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.

[Standardization of quantitative measurement of rheumatoid factor].

Recently many automated methods have been developed for a quantitative measurement of rheumatoid factor (RF). As they are widely used, it has been claimed that estimated values of RF are quite different among institutions. Standardization of quantitative measurement of RF is, therefore, required. The subcommittee for standardization of RF of the Ministry of Health and Welfare of Japan, worked from 1988 to 1994 in order to obtain an appropriate national standard of RF and has reached the conclusion that murine monoclonal rheumatoid factor (mRF) would be recommended as a new national standard of RF, which allows new expression of RF value (microgram/ml equivalent to the mRF instead of unit/ml referred to the WHO standard). There are two types of methods widely used for the quantification of RF. The first type is a method of light scattering analysis which is based on interaction of RF with particle-coated or aggregated human IgG (Fc) in nepherometry or turbidimetry, RF being expressed as unit/ml referred to the WHO standard. The second is a radioimmunoassay or an enzyme immunoassay in which RF is expressed as index compared to arbitrary standard. The former expression of RF is recognized to indicate agglutination titer of RF and the latter expresses the concentration of RF. The subcommittee decided to target on the former type of methods because of rapid prevalence with large inter-laboratory variations. The mRF is able to agglutinate proportionally IgG-complex with various features in wide range (x x microgram/ml to more than 1 mg/ml). It can be measured by nepherometry and turbidimetry with sufficient stability. The mRF has no misgivings in present and future supply and in immutability after a century. One of the largest advantages of the use of mRF national standard is expected to be able to eliminate the inter-laboratory variations of RF values which are considered to be mainly derived from different methods of laboratory instruments as well as from biological properties of polyclonal RF. The use of microgram/ml equivalent to mRF instead of unit/ml referred to the WHO standard makes it possible to determine the exact RF value of test serum since the mRF preparation could be adjusted to the best concentration to be requested by any available method of instrument to measure agglutination titer of RF.

Clinical features of patients with juvenile onset mixed connective tissue disease: analysis of data collected in a nationwide collaborative study in Japan.

OBJECTIVE: To analyze the clinical features and outcome of juvenile-onset mixed connective tissue disease (MCTD). METHODS: Clinical and laboratory findings were compared in 2 groups of MCTD patients divided according to age at onset: juvenile onset: under 16 yrs: adult onset: 16 yrs or older). RESULTS: Systemic lupus erythematosus-like symptoms, such as facial erythema, photosensitivity. LE cells, lymphadenopathy, and cellular casts, were more frequent in juvenile onset MCTD than in the adult form of the disease. On the other hand, scleroderma-like symptoms, such as esophageal hypomotility, scleroderma-like lesions evident on skin biopsy, pulmonary involvement, proximal scleroderma, and pitting scars, were less frequent in juvenile onset MCTD than in the adult form. Patients with juvenile onset MCTD more frequently met the classification criteria for systemic lupus erythematosus (SLE) and less frequently met those for progressive systemic sclerosis (SSc), compared to patients with adult onset MCTD. At disease onset, hand edema and stiffness were observed less frequently in juvenile onset MCTD than in the adult form. Furthermore, the mortality rate was lower in the former than in the latter (2.8% vs 8.4%). CONCLUSION: Although previous studies have reported severe symptoms and adverse outcome for juvenile onset MCTD, we conclude from this nationwide study in Japan that patients with juvenile onset MCTD exhibit more SLE-like and fewer SSc-like features and have a relatively favorable outcome.

C1q-binding immunoglobulin G in MRL/l mice consists of immune complexes containing antibodies to DNA.

Previous studies have shown that the majority of C1q-binding IgG in patients with systemic lupus erythematosus (SLE) is composed of autoantibodies to the collagen-like region of C1q. Mice of the MRL/l strain are considered as a murine model of human SLE and possess autoantibodies to nuclear antigens as well as IgM and IgG rheumatoid factors (RF). This study was undertaken to characterize the C1q-binding IgG in MRL/l mice. In contrast to human SLE, C1q-binding IgG in MRL/l mice showed immunochemical characteristics of immune complexes rather than those of autoantibodies to C1q. Namely, C1q-binding IgG in MRL/l mice was large-sized upon HPLC gel filtration and abolished by digestion with pepsin or by high salt concentration, and bound to the globular region of C1q. The C1q-binding activity in MRL/l mice was absorbed by double-stranded DNA- or single-stranded DNA-cellulose. The medium-sized immune complexes containing RF have been well documented in MRL/l mice. In this study, however, mouse IgG-Sepharose failed to absorb fully C1q-binding IgG. We conclude that the majority of C1q-binding IgG in MRL/l mice consists of large-sized immune complexes containing antibodies to DNA.

