系统性红斑狼疮患者外周血干扰素基因的定量表达

目的　探讨系统性红斑狼疮(SLE)患者外周血中IFN α、 β、 ω和 γmRNA与蛋白的定量表达水平与SLE疾病特异性和病情活动性是否相关。方法　对 144例SLE患者、27例非SLE患者与 59例正常人取外周血抽提总RNA并逆转录成cDNA,运用实时定量PCR法在基因测序仪上检测 4种IFN的mRNA表达水平,分离血浆,用ELISA法检测 4种蛋白水平,并与病情特异性和活动性进行分组比较,分析其意义。结果　(1)SLE患者组的 4种IFNmRNA定量表达水平均显著低于正常对照组, 差异有统计学意义 (P值均<0 01);SLE患者组的IFN αmRNA定量表达水平同时显著高于非SLE对照组 (P<0 01 )。(2)SLE活动组与非活动组之间的 4种IFN表达水平差异无统计学意义(P值均>0 05),SLE患者组的 4种IFNmRNA表达水平与有无肾、肺、脑损害无相关性。(3)SLE患者组的IFN积分显著低于正常对照组, 差异有统计学意义 (P<0 01 )。(4)SLE患者组和正常对照组组内的 4种IFN表达水平之间均呈强正相关 (各组间P<0 01 )。(5)IFN α、 β和 ω的蛋白水平在正常对照和SLE患者中差异均无统计学意义 (P值均 >0 05),IFN α的蛋白水平在活动期SLE患者比非活动期显著增高 (P<0 01 )。IFN γ的蛋白水平在SLE患者中有升高趋势。结论　IFN α、 β、 ω和 γ的mRNA定量表达水平的减低     

Interferon-alpha and gamma product in peripheral blood of patients with thyroid diseases

To investigate the immunological process in various thyroid disease, we measured interferon-alpha, -gamma (IFN-alpha,-gamma, natural killer (NK) activity, and lymphocyte subsets in the peripheral blood of 27 patients with Basedow's disease (BD) (M:F = 9:18), 8 with Hashimoto's thyroiditis (HT) (2:6), 5 with idiopathic hypothyroidism (1HT) (1:4), and normal controls (C). IFN-alpha, -gamma levels were measured by bioassay with Dye-uptake method, and NK activity was measured by the LDH method. The mean +/- SD levels of IFN-gamma in BD, HT, IHT, and C (N = 217) were 173.9 +/- 88.0, 288.0 +/- 134.9, 120.4 +/- 38.0 and 173.9 +/- 88.01U/ml, respectively. The IFN-gamma level was higher in HT (p less than 0.05) than in controls, and lower in BD (p less than 0.02). Moreover, this IFN-gamma level did not correspond with the titers of thyroid hormones; TSH, anti-microsome antibody, and anti-TSH-receptor antibody in peripheral blood. However, IFN-alpha levels and NK activity in the patients of every group were similar to those in controls. The ratios of lymphocyte subsets of peripheral blood were measured by cytofluorometry with monoclonal antibodies. The mean +/- SD levels of T cells in BD, HT, IHT and C were 75.9 +/- 7.0, 83.4 +/- 7.2, 85.2 +/- 5.7, 82.3 +/- 5.8%, and those of B cells were 17.5 +/- 6.1, 10.9 +/- 4.2, 8.0 +/- 5.8, 10.9 +/- 5.0%, respectively. The ratio of T cells was higher in IHT (p less than 0.05) and lower in BD (p less than 0.01) than in controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Surface characteristics of synovial fluid and peripheral blood lymphocytes in inflammatory arthritis

