggest the role of adrenal androgens in the onset and development of chronic inflammatory process due to autoantigens. Dehydroepiandrosterone (DHEA) and dehydroepiandrosterone-sulphate (DHEA)--the major androgen products of the adrenal gland--have immunosuppressive effect inhibiting interleukin-6 production and substantially determining acute phase reaction. Decreased serum levels of DHEA and DHEAS has been observed in most of autoimmune diseases. Recent data suggest that adrenal hypoandrogenism comes from disturbed neuroendocrine, regulation due to hypothalamic effect of the inflammatory cytokines. On the other side, decreased adrenal androgen activity negatively influences the anabolic tonus of steroid hormone system while a relative enhancement of catabolic pressure occurs by the glucocorticoids. Moreover, the hypothalamus-hypophysis-gonadal axis can also be involved, resulting shifts in serum levels of prolactin, estrogens and gonadal androgens. All these hormonal changes can be summarised in decreasing the immunosuppressive tonus. This hypothesis connects the endocrine dysregulation with the development of autoimmune disorders. The new results promise not only a basically different theory of chronic inflammation but they will permit using new diagnostic tools as well as inducing substantially new and more effective therapeutic approaches.

Twenty-four hour intraesophageal pH monitoring in children and adolescents with scleroderma and mixed connective tissue disease.

OBJECTIVE: To investigate esophageal dysmotility and gastroesophageal reflux, which are important, but often occult features of gastrointestinal (GI) involvement in children and adolescents with mixed connective tissue disease (MCTD) and scleroderma. METHODS: We carried out intraesophageal 24 h pH monitoring of 14 patients with scleroderma and MCTD. RESULTS: We observed an elevated reflux index in 64% of the patients. An increased number of reflux events was found in 85%. Reflux events > 5 min were noted in 50% of the patients, which is indicative of possible development of esophagitis. Only 3 patients presented clinical symptoms. Four of 5 patients with localized scleroderma revealed a pathological reflux index. We found no relationship between gastroesophageal reflux, age of patients, duration of disease, and Raynaud's phenomenon. CONCLUSION: GI involvement in children with scleroderma or MCTD is more frequent than clinical symptoms indicate. An active diagnostic investigation for GI dysmotility and gastroesophageal reflux is necessary to detect these complications.

Enhanced synthesis of cytokines by peripheral blood monocytes cultured in the presence of autoantibodies against U1-ribonucleoprotein and/or negatively charged molecules: implication in the pathogenesis of pulmonary hypertension in mixed connective tissue disease (MCTD).

An attempt was made to determine whether addition of purified autoantibodies against U1-ribonucleoprotein (RNP) and negatively charged molecules (cardiolipin and double-stranded (ds) DNA) to cultures of peripheral blood monocytes could enhance the synthesis of cytokines in patients with MCTD and normal healthy volunteers. It was found that: (i) at the baseline, levels of cytokines such as IL-1 alpha, IL-1 beta and IL-6 extracellularly released by or associated with monocytes were significantly higher in MCTD patients than in normal subjects; (ii) addition of antibodies against U1-RNP to cultures of MCTD monocytes resulted in a significant overall increase of the released and cell-associated IL-1 alpha, IL-1 beta and IL-6. On the other hand, addition of antibodies against cardiolipin or dsDNA to cultures of MCTD monocytes resulted in a significant increase of released and/or cell-associated IL-1 alpha and IL-1 beta; (iii) addition of these autoantibodies to cultures of normal monocytes resulted in a significant overall increase of released and cell-associated IL-1 alpha, IL-1 beta and IL-6. The extent of enhancement of cytokines released by or associated with monocytes was greater in normal subjects than in MCTD patients; (iv) a F(ab')2 preparation of autoantibodies against U1-RNP also enhanced the level of released and cell-associated IL-1 alpha. Our findings that both autoantibodies against U1-RNP and negatively charged molecules were able to enhance the synthesis of cytokines by monocytes suggest that these autoantibodies might cause derangement of endothelial cells and lead to proliferative vasculopathy, which is a characteristic of pulmonary hypertension in MCTD.

Effects of nutritional supplementation on bone mineral status of children with rheumatic diseases receiving corticosteroid therapy.

OBJECTIVE: Because children with rheumatic disease receiving longterm corticosteroids are at high risk for developing osteoporosis, we attempted to determine whether nutritional supplementation would improve bone status in this group of children. METHODS: In a crossover design study, 10 corticosteroid treated children with rheumatic disease and osteoporosis received calcium and vitamin D supplementation for 6 months to determine their effect on bone density. They were then studied for 6 months without added nutrition supplements. The mean age was 13.1 years with a mean duration of disease of 4.2 years. Six patients had juvenile rheumatoid arthritis, 2 had systemic lupus erythematosus and 2 had mixed connective tissue disease. These children obtained a minimum of 1 g of calcium and 400 IU of vitamin D daily from diet and added supplements. Dual photon absorptiometry, laboratory and dietary data were obtained at baseline, 6 months, and one year. RESULTS: Spinal bone density significantly improved with supplementation. Osteocalcin values remained low throughout the study. CONCLUSION: Our results suggest some children with rheumatic disease receiving corticosteroids would benefit from calcium and vitamin D supplementation.