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.

Calcitonin inhibits production of immunoglobulins, rheumatoid factor and interleukin-1 by mononuclear cells from patients with rheumatoid arthritis.

OBJECTIVES: Elcatonin (eCT), an eel calcitonin derivative, is shown to considerably improve the clinical signs and symptoms, as well as laboratory data, in patients with rheumatoid arthritis (RA). The therapeutic efficacy of eCT, however, is reduced by preceding and/or concomitant use of corticosteroid. Thus the effects of eCT on the production of immunoglobulins, IgMRF and interleukin-1 (IL-1) by mononuclear cells (MNCs)/monocytes were studied, and compared among patients with RA that received three kinds of treatment and also normal volunteers (NV). METHODS: Ten patients with RA had been treated with a non-steroidal anti-inflammatory drug only (NSAID group), 11 with oral prednisolone (PSL group), and eight with intramuscular eCT (eCT group). MNCs/monocytes from these patients, and also 10 from the NV group, were collected and cultured. IgG, IgA, IgM, IgMRF, IL-1 alpha and IL-1 beta in the supernatants were measured by enzyme-linked immunosorbent assay (ELISA). In the NSAID, PSL and NV groups, eCT was added to the culture medium, and the effects of eCT on production of these substances were studied. RESULTS: Baseline production of IgM, IL-1 alpha and IL-1 beta by MNCs/monocytes in the eCT and NV groups was significantly lower than that in the NSAID group. Furthermore, addition of eCT to the culture medium significantly inhibited the productions of IgG, IgMRF, IL-1 alpha and IL-1 beta by MNCs/monocytes in the NSAID group, whereas production of neither IgG, IgA, IgM, IgMRF nor IL-1 by MNCs/monocytes in the PSL and NV groups was affected by eCT. CONCLUSION: eCT may regulate immune responses through MNC/monocyte function in patients with RA. The present results support our proposal that eCT is an effective agent for the treatment of RA.

Down-regulation by eel calcitonin of interleukin-1 release and production by peripheral blood monocytes from patients with rheumatoid arthritis.

To clarify the reason for the therapeutic efficacy of Elcatonin (eCT), an eel calcitonin derivative, in rheumatoid arthritis (RA), we studied the effect of eCT on the extracellular (EC) release and intracellular (IC) production of interleukin-1 (IL-1) by peripheral blood monocytes from patients with RA. In vitro treatment of RA monocytes with eCT reduced predominantly the EC release of both IL-1 alpha and IL-1 beta. Moreover, EC release and IC production of IL-1 alpha and IL-1 beta by monocytes from RA patients who received non-steroidal anti-inflammatory drugs (NSAIDs) plus eCT (CT group) were significantly lower than in those who received NSAIDs only (NSAID group). The anti-rheumatic effect of eCT may be mediated via the inhibition of EC release and the IC production of IL-1 from RA patients.

The B cell repertoire in patients with systemic autoimmune diseases: analysis of Epstein-Barr virus (EBV)-inducible circulating precursors that produce autoantibodies against nuclear ribonucleoprotein (nRNP).

Peripheral blood B cells from patients with systemic autoimmune disease and healthy volunteers were immortalized using EBV and the frequencies of B cell precursors that produced immunoglobulin class-specific antibodies against anti-nRNP, a specific marker for mixed connective tissue disease, were assessed using limiting dilution analysis. The frequencies of EBV-induced B cell precursors that produced IgG anti-nRNP were correlated closely with the serum titres of the corresponding autoantibodies, which indicates that B cell precursors that produced potentially pathogenic autoantibodies could be immortalized from the peripheral blood of the patients by EBV. In contrast, the frequency of EBV-induced B cell precursors that produced IgM anti-nRNP in patients with systemic autoimmune disease was comparable to that in healthy volunteers and greater than those that produced IgG and IgA anti-nRNP. Moreover, many of the clones that produced IgM antibodies against nRNP reacted with other autoantigens, such as double-stranded DNA, single-stranded DNA and rabbit IgG. These polyreactive IgM antibodies are believed to belong to the 'natural antibodies', to be coded by the germline immunoglobulin V genes, and to react with evolutionarily conserved structural cellular components, including nRNP. Our finding that nRNP is one of the target antigens for this polyreactive autoantibody may lead to the elucidation of the origin of the pathogenic IgG and IgA anti-nRNP antibodies found in sera from patients with systemic autoimmune diseases.

Correlation of antibodies to ribosomal P protein with psychosis in patients with systemic lupus erythematosus.

