Role of pathogenic auto-antibody production by Toll-like receptor 9 of B cells in active systemic lupus erythematosus.

OBJECTIVES: Toll-like receptor 9 (TLR9) is a pattern-associated receptor functioning in innate immunity that may be involved in the recognition of self-antigens and the production of pathogenic auto-antibodies. Therefore, we examined the expression of TLR9 in systemic lupus erythematosus (SLE) to determine whether TLR9 is involved in the production of pathogenic auto-antibodies. METHODS: B cells were collected from patients with active SLE, and subjected to analysis of the TLR9 molecule using flow cytometry fluorescence activated cell sorting (FACS) and TLR9 mRNA by reverse-transcriptase polymerase chain reaction. SLE B cells were stimulated with CpG-ODN, and subsequent cytokine and anti-dsDNA antibody production was measured by enzyme-linked immunosorbent assay. RESULTS: The expression and mRNA level of TLR9 on B cells was up-regulated in SLE patients, and SLE disease activity index (SLEDAI) and CH50 were correlated with TLR9 expression on CD20+ B cells. Moreover, TLR9-CpG interaction enhanced the production of anti-dsDNA antibody and IL-10. CONCLUSIONS: The present study demonstrated that higher expression of TLR9 on peripheral blood B cells from patients with active SLE was significantly correlated with CH50 and SLEDAI to TLR9, and induced the production of anti-dsDNA antibody and IL-10 by TLR9-CpG ligation. These results suggest that an abnormality of innate immunity plays a crucial role in the pathology of SLE, and that blockade of CpG-TLR9 interaction may be a new therapeutic approach for SLE.

Determination of IgG subclasses of immunoglobulin preparations for intravenous injection and the problems involved.

The IgG subclasses of immunoglobulin for intravenous injection (IVIg) were investigated after various different types of treatment. The level of IgG2 was relatively low in preparations treated with pepsin, but by changing the clone producing anti-IgG2 antibody, the level approached that of normal human serum. Though undetectable in sulfonated immunoglobulin preparations, IgG3 was detected after reoxidation. When determining the IgG subclasses of IVIg, it must be kept in mind that the apparent value may subsequently become lower due to the depressed reaction of the antibody.

Analysis of T cell responses to the beta 2-glycoprotein I-derived peptide library in patients with anti-beta 2-glycoprotein I antibody-associated autoimmunity.

Antiphospholipid syndrome (APS) is an autoimmune disease that accompanies anti-phospholipid antibodies measured as either anti-cardiolipin antibodies (aCL) or lupus anticoagulant. beta(2)-glycoprotein I (beta(2)GPI) is the most common and apparently the best-characterized antigenic target for aCL. To investigate T-cell responses to beta(2)GPI, we stimulated PBMC of 18 APS or systemic lupus erythematosus (SLE) patients carrying anti-beta(2)GPI and 10 healthy controls, using a peptide library covering the beta(2)GPI sequence. We established seven CD4(+) T cell lines reactive with beta(2)GPI peptide. Three of four epitopes for patient-derived T cell lines were p244-264, whereas one T cell line from a control subject also recognized p244-264. Furthermore, there was no tendency for particular HLA class II molecules to present beta(2)GPI peptides. However, cytokine producing patterns were significantly different between T cell lines from patients and those from healthy individuals (p =.028); patients' T cells tend to exhibit higher IL-4 and lower IFN-gamma responses. These T cell lines did not react to beta(2)GPI purified from human plasma. These results indicate that beta(2)GPI-reactive CD4(+) T cells of APS/SLE patients mainly recognize cryptic p244-264 in the context of various HLA class II molecules, and exhibit Th0-Th2-type responses. Our findings may provide a clue to the pathogenesis of APS.

The increased interleukin-13 in patients with systemic lupus erythematosus: relations to other Th1-, Th2-related cytokines and clinical findings.

The levels of interleukin-13 (IL-13) in patients with systemic lupus erythematosus (SLE) were examined and related to other Th1-/Th2- related cytokines, clinical manifestations and other markers. Serum levels of IL-13 and other cytokines, soluble markers were measured by enzyme-linked immunosorbent assay (ELISA). Patients with active SLE had a significantly increased level of IL-13. Most patients with high levels of IL-13 had higher levels of IL-6, and some patients had high levels of gammaIFN. These patients were divided into two groups according to the patterns of these increased cytokines; one with a high level of only Th2 related cytokines (IL-13 or IL-6) and another with high levels of both Th2 related and Th1 related cytokines (gammaIFN or IL-2). The latter patients had high levels of soluble CD8 and CD23, and some of them had hemolytic anemia or pulmonary involvement, while most of the former patients had nephropathy. Thus, in SLE, the levels of IL-13 were increased, and the heterogeneity of increased Th2- and Th1-related cytokines was related to that of activation markers and clinical manifestations.

HLA-DP-positive T cells in patients with systemic lupus erythematosus.

