Expression of BAFF and BAFF-R in the synovial tissue of patients with rheumatoid arthritis.

OBJECTIVE: The elevated expression of B-cell-activating factor belonging to the TNF family (BAFF) is associated with systemic autoimmune disease, including rheumatoid arthritis (RA). The present study was undertaken to determine the distribution of BAFF and its receptor BAFF-R in the cells residing in the rheumatoid synovium. METHODS: The expression of BAFF and BAFF-R in synovial tissues obtained from 12 RA patients was examined by immunohistochemistry and flow cytometry. The mRNA expression of these molecules was determined by reverse transcriptase polymerase chain reaction (RT-PCR). Soluble BAFF levels were measured with an enzyme-linked immunosorbent assay (ELISA). Fibroblast-like synoviocytes (FLS) purified from the RA (RA-FLS) were co-cultured with peripheral B cells. The degree of apoptosis in the B cells was measured to assess the effects on the viability of the B cells. RESULTS: The RA synovium showed focal or diffuse infiltration of mononuclear cells (MNCs), and one specimen showed germinal centre (GC)-like structures. Synovial sublining cells, but not lining cells, expressed BAFF. These sublining cells were negative for BAFF-R. BAFF and BAFF-R were expressed in B and T cells extracted from the RA synovium. Notably, RA-FLS spontaneously expressed cytoplasmic BAFF after 4-6 passages; however, they did not express BAFF or BAFF-R on their cell surface. RA-FLS could support the survival of B cells by preventing their apoptosis, but its effect on B cells might not be BAFF dependent. CONCLUSIONS: BAFF and BAFF-R are widely expressed in the RA synovium. The cells residing in the RA synovium might affect each other through BAFF.

A retrospective study of the fluctuation in serum levels of anti-cyclic citrullinated peptide antibody in patients with rheumatoid arthritis.

OBJECTIVE: To investigate the fluctuation in serum levels of anti-cyclic citrullinated peptide antibody (anti-CCP) retrospectively in patients with rheumatoid arthritis (RA). METHODS: Serum levels of anti-CCP were measured retrospectively in 131 patients with RA and 90 patients with non-RA rheumatic diseases using a commercially available kit. All sera were collected from patients during the 22-year period, 1982-2004. To analyze the fluctuation in anti-CCP levels, 17 RA patients were selected on the basis of showing a significantly higher anti-CCP level in a serum sample taken at the first visit (> 80 U/ml), and availability of preserved serum samples that had been taken from each patient at 10 time points. RESULTS: The test gave a sensitivity of 88% (115/131) and a specificity of 81% (73/90). The longitudinal study of 17 RA patients showed that anti-CCP levels were elevated at the first visit in 12 (71%) patients and then decreased gradually, whereas those in the other five (29%) patients fluctuated substantially. In both cases, anti-CCP levels tended to fluctuate in parallel with the erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) level, reflecting the spontaneous aggravation of arthritis and the efficacy of anti-rheumatic drugs. The courses of three representative RA patients are illustrated in detail along with their therapeutic regimens, and these further confirm the correlation of anti-CCP levels with laboratory parameters (ESR and CRP) as well as the activity of arthritis. CONCLUSION: Measurement of serum anti-CCP levels was found to be useful for not only the diagnosis but also the management of RA.

Elevated immunoglobulin G antibodies to the proline-rich amino-terminal region of Epstein-Barr virus nuclear antigen-2 in sera from patients with systemic connective tissue diseases and from a subgroup of Sjögren's syndrome patients with pulmonary involvements.

Associations of Epstein-Barr virus (EBV) and autoimmune diseases have been hypothesized. We have analysed IgG antibodies to EBV nuclear antigen (EBNA)-2 in sera from Japanese patients with autoimmune systemic connective tissue diseases (CTD), exemplified by systemic lupus erythematosus (SLE), primary Sjogren's syndrome (SS), rheumatoid arthritis (RA), systemic sclerosis (SSc) and secondary SS (classical CTDs complicated with SS). An enzyme-linked immunosorbent assay (ELISA) which uses glutathione-S-transferase polypeptides fused to EBV nuclear antigen (EBNA)-2 and EBNA-1 was developed. Ratios of IgG antibody reactivity to whole IgG concentrations of sera were calculated to normalize EBNA-2 and EBNA-1 antibody levels to the hypergammaglobulinaemia that occurs in CTD. The ELISA optical density OD(450) readings of IgG antibodies to both the amino-terminal aa 1-116 of EBNA-2 and carboxyl-terminal aa 451-641 of EBNA-1 were elevated significantly in patients with SLE, primary SS, RA, SSc and secondary SS when compared to EBNA-1. The OD readings were divided by serum IgG concentrations to normalize for the hypergammaglobulinaemia. The specific levels of IgG antibodies to the amino-terminal region of EBNA-2 were elevated in patients with SLE, primary SS or RA, as well as those with secondary SS complicated with SLE or RA. The EBNA-2 amino-terminal region contains a polyproline tract and a proline-rich sequence and has considerable amino acid sequence homology with many cellular proline-rich proteins. High ratios of EBNA-2 aa 1-116 to EBNA-1 aa 451-641 IgG antibody levels which probably suggest reactivation of EBV latent infection were associated significantly with pulmonary involvement in SS patients. These results are consistent with the hypothesis that the sequence similarity between the amino-terminal region of EBNA-2 and proline-rich cellular proteins is associated with pathogenesis in a subpopulation of CTD patients, possibly by the molecular mimicry-epitope shift mechanism.

