Mixed connective tissue disease: what is behind the curtain?

Although there is still an emotional debate over the existence of mixed connective tissue disease, the evidence from animal models suggests that anti-U1RNP antibodies, similar to other autoantibodies in other connective tissue diseases (such as antisynthetase, anticentromere, and antitopoisomerase), play a pathophysiological role in this disease. Despite an antiendothelial effect of anti-U1RNP antibodies, which is reminiscent of anticentromere antibodies, patients with high-titer autoantibodies to U1RNP in the absence of anti-Sm antibodies do not usually have or develop typical systemic sclerosis. Instead, their severe Raynaud's syndrome is commonly accompanied by arthritis, which can be erosive, and by swollen/puffy hands and myositis. Pulmonary arterial hypertension is the major life-threatening complication in these patients and regular screening for this condition is essential.     

Overlap syndromes and mixed connective tissue disease.

While the etiology of connective tissue diseases remains unknown, the classification of individual cases will continue to depend on identifying certain patterns of clinical and laboratory features. As many as 25% of connective tissue disease patients present with an overlap syndrome with features of systemic lupus erythematosus, systemic sclerosis, polymyositis, or dermatomyositis, with rheumatoid arthritis and Sj?gren's syndrome evolving concurrently or consecutively during the course of the disease. The term overlap syndrome is applied to what appears to be a heterogeneous group of disorders, but in recent years there have been attempts to identify antibody markers within this population to identify subsets with particular patterns of disease expression. Thus, anti-U1-ribonucleoprotein is associated with overlap syndromes in which features of systemic lupus erythematosus are accompanied by features of systemic sclerosis or myositis; antibodies to polymyositis-scleroderma, Ku, and U2-ribonucleoprotein are associated with overlaps of systemic sclerosis and polymyositis, and anti-Jo-1 is associated with polymyositis and pulmonary fibrosis. A practical reason for subdividing cases in this way relates to prognosis and treatment, but at a more fundamental level it is hoped that the study of the origin of these antibodies and their antigen targets will provide clues to pathogenesis and even etiology.     

Immunological analysis of pulmonary hypertension in connective tissue diseases.

OBJECTIVE: To analyze the immunological characteristics of the sera of patients with inflammatory connective tissue diseases complicated by pulmonary hypertension (PH). METHODS: Sera of 24 patients with mixed connective tissue disease complicated by PH (MCTD-PH), sera of 11 patients with other connective tissue diseases complicated by PH (Other-PH; 6 systemic sclerosis, 3 systemic lupus erythematosus, 2 rheumatoid arthritis), and sera of 15 patients with MCTD not complicated by PH (MCTD-non-PH) were tested for IgG antibodies against U1RNP proteins, U1RNP-70K protein, U1RNP-A protein, and U1RNP-C protein, and for IgG and IgM antibodies to beta2-glycoprotein I dependent cardiolipin (CL) and human umbilical vein endothelial cells. We also measured the serum levels of von Willebrand factor related antigens and interleukin 6 (IL-6). RESULTS: (1) The titers of the anti-U1RNP, anti-U1RNP-70K, anti-U1RNP-A, and anti-U1RNP-C antibodies were significantly higher in the MCTD-PH and MCTD-non-PH groups than in the Other-PH group. However, there were no statistically significant differences in the titers of these 4 antibodies between the MCTD-PH and MCTD-non-PH groups. (2) The titers of the IgG aCL and the serum IL-6 levels were significantly higher in the MCTD-PH group than in the MCTD-non-PH group. (3) Statistically significant correlations between the anti-U1RNP and IgG anti-CL antibody titers, and between the IgG anti-endothelial cell and IgG anti-CL antibody titers were observed within the MCTD-PH and Other-PH groups, but not within the MCTD-non-PH group. CONCLUSION: The occurrence of anti-U1RNP, anti-endothelial cell, and anti-CL antibodies is associated with PH in certain patients with connective tissue disease.     