Lupus anticoagulant: interference with in vivo prostaglandin production and with platelet sensitivity to prostacyclin.

The hypothesis has been made that inhibition of prostacyclin (PG12) production may play a role in the pathogenesis of thrombosis in patients with the lupus anticoagulant (LA), but so far no evidence of reduced PG12 levels in vivo has been produced. We have tested the plasma levels of PG12 and thromboxane A2 (TXA2) and the platelet sensitivity to PG12 in 14 patients with and without LA and in 14 healthy controls. No significant difference in the prostanoid basal levels was detected among the groups; however, in some patients PG12 increments seemed to parallel the clinical course of the disease. Platelet sensitivity to exogenous PG12 was significantly enhanced in the LA + patients and correlated with PG12 values. We suggest that in these subjects additional factors, other than reduced PG12, may predispose to thrombosis.

Transforming growth factor-beta 1 in rheumatoid synovial membrane and cartilage/pannus junction.

Transforming growth factor (TGF)-beta has been shown to promote tissue repair and have immunosuppressive actions, and has been proposed to have a role in rheumatoid arthritis (RA). Using immunohistochemical techniques with rabbit F(ab')2 antibodies raised against recombinant human TGF-beta 1, we have detected TGF-beta 1 in the synovial tissue and cartilage/pannus junction (CPJ) from 18/18 patients with RA. TGF-beta 1 was found predominantly in the thickened synovial lining layer in RA, but also detected in a perivascular pattern in the synovial interstitium as well as in occasional cells in the lymphoid aggregates. At the CPJ it was found both in cells at the distinct junction as well as in the transitional region of the diffuse fibroblastic zone. The cells staining for TGF-beta 1 were identified by double immunofluorescence staining as being from the monocyte/macrophage series as well as the type B synovial lining cells. TGF-beta 1 was also detected in the synovial membrane sections from 4/4 patients with systemic lupus erythematosus/mixed connective tissue disease and 5/8 patients with osteoarthritis, in a similar distribution to that seen in RA, and in the lining layer of 1/7 normal synovial membranes. These results add to histological evidence confirming that TGF-beta 1 is present in RA synovial cells and those from other arthritides. The distributions of TGF-beta 1 in RA synovial membrane reflects its known actions, as it can be detected at the CPJ, where it could induce repair, and close to activated cells upon which it may exert an immunosuppressive action.

Intra-articular apatite deposition in mixed connective tissue disease: crystallographic and technetium scanning characteristics.

An acute arthritis in a patient with mixed connective tissue disease (MCTD) was found to be associated with intra-articular deposition of carbonated hydroxyapatite crystals. A technetium hydroxymethylene diphosphonate bone scan showed intense uptake in the delayed phase scan of the affected joints. Synovial fluid analysis demonstrated uptake of the radiopharmaceutical drug directly onto the crystals.

HLA and immunoglobulin allotypes in mixed connective tissue disease.

A group of patients with mixed connective tissue disease (MCTD) were HLA and immunoglobulin allotyped. We found that the incidence of DR4 in the patient group was increased compared with that in the normal controls, but the increase was restricted to the subgroup of patients with arthritis. The age at onset of MCTD was lower in patients with DR4 and higher in patients with DR2 compared with patients who did not have these antigens. A1, B8, and DR3 were more frequent, but not significantly so, in the MCTD patient group. We also found that there was a significant perturbation of the Gm allotype frequencies in patients with MCTD.

Inhibition of complement-mediated solubilisation of antigen-antibody complexes by serum factor(s) in patients with various connective tissue diseases.

Control of immune complex formation is important to limit disease resulting from their deposition in tissues. Any inhibition of immune complex solubilisation is thus significant in the pathogenesis of immune complex diseases. More than half of our patients with various rheumatic connective tissue diseases were demonstrated to have serum inhibition of immune complex solubilisation (12/16 rheumatoid arthritis, 22/37 systemic lupus erythematosus, 16/29 primary Sjogren's syndrome, and eight of nine with mixed connective tissue disease). This serum inhibitory activity did not correlate well with serum levels of IgM rheumatoid factor or circulating immune complexes, and its nature remains to be elucidated.

Pulmonary involvement in mixed connective tissue disease.

To determine the frequency of pulmonary function abnormalities in mixed connective tissue disease (MCTD), we studied 16 consecutive patients. Spirometry, total lung capacity (TLC), diffusing capacity (Dco), static lung compliance (Cst), and tests of respiratory muscle function (peak inspiratory pressure [PIP] and the maximum change in transdiaphragmatic pressure [delta PDI] during a diaphragmatic Müeller maneuver) were obtained. Airway resistance (RAW) and frequency dependence of compliance were also measured. Static lung compliance was less than 0.2 L/cm H2O in four of nine patients. The PIP was less than 75 cm H2O in only one patient and delta PDI was greater than 45 cm H2O in all ten patients evaluated. Frequency dependence of compliance was abnormal in seven of ten patients, whereas RAW was increased in only three of eight patients. The TLC and DCO were less than 80 percent of the predicted values in ten of 16 and 14 of 16 patients respectively. There was no correlation between the level of extractable nuclear antigen and pulmonary function abnormalities. The study did not identify significant abnormalities in respiratory muscle function.