Ninety one Japanese patients with systemic lupus erythematosus (SLE) were studied to determine the clinical significance of antibodies to ribosomal P protein (anti-P). Anti-P was detected by western blotting in 38 of 91 patients (42%). Clinical symptoms of SLE were compared between patients with and without anti-P. The occurrence of lupus psychosis was significantly higher in patients with anti-P than in those without anti-P (9/38 v 1/53). No significant association was found between anti-P and other symptoms of SLE. These data strongly support the suggestion proposed by previous workers that anti-P is a marker autoantibody for the development of lupus psychosis.

Risk factors in the pregnancy of patients with systemic lupus erythematosus: association of hypocomplementaemia with poor prognosis.

Fetal wastage is still high in the pregnancies of patients with systemic lupus erythematosus (SLE). We examined retrospectively the cases of 38 patients with inactive SLE in whom pregnancy was either desired or had already been obtained. The prevalence of antiphospholipid antibodies in the group with fetal loss was high. The antibodies were, however, also detected in five of 14 patients who had had a live birth. It was noted that low levels of serum complement activity (CH50 less than 25 U/ml) occurred in five of six patients with fetal loss, but in only two of 22 with a live birth. Serial studies also confirmed a close association between decreased serum complement activity and poor fetal prognosis in lupus pregnancy. Treatment with increased doses of prednisolone may help to achieve successful live births. Thus hypocomplementaemia may be associated with a worse prognosis for the fetus in the pregnancies of some patients with SLE in remission.

Antibodies against sulphatide in sera from patients with autoimmune rheumatic diseases.

We tested sera of patients with various autoimmune rheumatic diseases for the presence of antibodies against sulphatide (an acidic glycosphingolipid), identified as a target antigen for antibodies against the liver cell membrane. Thirty-five percent (7/20) of patients with lupus in the active stage possessed anti-sulphatide antibodies, whereas 10% (2/20) of those in the inactive stage and 20% (4/20) of those in the stationary stage possessed such antibodies. Moreover, 10% (3/29) of patients with other autoimmune rheumatic diseases also possessed anti-sulphatide antibodies. The level of anti-sulphatide antibodies was significantly correlated with the levels of anti-double-stranded (ds) DNA antibodies (r = 0.634, P less than 0.001) and dextran sulphate-binding IgG (r = 0.407, P less than 0.001). The serum levels of antibodies against sulphatide were correlated with a history of seizures or psychosis in patients with autoimmune rheumatic diseases. Gels coupled with polyanionic dextran sulphate, monoanionic sulphanilic acid and DNA were shown effectively to adsorb anti-sulphatide antibodies in the sera of patients with active systemic lupus erythematosus (SLE) and autoimmune chronic active hepatitis (AI-CAH). These results suggest that the observed reactivity with sulphatide is due to the presence of antibodies capable of reacting with various anionic molecules in the sera of patients with autoimmune rheumatic diseases as well as those with AI-CAH.

Circulating immune complex levels measured by new ELISA kits utilizing monoclonal anti-C1q and anti-C3d antibodies correlate with clinical activities of SLE but not with those of RA.

The authors studied sera from both patients with SLE and from those with RA to evaluate clinical usefulness and significance of circulating immune complexes (CIC) detected with new ELISA kits utilizing monoclonal anti-C1q and anti-C3d antibodies. CIC values of patients with SLE significantly correlated to severity of disease activities evaluated by clinical symptoms and laboratory tests, especially for serum complement levels. Since substances detected with the ELISA kits were closely related to serum complement components, it was determined that a direct relationship exists between clinical activities and CIC values appearing in SLE patients with hypocomplementemia. In RA patients, CIC values did not correlate to clinical activities evaluated by Lansbury's index, anatomical bone damage with X-ray or functional assessment of activities of daily living, but did significantly correlate to levels of IgM-RF, serum IgG concentrations and some markers of systemic inflammation. Detection of IC after fractionation of RA sera revealed a broad range of molecular sizes detectable with the ELISA kits, which indicated that CIC in vivo were heterogenic and complicated in formation, degradation and interaction with serum complements.

[Preliminary criteria for the diagnosis of pulmonary hypertension in mixed connective tissue disease].

Based on analysis of data obtained from multicenter patients with collagen diseases (83 with pulmonary hypertension(PH) and 472 without PH), preliminary criteria for the diagnosis of PH in mixed connective tissue disease (MCTD) were proposed by the Research Committee for MCTD of the Ministry of Health and Welfare of Japan. The diagnosis of PH requires four or more out of six clinical and laboratory findings, including exertional dyspnea, systolic pulsation on the left sternum, increased 2nd pulmonary sound, dilatation of the pulmonary artery on chest X-ray, right ventricular hypertrophy on the electrocardiogram, and right ventricular enlargement on the echocardiogram. Alternatively, either an increase of mean pulmonary artery pressure over 25 mmHg measured by right ventricle catheterization, or the corresponding finding on Doppler echocardiography is also valid for the diagnosis of PH. When these criteria were applied to the patients in this study, the sensitivity was 92% and the specificity 100%, showing that PH may be adequately diagnosed using non-invasive methods. The number of criteria satisfied by patients with PH was well correlated with their mean pulmonary artery pressure measured by heart catheterization.