Antigen specificity of antihistone antibodies in connective tissue disease patients with anti-U1RNP antibodies

To determine the prevalence of antibodies to individual histone components in collagen disease patients with anti-U1RNP antibodies. Serum samples were examined by enzyme-linked immunosorbent assay. Patients with mixed connective tissue disease (MCTD) and systemic sclerosis (SSc) showed similar levels and patterns of antihistone antibody (AHA) reactivities to individual histones: IgG responses to H2B or H3 and IgM responses to H2B were highest. However, both IgG and IgM AHAs against outer portion of chromatin (H1, H2A, or H2B) were generally higher in SLE compared with other diseases. SLE or SSc patients with anti-U1RNP antibodies showed generally higher AHA levels than in those without them. Thus, the pattern of reactivities to each histone component was dependent on the disease, while the intensity was dependent on both the disease and anti-U1RNP antibodies. The antigenic stimulus in SLE may be different from other connective tissue diseases and is more likely to be native chromatin.     

In vitro production of autoantibodies to U1 ribonucleoproteins by peripheral blood mononuclear cells from patients with connective tissue diseases

Antibodies to U1 ribonucleoproteins produced by peripheral blood mononuclear cells from patients with connective tissue diseases were measured by a sensitive enzyme-linked immunosorbent assay and by immunoblotting. Mononuclear cells from patients with high-titer serum antibodies spontaneously started secreting IgG anti-U1 RNP antibodies on the second day after culture. The amount of anti-U1 RNP in the culture supernatants reached maximum level on day 8. The most effective production of anti-U1 RNP by B cells was observed when they were cultured in the presence of T cells and adherent cells. Mononuclear cells from patients without anti-U1 RNP antibodies or from normal subjects did not produce a measurable amount of anti-U1 RNP. Treatment of mononuclear cells by cycloheximide resulted in complete inhibition of anti-U1 RNP secretion, which indicates that antibody in the culture supernatants reflects the active biologic phenomenon in vitro. The methods described should be useful in the study of the cellular mechanisms involved in antinuclear antibody production of connective tissue diseases.     

Inflammatory myositis associated with anti-U1-small nuclear ribonucleoprotein antibodies: a subset of myositis associated with a favourable outcome

OBJECTIVES: Inflammatory myositides are rare chronic disorders which may be either isolated or associated with other conditions such as connective tissue diseases or neoplasia. A large variety of autoantibodies can be detected in patients with myositis, some of which have a diagnostic and/or a prognostic value. Myositis associated with anti-U1-small nuclear ribonucleoprotein antibodies (anti-U1-snRNP Abs) are usually considered as overlapping syndromes, mainly mixed connective tissue diseases (MCTD) in which muscle symptoms occur insidiously during the disease course and are characterized by a favourable outcome. METHODS: The clinical, biological, immunological and pathological findings as well as the outcome of five patients with anti-U1-snRNP-associated myositis were retrospectively analysed. RESULTS: Patients were mainly black females. In all five patients, myositis was the predominant manifestation at presentation. Associated conditions consisted of interstitial lung disease (ILD) (three), arthritis (three) and neurological symptoms (two). No patient presented Raynaud's phenomenon nor met criteria for MCTD. Biological inflammatory features, rheumatoid factor and polyclonal hypergammaglobulinaemia were present in all cases. Besides anti-U1-snRNP Abs, one patient had anti-Ro/SSA and anti-La/SSB Abs at presentation and one additional patient developed anti-double-stranded-DNA and anti-Sm Abs after a follow-up of more than 4 yr. No patient had anti-PM/sclerosis (Scl) nor anti-aminoacyl-tRNA synthetase Abs. All patients dramatically improved with steroids, and reached complete remission (CR) within 3 weeks. Two patients relapsed 18 months after CR. They both reached rapidly second CR using steroids associated or not with oral methotrexate. CONCLUSION: Our data suggest that anti-U1-snRNP Abs may define a subset of myositis characterized by a favourable outcome, though often associated with ILD and/or neurological manifestations.     