HLA-DP+ T cells in peripheral blood from 23 patients with systemic lupus erythematosus (SLE) were examined using two-colour flow cytometry analysis. A marked increase of HLA-DP+ T cells was observed in patients with SLE (20.5-98.7%; 59.8 +/- 20.8%) in comparison to normal subjects (1.3-20.6%; 11.1 +/- 7.2%), and the ratio of these cells greatly exceeded that of the HLA-DR+ T cells (6.5-49.1%; 21.5 +/- 12.7%). This high frequency of HLA-DP+ T cells in patients with active SLE decreased with prednisolone therapy. When the lymphocytes from normal subjects were stimulated with PHA in vitro, HLA-DP+ T cells increased from 1.8 to 59.2%. Therefore, it appears that the HLA-DP antigen expression on T cells is a practical marker for monitoring changes in the proportion of activated T cells in patients with SLE during the course of therapy.

The distinct subgroup of patients with rheumatoid arthritis shown by Ig G3-reactive rheumatoid factor.

The reactivity of rheumatoid factor (RF) with immunoglobulins of the IgG3 subclass was examined in 49 patients with rheumatoid arthritis (RA) using two types of IgG3 myeloma (routine and IgG3m-15 allotype). Among 49 patients, serum from eight cases showed positive reactivity with both types of IgG3 myeloma by radio-immunoassay (RIA). The isotype of IgG3-reactive RF was not specific; it belonged to the IgM class as well as the IgG subclasses IgG1, IgG2 and IgG4. The patients with IgG3-reactive RF belonged to the clinically-severe classification of RA, having a high erythrocyte sedimentation rate (ESR), high titre in the RA hemagglutination (RAHA) test, and above all they had low levels of complement. Generally, it is concluded that patients with IgG3-reactive RF have serious arthritis and that IgG3-reactive RF might play an important role in the inflammatory process. Furthermore, it was also shown that the RF-reactive site was not associated with the protein-A binding site of IgG3, since RF reacting with IgG3m-15 reacted similarly with routine IgG3, regardless of the difference of the protein-A binding activity. This was confirmed by adding protein-A to the reaction of RF and IgG3m-15 which binds with protein-A. This suggests that the actual reactive site of RF is different to the site that binds protein-A.

HLA-DRB1 alleles and beta 2 glycoprotein I-dependent anticardiolipin antibodies in Japanese patients with systemic lupus erythematosus.

OBJECTIVE: To investigate the association of HLA DRB1 alleles with beta 2 glycoprotein I (beta 2 GPI)-dependent anticardiolipin antibodies (aCL) in Japanese patients with systemic lupus erythematosus (SLE). METHODS: One hundred and forty-five Japanese patients with SLE were studied. beta 2 GPI-dependent aCL was measured by enzyme-linked immunosorbent assays. DNA typing of the DRB1 alleles was performed by the polymerase chain reaction sequence specific oligonucleotide probe method. RESULTS: beta 2 GPI-dependent aCL was positive in 29 (20.0%) out of 145 SLE patients. SLE patients with beta 2 GPI-dependent aCL had a significantly higher frequency or one or more of the clinical manifestations assumed to be associated with aCL, compared to those without beta 2 GPI-dependent aCL (p < 0.05). The frequency of DRB1*0901 was lower in SLE patients than in healthy subjects. SLE patients with beta 2 GPI-dependent aCL were significantly associated with DRB1*0901 as compared to those without beta 2 GPI-dependent aCL (41.4% vs 15.5%, p < 0.005, R.R. = 3.8), although the corrected P value was not significant. CONCLUSION: A possible association of DRB1*0901 with Japanese SLE patients with beta 2 GPI-dependent aCL was found. This association indicates an association between the disease and the HLA-DR53 (DRB4)-bearing haplotypes in different ethnic groups.

Soluble CD4, CD8 in patients with polymyositis/dermatomyositis.

The concentrations of soluble CD4 (sCD4) and soluble CD8 (sCD8) were determined in 64 patients with polymyositis/dermatomyositis (PM/MD). The patients with PM/DM had significantly higher concentrations of sCD8, though the concentrations of sCD4 did not significantly increase. Patients with high concentrations of sCD8 tended to have too high concentrations of soluble interleukin-2 receptor (sIL-2R). The patients with high levels of myogenic enzymes tended to have high concentrations of sCD8. The results of a serial study indicated that the concentrations of sCD8 decreased simultaneously with the decrease of the myogenic enzymes. These results may suggest that the activation of CD8+ cells are related to muscular involvement.

Soluble CD4 and CD8 molecules in patients with systemic sclerosis.

The concentrations of sCD4 and sCD8 in 69 patients with systemic sclerosis (SSc) were examined by using a sandwich enzyme-linked immunosorbent assay. The patients with SSc had significantly higher concentrations of sCD8 (mean 249.2 U/ml (SD 155.1), median 224 U/ml) than the normal subjects (mean 149.3 U/ml (SD 42.1), median 148 U/ml). The concentration of sCD4 in patients with SSc were significantly lower (mean 6.2 U/ml (SD 3.8), median 5.0 U/ml) than in the normal subjects (mean 10.9 U/ml (SD 4.1), median 10.3 U/ml). The concentration of sCD8 in patients with diffuse sclerosis tended to be higher than in those with sclerodactyly.