In order to characterize the T- and B-cell populations of inflammatory arthritides, synovial fluid and peripheral blood lymphocytes from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), juvenile rheumatoid arthritis (JRA), and rheumatoid variant diseases (RV) were studied. Normal peripheral blood lymphocytes were examined as controls. T cells were identified by spontaneous sheep red blood cell (E) rosette formation. B cells were determined by complement receptor lymphocyte (EAC) rosette formation and by the presence of surface immunoglobulins (SIg) utilizing fluoresceinated polyvalent antiglobulin. Synovial fluids were analyzed in terms of duration, mucin quality, white cell count, and differential. Synovial fluid and peripheral blood lymphocytes from patients with RA and RV were distributed in T- and B-cell percentages similar to those found in normal peripheral blood. In contrast, significant T-cell depression was observed in the percentage of synovial fluid and peripheral blood lymphocytes of SLE and JRA patients. This depression was apparent in comparison with normal peripheral blood and with the synovial fluid and peripheral blood from RA patients. B-cell percentages were similar in all patient groups and in comparison to normal peripheral blood lymphocytes. No differences were noted in B-cell percentages when the EAC and SIg techniques of identification were compared. The percentage of cells bearing neither T- nor B-cell markers (null cells) was enumerated for each patient group and found to be significantly elevated in the synovial fluid and peripheral blood of SLE and JRA patients. Though the mean synovial fluid and peripheral blood null cell percentages in RA patients were similar to those in controls, a definite bimodal distribution was found in the synovial fluids. These data suggest that evaluation of T-, B-, and null-cell populations may be clinically useful in differentiating patients with SLE and JRA from those with rheumatoid arthritis and variant diseases.     

Dual downregulation of microRNA 17-5p and E2F1 transcriptional factor in pediatric systemic lupus erythematosus patients

To investigate the relative expression of miRNA 17-5p and one of its target genes E2F1 in peripheral blood of systemic lupus erythematosus SLE pediatric patients. miRNA 17-5p and its target E2F1 miRNA expressions in SLE peripheral blood and controls were analyzed by TaqMan real-time qPCR. Peripheral blood mononuclear cells (PBMCs) of SLE and controls were transfected using a nucleofector device, and E2F1 protein expression was analyzed using luciferase reporter gene assays. Results showed significant downregulation of miRNA 17-5p in SLE patients compared to healthy controls; moreover, miRNA 17-5p was more downregulated in patients on no treatment compared to those on treatment. Relative expression of E2F1, which is target for miRNA 17-5p, was significantly downregulated as well on both miRNA and protein levels in SLE. Our data show an unexpected dual downregulation of both miRNA 17-5p and its target gene E2F1 on the mRNA and protein levels. This may suggest an expression pattern of miRNA 17-5p and its target E2F1 that may be specific to SLE.     

Quantitation of IL-2Rp75 (CD122) expression on mononuclear cells in rheumatic disease.

OBJECTIVE: To compare expression of the p75 chain of the interleukin-2 receptor (IL-2Rp75, CD122) on peripheral and synovial mononuclear cells in rheumatoid and non-rheumatoid inflammatory arthritis. METHODS: Peripheral blood (PBMC) and synovial (SFMC) mononuclear cells were isolated from subjects with rheumatoid arthritis (n = 16) and non-rheumatoid inflammatory arthritis (n = 12). PBMC were isolated from six healthy controls. Expression of CD122 was examined using indirect immunofluorescence and quantitative flow cytometry. RESULTS: There was no difference in IL-2Rp75 expression on PBMC from rheumatoid arthritis patients, non-rheumatoid arthritis patients, and controls. In subjects with rheumatoid arthritis there was no difference in IL-2Rp75 expression on PBMC and SFMC. However, in the non-rheumatoid arthritis group there was an increase in IL-2Rp75 expression on SFMC compared with PBMC (P = 0.0032). On SFMC there was a greater expression of IL-2Rp75 in non-rheumatoid arthritis than in rheumatoid arthritis (P = 0.0007). Expression was greater on CD8 positive cells and in subjects with shorter duration of disease. CONCLUSIONS: The p75 chain of the IL-2 receptor, an important T cell activation antigen, is not upregulated in synovial fluid. This appears to be a disease specific defect and provides further support for the concept of "frustrated" or incomplete T cell activation in this disease.     