Mixed connective tissue disease

A defining feature of mixed connective tissue disease (MCTD) is the presence of antibodies against the U1-ribonucleoprotein (RNP) complex, but other autoantibodies in MCTD have recently been described. Research has also further elucidated the immune responses directed against U1-RNP in humans and in murine models of disease. Hypotheses implicating modified self-antigens and/or infectious agents in the pathogenesis of MCTD have been advanced. Links between the immunologic and clinical phenomena in MCTD are emerging. Longitudinal study of patients with MCTD highlights the impact of pulmonary hypertension on disease outcome.     

Does Mixed Connective Tissue Disease Exist? Yes

For patients who have combined features of rheumatoid arthritis, the limited cutaneous form of systemic sclerosis, and inflammatory myopathies, the concept of mixed connective tissue disease (MCTD) often helps to predict and diagnose organ problems and to educate the patient accordingly. With high titer IgG antibodies to U1 ribonucleoprotein (U1-snRNP), this concept is supported by a specific serologic marker, and autoantibodies to U1-snRNP and to heterogeneous nuclear ribonucleoprotein (hnRNP)-A2 display MCTD specificity with regard to the recognized epitopes. In addition, the association of MCTD with HLA-DR4 distinguishes it from systemic erythematosus lupus and systemic sclerosis, and speaks to its being a disease entity, rather than a mixture of yet undifferentiated collagen vascular diseases. The authors believe that the concept is useful in daily practice and accurate in the idea that MCTD constitutes a disease entity of its own.     

Raynaud's phenomenon in mixed connective tissue disease

Raynaud's phenomenon affects most patients who have mixed connective tissue disease (MCTD) and frequently represents the initial manifestation of the disease. It is the cutaneous symptom of a systemic vasculopathy that is characterized by intimal fibrosis and blood vessel obliteration that frequently leads to visceral involvement, particularly pulmonary hypertension. An association between Raynaud's phenomenon and the characteristic autoantibody in MCTD, anti-U1-RNP (ribonucleoprotein), is found across the spectrum of rheumatic diseases, including undifferentiated connective tissue disease, scleroderma, and systemic lupus erythematosus. Capillary nailfold examination represents a valuable tool to identify patients who are at risk for MCTD. The goal in the therapy of Raynaud's phenomenon in MCTD is to decrease the frequency of attacks, to prevent digital ulceration, and to limit progressive vascular damage. Therapeutic regimens include the traditional use of calcium channel blockers and novel vascular therapies.     

Longitudinal study of interleukin-10, tumor necrosis factor-alpha, anti-U1-snRNP antibody levels and disease activity in patients with mixed connective tissue disease.

OBJECTIVE: To investigate the levels and relationship between IL-10, TNF-alpha, anti-U1snRNP antibodies and disease activity in longitudinally collected serum samples from patients with mixed connective tissue disease (MCTD). METHODS: Six patients followed for 17-138 months were investigated with ELISA for estimation of cytokine levels and antibodies to the different epitopes of the U1snRNP. Disease activity was assessed by systemic lupus activity measure (SLAM). RESULTS: IL-10 and TNF-alpha levels fluctuated with time in at least half of the patients. Three patients had increased IL-10 levels and two had increased TNF-alpha in all samples. There was no correlation between cytokine levels and disease activity or clinical manifestations. All patients had increased levels of antibodies to the main components of the U1snRNP. Both antibody levels and disease activity decreased with time. A correlation between TNF-alpha and U1snRNP antibody levels were observed in five patients. CONCLUSIONS: Increased and fluctuating levels of IL-10 or TNF-alpha without correlation to disease activity were observed in MCTD patients. In some patients increased cytokine levels were observed over several years irrespective of disease activity indicating that they could be constitutively increased in these individuals.     