Effects of intravenous anesthetics on interleukin (IL)-6 and IL-10 production by lipopolysaccharide-stimulated mononuclear cells from healthy volunteers.

BACKGROUND: Surgical trauma has been shown to augment the plasma concentrations of proinflammatory cytokines, which are important mediators of host defense mechanisms and the systemic inflammatory response syndrome (SIRS). Recently, it has been shown that certain kinds of surgery provoke not only a proinflammatory response (SIRS) but also a concurrent anti-inflammatory response. The aim of this study was therefore to examine the effects of intravenous anesthetics on the synthesis of interleukin (IL)-6 (a proinflammatory cytokine) and IL-10 (an anti-inflammatory cytokine) by lipopolysaccharide (LPS)-stimulated mononuclear cells from healthy volunteers. METHODS: Peripheral blood mononuclear cells (PBMCs) from 17 healthy volunteers, separated by centrifugation on a Ficoll-Hypaque gradient, were washed and suspended in RPMI containing 10% heat-inactivated fetal calf serum (FCS). After adding RPMI-FCS containing various concentrations of intravenous anesthetics (propofol, thiopental, ketamine and midazolam), the PBMCs were incubated overnight in the presence of a submaximal concentration of LPS. The supernatants were collected and their IL-6 and IL-10 contents were assayed using enzyme-linked immunosorbent assay kits. RESULTS: Propofol inhibited both IL-6 and IL-10 production at 0.5 microg/mL, 5 microg/mL and 50 microg/mL. Conversely, thiopental induced IL-10 production at 2 microg/mL and 20 microg/mL. CONCLUSION: Propofol appears to inhibit both IL-6 and IL-10 production by LPS-stimulated PBMCs in vitro. Further study is required to clarify the mechanism of the suppressive effect of propofol.

Endothelial cell-binding activity of anti-U1-ribonucleoprotein antibodies in patients with connective tissue diseases.

In order to elucidate the immunological properties of anti-U1-ribonucleoprotein (RNP) antibody, one of the autoantibodies detected in patients with connective tissue diseases (CTDs), we tested the endothelial cell-binding by anti-U1-RNP antibodies and epitopes on human pulmonary artery endothelial cells (HPAECs) to which the autoantibody bound. IgG fractions positive for anti-U1-RNP from patients with CTDs bound to the HPAECs. Furthermore, intact and F(ab')2 IgG anti-U1-RNP purified by affinity chromatography also bound to endothelial cells. The binding activity of IgG fractions positive for anti-U1-RNP to the endothelial cells could be effectively absorbed by U1-RNP-Sepharose. An immunoblotting assay of purified IgG anti-U1-RNP antibodies showed that these antibodies could bind to various membrane proteins of NP40-treated HPAECs such as 68, 48, 43, 38, 33, 29, 28 and 24 kDa. Some bands, 68, 33, 28 and 24 kDa, seemed to correspond to components of U1-RNP, i.e. 68 kDa, A, B' and C peptides, respectively. We confirmed that the anti-U1-RNP antibody from patients with CTDs can directly recognize a variety of antigens on the endothelial surface of the pulmonary artery, including the components of U1-RNP or other unknown polypeptides. These results suggest that binding to pulmonary artery endothelial cells of this autoantibody may be one of the triggers of endothelial cell inflammation in CTDs.

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.

Up-regulation of intercellular adhesion molecule-1 (ICAM-1), endothelial leucocyte adhesion molecule-1 (ELAM-1) and class II MHC molecules on pulmonary artery endothelial cells by antibodies against U1-ribonucleoprotein.