Expression of NKB1 on peripheral T cells in patients with rheumatoid arthritis.

NKB1 inhibits cytoxic activity of T lymphocytes mediated by superantigens, which is one of the contributing factors in the pathogenesis of rheumatoid arthritis (RA). In this study, we determined the expression of NKB1 on peripheral blood T cells in patients with RA. Our findings revealed that among patients with RA, NKB1(+) CD8(+ )T cells decreased significantly in comparison to controls (ratio: P < 0.05; absolute number: P < 0.005), and this decrease was not related to or influenced by HLA-Bw4 as a ligand of NKB1. This result may suggest that decreased expression of NKB1(+) CD8(+ )T cells contributes to the pathogenesis of RA mediated by the activation of CD8(+) T cells.

Significance of stem cell factor and soluble KIT in patients with systemic lupus erythematosus.

To determine the significance of stem cell factor (SCF) and soluble KIT (sKIT) in the serum of patients with systemic lupus erythematosus (SLE), levels of SCF and sKIT in patients with SLE were estimated, and their correlations with clinical parameters were examined. The sKIT levels in SLE patients (n = 106) were significantly lower than those in healthy controls (n = 40). A significant negative correlation was found between the SCF and sKIT levels of SLE patients. Although the SCF levels correlated with the titre of anti-RNP antibody, no significant relationship was found between SCF levels and blood cell counts, such as white blood cell, red blood cell and platelet counts. sKIT levels were significantly correlated with the platelet count, and were negatively correlated with the white blood cell count, titre of anti-DNA antibody, and SLE activity index (SLEDAI). sKIT levels were also negatively affected by high doses of corticosteroid. These results indicate that serum sKIT levels may be more closely related than SCF to the haematological abnormalities in SLE patients, and may reflect the clinical status of SLE patients and the effectiveness of high-dose corticosteroid treatment.

Increased soluble IL-2 receptor in serum of patients with systemic lupus erythematosus.

We estimated the concentration of soluble IL-2R (sIL-2R) in the serum of patients with systemic lupus erythematosus (SLE) and examined the relationship between the serum levels of sIL-2R and clinical features or laboratory data. We found that elevated levels of sIL-2R were present in the serum of SLE patients with discoid rash, and sIL-2R concentrations were correlated with the soluble CD4 and soluble CD8 concentrations but not with classical serological marker, anti-DNA antibody or complement titer.

Very low-dose cyclosporin treatment of steroid-resistant interstitial pneumonitis associated with Sjögren's syndrome.

Cyclosporin is known to be effective for both transplantation and a spectrum of immune-mediated diseases. Because this agent also causes severe adverse effects, especially nephrotoxicity, careful monitoring is required for the development of such reactions. Here we report the successful treatment with extremely low-dose cyclosporin (1 mg/kg/day) of a patient who had steroid-resistant interstitial pneumonitis and Sjögren's syndrome.

[The relation between IgG subclasses and clinical manifestations in patients with active systemic lupus erythematosus].

The levels of IgG subclasses were determined in patients with active systemic lupus erythematosus (SLE). The levels of all IgG subclasses in these patients were higher than normal controls. However, there were variations in the increase of IgG subclasses, especially IgG 1 or IgG 2. As concerning the relation to the clinical findings, the levels of IgG 1 significantly increased in patients with renal involvements and those of IgG 3 increased in patients with low complements. Thus, it was suggested that some IgG subclass related to some clinical findings.

The expression of lymphocyte function associated antigen-1, intercellular adhesion molecule-1 on peripheral blood lymphocytes in patients with systemic lupus erythematosus.

The ratios of CD11a, CD18, HLA-DP on T cells and CD54 on B cells of 54 patients with active systemic lupus erythematosus (SLE) were examined. The ratios of LFA-1 alpha, beta, ICAM-1, HLA-DP among SLE patients were significantly higher when compared with normal healthy controls, and the significant correlation between the ratio of LFA-1+ T cells and HLA-DP+ T cells, and LFA-1+ T cells and ICAM-1+ B cells was recognized. These results may suggest that LFA-1, ICAM-1 is related to the mutual action between activated T cells and B cells.

Effective treatment of autoimmune diseases with extremely low dose cyclosporine.

The dosage of cyclosporine administered in the treatment of autoimmune diseases has generally been comparable to those required in cases of transplantation. Here we report on the successful treatment using an extremely low dose cyclosporine (1 mg/kg/day) on four patients, involving thrombocytopenia with systemic lupus erythematosus, and interstitial pneumonitis with Sjögren's syndrome, and discuss the optimal dose of cyclosporine for autoimmune-mediated manifestations.

[A case of reactive arthritis fallen after shigellosis infection].

A 25-year-old woman was admitted for general arthralgia in July, 1989. Reactive arthritis with arthralgia after Shigellosis was diagnosed by sex, localization of arthralgia and positive for HLA-B 27. Within 3 weeks after starting diclifenac sodium 75 mg/day, the arthralgia remitted. It has been reported that patients who are positive for HLA-B 27 have a more severe acute or chronic sacroiliitis, and our case may support this report.