Increase of cytokine production by pulmonary artery endothelial cells induced by supernatants from monocytes stimulated with autoantibodies against U1-ribonucleoprotein

OBJECTIVE: To clarify the pathogenetic role of autoantibodies against U1-RNP (ribonucleoprotein) (anti-U1-RNP) in mixed connective tissue disease (MCTD), we examined whether supernatants of monocytes which were stimulated with anti-U1-RNP could induce the production of proinflammatory cytokines by human pulmonary artery endothelial cells (HPAECs). METHODS: Monocytes from MCTD patients (n = 11) and normal volunteers (n = 11) were stimulated with purified antibodies against U1-RNP or double-stranded DNA and their supernatants were added to cultures of HPAECs. Cell-associated cytokines were assayed by an enzyme-linked immunosorbent assay. RESULTS: The supernatants of anti-U1-RNP-stimulated MCTD monocytes significantly up-regulated the cell-associated production of IL-1 alpha (p < 0.01) and IL-6 (p < 0.01) by HPAECs compared with their production by normal IgG-stimulated MCTD monocytes, whereas the cell-associated production of IL-1 beta and TNF-alpha by HPAECs was not up-regulated. The supernatants of anti-U1-RNP-stimulated monocytes from normal volunteers similarly up-regulated the cell-associated production by HPAECs of IL-1 alpha (p < 0.01) and IL-6 (p < 0.01), but not of IL-1 beta and TNF-alpha. Supernatants of monocytes stimulated with the F(ab')2 preparation of anti-U1-RNP antibodies enhanced the amounts of both Il-1 alpha and IL-6 associated with HPAECs almost as effectively as those stimulated with intact autoantibody molecules. Inhibition experiments employing specific anti-cytokine antibodies of anti-U1-RNP-stimulated monocyte supernatants suggested that soluble factors, including cytokines, in monocyte supernatants could enhance the cytokine association with HPAECs. CONCLUSION: Up-regulation by anti-U1-RNP autoantibodies of proinflammatory cytokines associated with vascular endothelial cells may play a role in the immunopathological processes leading to proliferative vasculopathy, a characteristic of MCTD.     

A new conformational epitope generated by the binding of recombinant 70-kd protein and U1 RNA to anti-U1 RNP autoantibodies in sera from patients with mixed connective tissue disease

OBJECTIVE: To establish an enzyme-linked immunosorbent assay (ELISA) using a complex of in vitro-transcribed U1 RNA and recombinant 70-kd, A, and C proteins (C-ELISA) to detect anti-U1 RNP antibodies reactive in double immunodiffusion (DID), but not in ELISA using the proteins alone (P-ELISA). METHODS: Sera from 196 patients with mixed connective tissue disease were used to test reactivity in P- and C-ELISAs, and the specificity of the sera was also tested by DID and immunoprecipitation (IP). RESULTS: In P-ELISA, 15 of 196 sera positive for anti-U1 RNP in DID did not react, while all sera reacted in C-ELISA. The reactivity of 15 sera to the U1 RNA was tested by IP and ELISA, and only 3 sera reacted with the U1 RNA. These results indicated that the increased reactivity in C-ELISA was not due to the U1 RNA itself. We confirmed that the 70-kd and A proteins were bound directly to the U1 RNA by IP using antibodies to His-tag, and we tested the reactivity of the sera to the U1 RNA-70-kd protein complex and the U1 RNA-A protein complex by IP. All sera reacted with the U1 RNA-70-kd protein complex, and 1 sample reacted with the U1 RNA-A protein complex. CONCLUSION: These results suggest that some anti-U1 RNP-positive sera specifically recognize the conformational structure altered by the binding of U1 RNA to the proteins, and the ELISA using U1 RNA and recombinant proteins is as useful as the DID method for detecting anti-U1 RNP antibodies.     