In order to elucidate the pathogenic role(s) of autoantibodies in connective tissue disease (CTD), we examined whether autoantibodies against U1-ribonucleoprotein (RNP) and double-stranded (ds) DNA can up-regulate ICAM-1, ELAM-1 and class I and II MHC molecule expression on pulmonary artery endothelial cells (HPAEC). ICAM-1, ELAM-1 and class II MHC molecule expression on HPAEC cultured in the presence of anti-U1-RNP-containing and anti-dsDNA-containing IgG from CTD patients was up-regulated significantly in comparison with that on HPAEC cultured with IgG from normal healthy volunteers. Affinity chromatographic enrichment and depletion of the anti-U1-RNP antibody content of anti-U1-RNP-containing IgG confirmed that the anti-U1-RNP antibody did up-regulate ICAM-1, ELAM-1 and class II MHC molecule expression. The finding that an IgG F(ab')2-purified anti-U1-RNP antibody also up-regulated expression of these molecules may indicate that mechanisms other than Fc receptor-mediated stimulation are involved. These in vitro findings suggest that autoantibodies against U1-RNP and dsDNA play important roles in the immunopathological processes leading to the proliferative pulmonary arterial vasculopathy observed in CTD patients with pulmonary hypertension by up-regulating adhesion and class II MHC molecule expression on endothelial cells.

Increased levels of matrix metalloproteinase-3 in sera from patients with active lupus nephritis.

OBJECTIVE: To determine the matrix metalloproteinase-3 (MMP-3) levels in sera from patients with systemic lupus erythematosus (SLE) and to analyse the relationships between MMP-3 and clinical and laboratory features. METHODS: Serum MMP-3 levels were measured by an enzyme immunoassay in 124 patients with SLE and 237 patients with other systemic rheumatic diseases. RESULTS: The frequencies of patients with high MMP-3 levels were 76% in SLE and 82% in rheumatoid arthritis (RA). The level of MMP-3 in the SLE patients was 193.0 +/- 171.5 ng/ml (mean +/- SD) and was almost equal to the level in the RA patients (259.5 +/- 255.6 ng/ml). The MMP-3 levels were significantly higher in SLE patients who had a history of the following abnormalities: persistent proteinuria, cellular casts, anti-double stranded DNA antibodies, decreased C3, decreased creatinine clearance (p < 0.001), circulating immune complex (p < 0.01), malar rash, hypoalbuminemia, or decreased C4 (p < 0.05). The serum MMP-3 level in patients with SLE at admission showed direct correlations with serum uric acid, total cholesterol (p < 0.001), triglyceride, the white blood cell count, and the neutrophil count (p < 0.05), as well as inverse correlations with the total protein (p < 0.01), and IgG (p < 0.05). In SLE patients with active renal disease, the median MMP-3 level at admission and that at 6 months after admission were significantly higher than that at 6 months before admission. CONCLUSIONS: The increased level of serum MMP-3 in SLE is closely associated with clinical features relevant to lupus nephritis, suggesting that it plays a role in the pathogenesis of this condition.

Histological evaluation of destructive monoarthropathy in mixed connective tissue disease.

The synovium from a patient with mixed connective tissue disease and destructive ankle monoarthritis was studied in detail to determine its immunohistological characteristics. Fibrinoid necrotic tissue on the surface of the synovium, multi-layered lining cells, increased numbers of capillaries, interstitial edema, infiltration of macrophages, relatively small numbers of lympho-plasma cells and polymorphonuclear leukocytes, scattered bone fragments, and multinucleated giant cells were observed. Many cells in the lining and sublining area were positive for CD68 and MAC387. Lower layers of increased lining cells which had a spindle shape were positively stained with anti-HLA-DR antibody. The small arteries in the deeper part of the synovium revealed obstruction or highly stenotic change. These findings suggest that obstructive circulatory disturbance due to endothelial injury might influence the progression of arthritis.

Clinical significance of the serum lipoprotein(a) level in patients with systemic lupus erythematosus: its elevation during disease flare.