Presence of cutaneous interferon-alpha producing cells in patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) patients have increased levels of interferon-alfa (IFN-alpha) in the circulation but a reduced number of functionally intact natural IFN-alpha producing cells (IPC) in peripheral blood. In search for tissue localisation of activated IPC, we investigated skin biopsies from SLE patients for the occurrence of such cells. Eleven SLE patients with inflammatory skin lesions and six healthy controls were biopsied. An immunohistochemical technique (IH) and in situ hybridisation (ISH) were used to detect intracellular IFN-alpha protein and IFN-alpha mRNA, respectively. In all 11 biopsies from SLE lesions, a high number of IPC were detected by IH. In the nonlesional SLE biopsies we could also demonstrate IPC in 10/11 patients. In 6/11 SLE patients, IFN-alpha mRNA containing cells could be detected in the specimens. A low number of IPC were detected in 1/6 healthy controls by IH, but no ISH positive cells were seen. Our results demonstrate that SLE patients have active IPC in both dermal lesions and in noninflammatory skin. A recruitment of IPC from blood to peripheral tissues may explain the low number of circulating natural IPC in SLE patients. Because the type I IFN system is involved in the SLE disease process, these results are of interest for the understanding of the pathogenesis in SLE.     

Overexpression of osteopontin in rheumatoid synovial mononuclear cells is associated with joint inflammation, not with genetic polymorphism

OBJECTIVE: Osteopontin (OPN) is thought to play an important role in rheumatoid synovitis. We investigated the expression of OPN in rheumatoid synovial fluid mononuclear cells (SFMC) and its potential association with genetic polymorphism of the OPN gene and joint inflammation in rheumatoid arthritis (RA). METHODS: 1. The expression of OPN mRNA in peripheral blood mononuclear cells (PBMC) and SFMC of patients with RA was analyzed quantitatively by real-time polymerase chain reaction (PCR). Results were analyzed in paired PBMC and SFMC and control PBMC. 2. Six single nuclear acid polymorphisms of the OPN gene were genotyped in a cohort of 192 Chinese patients with RA and controls (n = 288) by restriction fragment length polymorphism PCR or direct DNA sequencing. 3. SF derived from RA patients was examined for the stimulating effect on mRNA expression of the OPN gene in PBMC. RESULTS: The expression of OPN gene was significantly increased in SFMC and, to a lesser degree, in PBMC of patients with RA compared to control PBMC (p < 0.01). However, the prevalence of OPN genotype and allele frequencies at the selected positions did not differ significantly between RA patients and the control group (p > 0.05). Further characterization indicated that SF known to contain a variety of proinflammatory factors significantly stimulated mRNA expression of OPN in PBMC obtained from RA patients or healthy controls. CONCLUSION: Overexpression of OPN mRNA in SFMC is associated with proinflammatory factors produced in inflamed joints, but not with OPN genetic polymorphisms. OPN gene polymorphisms do not correlate with susceptibility to RA.     

Monocyte cytotoxicity in connective tissue diseases. Correlation with disease groups

The proportion of antibody-dependent cytotoxic plaque-forming cells (PFC) with monocyte characteristics was estimated in peripheral blood mononuclear leukocyte suspensions from 40 patients with rheumatoid arthritis (RA), (29 seropositive and 11 seronegative), 7 with ankylosing spondylitis (AS), 14 with juvenile rheumatoid arthritis (JRA) and 7 with systemic lupus erythematosus (SLE). In RA and also in AS patients, higher mean proportions of PFC were found when compared with those of normal adult blood donors. Seropositive RA patients had a significantly higher proportion of PFC than seronegative RA patients. SLE patients showed values equal to those of the controls. Patients with JRA had a lower percentage of these cells than age-matched healthy children.     

Expression of costimulatory molecules on peripheral blood lymphocytes of patients with systemic lupus erythematosus

OBJECTIVE: In systemic lupus erythematosus (SLE) autoantibody production is T cell dependent. For a proper T and B cell interaction, signalling of costimulatory molecules on these cells is necessary. The expression of costimulatory molecules on peripheral blood lymphocytes in patients with SLE in conjunction with disease activity was measured to evaluate whether expression of costimulatory molecules in SLE is increased. METHODS: Thirteen patients with SLE with active disease, 10 patients with inactive disease, and 14 controls entered the study. In addition, samples from 10 of the 13 patients with active disease could be studied at a moment of inactive disease as well. Isolated peripheral blood lymphocytes were stained for the lymphocyte subset markers CD4, CD8, CD19, their respective activation markers CD25, HLA-DR, CD38, and the costimulatory molecules CD40L, CD28, CD40, CD80, and CD86. Expression was measured by flow cytometry. RESULTS: Peripheral blood lymphocytes of patients with SLE showed signs of increased activation at the moment of active disease. Almost all CD4+ T cells expressed CD28, both in patients and in controls. CD80 expression on CD19+ B cells was low in both groups and did not correlate with disease activity. In contrast, the percentage of CD19+ B cells expressing CD86 was increased in patients with SLE even in patients with inactive disease (p=0.04) and correlated with the SLEDAI score (p=0.0005) and levels of anti-dsDNA (p=0.006). No changes in CD40 or CD40L expression were found in the patients with SLE. CONCLUSION: In patients with SLE the expression of CD86 on CD19+ B cells is increased and is associated with disease activity, B cell activation, and levels of anti-dsDNA. The increased CD86 expression will render (autoreactive) B cells more susceptible for T cells. This can facilitate autoantibody production and might be a target for immunosuppressive treatments.     