A recombinant autoantigen derived from the human (U1) small nuclear RNP-specific 68-kd protein. Expression in Escherichia coli and serodiagnostic application

A human liver complementary DNA expression library was screened using sera from patients with high titers of autoantibodies, to search for clones expressing major autoantigens that are relevant in connective tissue diseases. One of the clones isolated expressed a major epitope(s) that was immunoreactive with anti-U1 RNP sera, as shown by several techniques. Affinity-purified autoantibodies from the cloned RNP protein specifically recognized the 68-kd U1 RNP protein of HeLa cell nuclear extracts. All sera containing anti-U1 RNP antibodies detected by immunodiffusion, counterimmunoelectrophoresis, or immunoblotting also recognized the cloned RNP protein. The RNP antigen-expressing bacterial colonies and the partially purified cloned RNP fusion protein have been applied to fast and sensitive immunologic assays for the detection and quantification of anti-U1 RNP antibodies.     

Human autoantibodies against the 70-kd polypeptide of U1 small nuclear RNP are associated with HLA-DR4 among connective tissue disease patients

Serum samples from patients with connective tissue disease (CTD) were characterized using a recently developed enzyme-linked immunosorbent assay for reactivity with individual specific polypeptides of U1 small nuclear ribonucleoproteins, including the U1-70-kd protein. The distribution of HLA antigens was compared in CTD patients with and in those without anti-U1-70-kd autoantibodies and in normal controls. The frequencies of HLA-DR4 and HLA-DRw53 were increased among the anti-U1-70-kd autoantibody positive CTD group compared with the frequencies in anti-U1-70-kd autoantibody negative systemic lupus erythematosus patients and compared with normal controls. We conclude that the presence of autoantibodies reactive with the anti-U1-70-kd protein antigen is associated with HLA-DR4 and HLA-DRw53.     

Cardiovascular autonomic function, autoantibodies, and esophageal motor activity in patients with systemic sclerosis and mixed connective tissue disease

OBJECTIVE: To study cardiovascular autonomic nerve function and presence of autoantibodies in relation to esophageal motor activity in patients with systemic sclerosis (SSc) and mixed connective tissue disease (MCTD). METHODS: Twenty-five patients with SSc (13 limited, 12 diffuse cutaneous disease; disease duration 1-19 yrs) and 6 patients with MCTD (disease duration 1-10 yrs) were studied. Cardiovascular autonomic function was assessed using 5 standard tests and autoantibody status determined. Esophageal motor activity and lower and upper esophageal sphincter pressures were recorded manometrically. RESULTS: Five patients with SSc had definite, 7 borderline, and 13 no autonomic dysfunction; 23 had antinuclear. 9 anti-Sc170, 4 anticentromere, and 1 U1snRNP antibodies. Contraction amplitudes in the smooth muscle as well as the striated muscle esophagus and lower esophageal sphincter pressures were significantly lower and autonomic dysfunction more frequent in patients with than in those without anti-Sc170 (6 of 9 vs 6 of 16 patients); upper esophageal sphincter pressures did not differ. All patients with MCTD had antinuclear antibodies, 5 had definite autonomic dysfunction; their lower esophageal sphincter pressures were significantly lower than in SSc patients without anti-Sc170 and anti-U1snRNP. CONCLUSION: Esophageal motor dysfunction may be associated with the presence of anti-Sc170 and anti-U1snRNP autoantibodies and prevail in patients with cardiovascular autonomic neuropathy.     

Impacto clínico de la capilaroscopia periungueal en la práctica clínica diaria

IntroductionNailfold capillaroscopy (NC) is useful in the evaluation of Raynaud's phenomenon, associated with some connective tissue diseases and in the follow-up of patients with systemic sclerosis. Our study evaluates the impact of NC in the diagnosis, according to the reason for the request and profile of autoantibodies in daily clinical practice.Material and methodsAll patients that undergone at least one NC between June 2012 and December 2017 were included. Clinical records were reviewed and analysed in a dichotomous way (yes/no), to see whether the NC contributed to a change of diagnosis in subsequent consultations. In addition, demographic, clinical and laboratory data were collected, and the relationship with NC patterns evaluated.ResultsOf the 530 patients who had undergone at least one NC, 266 had Raynaud's phenomenon as primary indication for the technique. Of those, 20 patients (3.8%) had a diagnostic change in the post-NC consultation; 15 were diagnosed with systemic sclerosis, 4 with undifferentiated connective tissue disease and one with mixed connective tissue disease. All patients had, except for one patient diagnosed with undifferentiated connective tissue disease, positive antinuclear antibodies titres, 11 of them had disease specific antibodies (9 anti-centromere, one anti-Scl70 and other anti-RNPC). The positivity of antinuclear antibodies titres was associated with a higher probability of presenting a scleroderma pattern in the NC, and all patients with a specific rheumatological diagnosis had an abnormal NC.ConclusionNC is a useful technique, but with limited impact in the diagnosis of connective tissue diseases. Autoantibody positivity is associated with a greater likelihood of presenting pathological NC patterns.     