Selective removal of anti-DNA and anticardiolipin antibodies by adsorbent plasmapheresis using dextran sulfate columns in patients with systemic lupus erythematosus.

The effect of adsorbent plasmapheresis using dextran sulfate columns on anti-DNA and/or anticardiolipin antibodies (aCL) in 6 patients with systemic lupus erythematosus (SLE) was studied by multicenter clinical trials. The titers of anti-DNA (RI assay), IgG anti-dsDNA (ELISA), IgG and/or IgM anti-ssDNA, (ELISA), and IgG aCL (ELISA) significantly decreased or normalized after 4 treatments of plasmapheresis during a 2 to 4 week period. A patient with SLE with recurrent abortion and aCL who was successfully treated by adsorbent plasmapheresis is reported. We expect that adsorbent plasmapheresis will be an influential treatment for patients with not only SLE but aCL syndrome.

Production of intracellular and extracellular interleukin-1 alpha and interleukin-1 beta by peripheral blood monocytes from patients with connective tissue diseases.

An enzyme linked immunosorbent assay (ELISA) was used to estimate the production of intracellular and extracellular interleukin-1 (IL-1) alpha and beta by peripheral blood monocytes from 26 patients with various connective tissue diseases (CTD), including 19 with systemic lupus erythematosus, four with progressive systemic sclerosis, two with mixed connective tissue disease, and one with Sjögren's syndrome. Monocytes obtained from patients with CTD with serum antibodies to nuclear ribonucleoprotein (nRNP) released significantly higher concentrations of extracellular IL-1 alpha and IL-1 beta, whereas intracellular IL-1 alpha and IL-1 beta production was similar to that by monocytes from patients with CTD without antibodies to nRNP. Furthermore, the concentrations of extracellular IL-1 alpha correlated significantly with those of extracellular IL-1 beta. There was no significant correlation between the concentrations of extracellular and intracellular IL-1 alpha, and those of extracellular and intracellular IL-1 beta, indicating that synthesis and secretion of IL-1 by human monocytes may be two distinct biological events. It seems that enhanced extracellular release of both IL-1 alpha and IL-1 beta contributes to the excessive anti-nRNP production in CTD.

Suppressive effect of anti-rheumatic drugs on interleukin-1 beta release from human peripheral blood monocytes.

We developed an ELISA system for human IL-1 alpha and -beta release from silica-stimulated monocytes from healthy volunteers and tested the effect of several anti-rheumatic drugs including nonsteroidal anti-inflammatory drug (Ibuprofen). Anti-rheumatic drugs including Auranofin and Sulphasalazine suppressed IL-1 beta release significantly at therapeutic concentrations, whereas Bucillamine, Lobenzarit, D-Penicillamine and Ibuprofen did not. These results suggest a possible immunotherapeutic effectiveness of some anti-rheumatic drugs on rheumatoid arthritis through their inhibition of IL-1 beta release.

Interleukin-1 beta release from human peripheral blood monocytes associated with phagocytosis of carbonyl-iron or erythrocytes.

Interleukin-1 beta (IL-1 beta) release from human peripheral blood monocytes during the incubation with carbonyl-iron or sheep red blood cells was investigated. The incubation of purified monocytes with carbonyl-iron or sheep red blood cells enhanced IL-1 beta release, while their compounds, hemoglobin, globin and ferric citrate did not. The mechanisms of IL-1 beta release by carbonyl-iron or sheep red blood cells may be related to their phagocytosis, as non-phagocytic monocytes did not release IL-1 beta.

Adherent synovial cells from nonrheumatoid arthritis do not release interleukin 1 beta and prostaglandin E2 spontaneously in longterm culture.

Adherent synovial cells from both 13 patients without rheumatoid arthritis (RA) (gout, osteoarthritis and meniscal lesion) and 8 patients with RA consisted of dendritic cells, macrophage-like cells and fibroblast-like cells after cloning in a similar fashion as reported in our previous paper. All the adherent synovial cells from patients without RA did not release interleukin 1 (IL-1) beta and prostaglandin E2 (PGE2) spontaneously, while those cells released comparable amounts of IL-1 beta, but not PGE2 to RA cells after type II collagen stimulation. Only the synovial cells from RA, irrespective of morphology and cloning, released IL-1 beta and PGE2 without stimulation. Nonrheumatoid synovial cells may differ functionally from RA cells.