Ethnic differences in risk for pediatric rheumatic illness in a culturally diverse population

OBJECTIVE: To analyze the differences of occurrence of pediatric rheumatic disease among various ethnic groups in a culturally diverse isolated geographic area. METHODS: A retrospective study of pediatric rheumatic diseases in a multiethnic area during a 6 year period. RESULTS: A group of 922 patients was categorized based on predominant ethnicity, and their risk of having acute rheumatic fever (ARF), juvenile rheumatoid arthritis (JRA), and systemic lupus erythematosus (SLE) was studied. Odds ratios (OR) were computed for each illness with Caucasians as the reference group. Results indicated that Polynesians were overrepresented among patients with ARF, having elevated OR that were significantly different from Caucasians (22.5-120.7, p < 0.0001). For SLE, the highest OR were obtained for Samoans, Filipinos, and Japanese. In contrast, for JRA, Filipinos and Japanese had OR less than one, and no Samoans were diagnosed with JRA, possibly indicating a protective effect against developing JRA. CONCLUSION: This unique retrospective study examined the ethnic variations of expression of certain rheumatic diseases in an isolated region. Results reveal that certain ethnic groups are at risk for ARF and SLE, but are protected against JRA. These findings suggest investigating possible immunogenetic similarities and differences in these illnesses.     

Tumor necrosis factor induced adhesion molecule serum concentrations in Henoch-Schönlein purpura and pediatric systemic lupus erythematosus

OBJECTIVE: Animal models of immune complex mediated tissue injury have shown that tumor necrosis factor (TNF) and TNF induced adhesion molecules play an important role in the pathogenesis of tissue damage mediated by IgG, but not in that mediated by IgA, immune complexes. We compared possible differences in the behavior of 2 TNF induced adhesion molecules (VCAM-1 and ICAM-1) in Henoch-Sch?nlein purpura (HSP), which is characterized by the formation of IgA immune complexes, versus systemic lupus erythematosus (SLE), which is mostly associated with the vascular deposition of IgG immune complexes. METHODS: Serum concentrations of soluble (s)VCAM-1 and ICAM-1 were determined by ELISA methods in 20 patients with pediatric SLE showing variably active disease, 20 active patients with active HSP, and 19 healthy controls. TNF-alpha as well as p55 and p75 soluble receptors (sTNF-R) were simultaneously tested by enzyme amplified sensitivity immunoassay in 22 patients (12 SLE, 10 HSP). RESULTS: Serum sVCAM-1 concentration was significantly higher in patients with SLE (mean +/- SD, 608 +/- 76 ng/ml), than in patients with HSP (501.9 +/- 63.3 ng/ml) and controls (446.8 +/- 139.2 ng/ml) (p < 0.001). In SLE patients, sVCAM-1 correlated positively with ESR (r = 0.45, p = 0.02) and negatively with C4 serum levels (r = -0.57, p = 0.004), platelets (r = -0.38, p = 0.03), and lymphocyte count (r = -0.42, p = 0.03). No differences in sICAM-1 serum concentrations were detected among SLE, HSP, or control groups. Soluble VCAM, but not sICAM-1, showed a positive correlation with TNF-alpha (r = 0.71, p = 0.01), p55 (r = 0.63, p = 0.02), and p75 (r = 0.7, p = 0.01) sTNF-R serum concentrations in SLE, but not in patients with HSP. CONCLUSION: Our study provides additional evidence of a possible differential involvement of TNF and TNF induced adhesion molecules in the pathogenesis of tissue damage between pediatric SLE and HSP.