Mixed connective tissue disease: a case with scleroderma renal crisis following abortion

The term mixed connective tissue disease (MCTD) has been applied to a particular subset of patients with overlapping clinical features of systemic sclerosis, systemic lupus erythematosus, and polymyositis. Immune response to U1-ribonucleoprotein is the defining serological feature of MCTD. There are different organ and system involvements in MCTD including the heart, lung, kidney, muscle, joints, gastrointestinal, and hematologic involvements. Reports describing pregnancies in patients with MCTD are rare, and the results have been contradictory: a high risk of fetal loss and of disease exacerbation or no influence on fetus or mother. In MCTD, simultaneous pulmonary and renal involvement is very rare. In this paper, we report a case of MCTD with pulmonary involvement that developed scleroderma renal crisis after an abortion.     

A longitudinal study of autoantibodies against central nervous system tissue and gangliosides in connective tissue diseases

Our objective was to investigate longitudinally, antibodies against central nervous tissue (anti-CNS) derived from bovine brain and gangliosides GM 1, GD1a, GD1b, and GT1b in 91 patients with connective tissue diseases (systemic lupus erythematosus, n = 38; mixed connective tissue disease, n = 16; primary Sjogren's syndrome, n = 7; progressive systemic sclerosis, n= 13; polymyositis/dermatomyositis, n=4; overlap syndrome, n = 5; undifferentiated connective tissue disease, n = 8). Anti-CNS and anti-ganglioside antibodies, measured by enzyme-linked immunosorbent assay, were found in 73% and 63% of patients, respectively. Anti-CNS positive sera were also reactive in Western blotting in 74% of cases and recognized up to 14 different polypeptides from 29 to 130 kDa. Anti-CNS and anti-ganglioside antibodies reflected only in a limited extent the disease activity. In 27 of 58 patients, anti-CNS antibodies remained positive independently of disease activity and antibody levels did not correlate with the phases of exacerbations. A total of 36 of 60 anti-CNS-positive patients, in contrast to two of 22 anti-CNS-negative patients, had major neuropsychiatric manifestations (P < 0.001). Anti-ganglioside antibodies were not significantly associated with neuropsychiatric manifestations. In conclusion, our longitudinal data suggest that anti-CNS antibodies may be an important marker for the diagnosis of cerebral involvement in connective tissue diseases, but the pathogenic role of these autoantibodies remains to be determined.     

Antibodies to Sm and RNP. Prognosticators of disease involvement

The charts of 150 consecutive patients found to have antibodies to Sm, ribonucleoprotein (RNP), or both were examined to determine these antibodies' possible associations with certain clinical conditions as well as their diagnostic specificities. Patients with anti-Sm were more likely to have renal disease and antibodies to double-stranded DNA, single-stranded DNA, and nuclear protein than were patients with anti-RNP. No clinical associations were found for anti-RNP. Although most of the patients with antibodies to Sm, RNP, or both had systemic lupus erythematosus, some had other diagnoses, including cutaneous lupus, drug-induced lupus, rheumatoid arthritis, juvenile arthritis, mixed connective tissue disease, Raynaud's disease, progressive systemic sclerosis, miscellaneous rheumatic and nonrheumatic diseases, and undifferentiated connective tissue disease syndromes. These findings suggest that these antibodies may be associated with some diseases, but are not disease-specific.