OBJECTIVE: To determine the clinical significance of serum lipoprotein(a) [Lp(a)] levels in patients with systemic lupus erythematosus (SLE). METHODS: Serum Lp(a) levels in 77 patients with SLE were measured by turbidimetric immunoassay. RESULTS: The median serum Lp(a) levels in all the SLE patients (14.4 mg/dl) and in those with active disease (Group A; 19.6 mg/dl, n = 39) at admission were significantly higher than those in healthy subjects (11.9 mg/dl, p < 0.05 and p < 0.001, respectively). The serum Lp(a) levels in SLE patients correlated directly with the serum cholesterol (p < 0.001) and urinary protein (p < 0.001) levels and inversely with the serum albumin levels (p < 0.02). Analysis limited to Group A patients with renal disease (Group A + RD, n = 28) revealed that the median serum Lp(a) level at the time of admission (OM) was significantly higher than those at 6 months before (-6M, p < 0.01) and at 6 months after admission (+6M, p < 0.01). Moreover, the serum Lp(a) level decrease from 0M to +6M in Group A+RD correlated significantly with the serum albumin level increase (p < 0.05). Multiple regression analysis demonstrated that the serum albumin level increment, the SLEDAI score decrement, the cholesterol level at 0M and the total dose of oral corticosteroids administered during the 0M to +6M period contributed independently and significantly to the serum Lp(a) level decrement from 0M to +6M in Group A + RD. CONCLUSION: Our study is the first to reveal that hypoalbuminemia appearing during disease flare plays an important role in increasing the serum Lp(a) levels in lupus patients with renal disease and shows that corticosteroid treatment reduced the elevated serum Lp(a) levels almost to original levels.

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.

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.

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.

Adsorption of anti-dsDNA antibodies by immobilized polyanionic compounds.

Antibodies to dsDNA are characteristically present in serum from patients with systemic lupus erythematosus (SLE), and have been shown to have the capacity to react with various molecules bearing repeating negative charges. After a number of polymeric or monomeric molecules with differently charged groups and hydrophobic molecules had been coupled covalently as ligands on cellulose gel, the adsorption capacities of the ligands for anti-dsDNA antibodies were evaluated. It was found that gels coupled with polyanionic dextran sulphate (DXS) and polyacrylic acid (PA) and monoanionic sulphanilic acid (SA) absorbed anti-dsDNA antibodies effectively. DXS gel also adsorbed antibodies to ssDNA and heparan sulphate, antigens with repeating negatively charged moieties, while no ligand was able to adsorb anti-nRNP antibodies. The finding that DXS gels adsorbed anti-dsDNA antibody in proportion to their charge density, and that the interaction between anti-dsDNA and DXS gel is broken readily by an increase in ionic strength, indicated that the binding is ionic in nature. Moreover, virtually all F(ab')2 anti-dsDNA became adsorbed onto the DXS gels, suggesting that the binding occurred via specific antigen-binding sites on the antibody molecule. Binding of these polyanion-binding autoantibodies with anionic sites in the glomerular basement membrane may therefore cause the tissue damage observed in SLE.

Monocyte-mediated suppression of anti-DNA antibody production in healthy subjects.

Monocyte-depleted mononuclear cells (MNC) from healthy subjects synthesized IgM-class antibody to single-stranded DNA (anti-ssDNA) at significantly higher levels than unfractionated MNC. The increased production of IgM anti-ssDNA by monocyte-depleted normal MNC was inhibited to a greater degree than total IgM by addition of supernatants from autologous monocytes. Moreover, supernatants obtained from normal monocytes cultured with indomethacin retained this suppressive effect on IgM anti-ssDNA antibody production, suggesting that prostaglandin E2 may not be involved in the suppression. These results indicate that B cells programmed to produce IgM anti-ssDNA are present in the normal B cell repertoire, but may be suppressed by monocytes or by a soluble factor (or factors) from the monocytes.

Analysis of negatively charged dye-binding antibodies reactive with double-stranded DNA and heparan sulfate in serum from patients with rheumatic diseases.

Antibodies to double-stranded (ds) DNA are characteristically present in serum from patients with systemic lupus erythematosus (SLE). Recently, anti-dsDNA antibodies have been shown to have the capacity to react with a diversity of molecules with repeating negative charges. Using the anionic dye Cibacron blue F3GA, bound to crosslinked agarose, we analysed the nature of antibodies capable of reacting with this dye in serum samples from patients with various rheumatic diseases. The dye-antibody complex could easily be split by eluting with solutions of increasing ionic strength, suggesting that the interaction is ionic in nature. Pepsin-digested F(ab')2 antibodies retained the capacity to bind Cibacron blue, confirming that the binding occurred via antigen-binding sites on the antibody molecule. The eluates obtained from dye-ligand chromatography of active SLE sera contained antibodies to both dsDNA and heparan sulfate, while those of sera from patients with other non-SLE rheumatic diseases contained antibodies only against heparan sulfate. Furthermore, the dye-ligand eluates of sera from patients with active SLE and other non-SLE rheumatic diseases were found to contain increased amounts of IgG. In one patient with SLE, levels of antibodies to dsDNA and heparan sulfate, and the amounts of total IgG in dye-ligand eluates, were shown to be correlated with disease activity.