Expression of human B-Cell specific receptor FCRL1 in healthy individuals and in patients with autoimmune diseases

The expression levels of the FCRL1 gene, which encodes a human B-cell surface receptor, were compared in healthy individuals and patients with autoimmune diseases. The expression levels were evaluated using DNA dot hybridization on membranes with spotted cDNA samples derived from blood-cell sub-populations of patients with autoimmune diseases. Quantification of the hybridization signals showed that FCRL1 expression in peripheral blood B-lymphocytes of patients with multiple sclerosis, lupus anticoagulants, Takayasu’s arteritis, and von Willebrand disease was significantly higher than in healthy individuals. Monoclonal and polyclonal FCRL1-specific antibodies that enable FCRL1 detection in Western blotting, immunohistochemistry, and flow-cytometry assays were generated. It was found that FCRL1 is expressed on the surfaces of mature CD19+ B-cells. In the tonsils, FCRL1-positive cells were located in the crypt area, i.e., in the mantle zone of secondary lymphoid follicles and among the cells of lymphoid epithelium. FCRL1-positive cells were also found in B-cell follicles of the spleen.     

Enhanced RIG-I expression is mediated by interferon regulatory factor-2 in peripheral blood B cells from hepatitis C virus-infected patients

Chronic hepatitis C patients carry the risk of developing into B-cell non-Hodgkin’s lymphoma (B-NHL). To clarify the mechanisms underlying this association, we first investigated the molecular markers of B cells from hepatitis C virus (HCV)-infected patients. CD19-positive cells were isolated as B cells from the peripheral blood mononuclear cells of patients infected with the hepatitis C virus and IFN-related gene expression was analyzed. We found that RIG-I and IRF-2 expression were up-regulated in CD19-positive cells from the infected patients. In vitro luciferase reporter analysis using human cell lines indicated that IRF-2 activates the human RIG-I promoter. IRF-2 expression levels were enhanced by HCV cDNA transfection in Huh7 cells. In addition, we observed much less induction in the interferon stimulated gene 15 (ISG15) after Sendai virus (SenV) stimulation of CD19-positive cells from infected patients versus healthy controls, thereby suggesting an impairment of RIG-I downstream signaling in HCV-infected patients. Hence, we found that the failure of the anti-viral response with enhanced IRF-2 oncogenic protein expression in blood B cells from HCV-infected patients. Our results provide important information to better understand the role of IRFs in the cause of HCV chronic infection.     

FCRL3 promotes IL‐10 expression in B cells through the SHP‐1 and p38 MAPK signaling pathways

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system that is caused by the interaction of genetic and environmental factors. Current studies have shown that Fc‐receptor like‐3 (FCRL3) is closely related to MS, but the specific role of FCRL3 in MS has not yet been clarified. This study further found that FCRL3 and interleukin 10 (IL‐10) expression was downregulated in MS patients, but the expression of these proteins was higher in the remission phase than that in the acute phase. The C allele of rs7528684 was associated with MS, and the CC genotype could lead to the upregulation of FCRL3 expression and the increase in IL‐10 secretion. Further in vitro experiments with B cells found that lipopolysaccharide (LPS) promoted FCRL3 expression in a dose‐ and time‐dependent manner, thereby promoting IL‐10 secretion. LPS regulated Src homology region 2 domain‐containing phosphatase‐1 (SHP‐1) expression and p38 mitogen‐activated protein kinase (MAPK) pathway activation through FCRL3, and FCRL3 upregulated the SHP‐1 expression and p38 phosphorylation levels. When SHP‐1 small interfering RNA or a p38 pathway inhibitor was added, the effect of FCRL3 on IL‐10 secretion was significantly inhibited. In addition, FCRL3 inhibited the secretion of inflammatory factors (tumor necrosis factor‐α, IL‐1β, IL‐6, and IL‐8); after inhibiting the expression of IL‐10, the abovementioned effects of FCRL3 were blocked. These results suggest that FCRL3 can activate the SHP‐1 and p38 MAPK pathways and then promote the secretion of IL‐10 in B cells, thus inhibiting the secretion of inflammatory factors. Therefore, FCRL3 may play an immunoprotective role in MS, and it will be an effective target for the diagnosis and treatment of MS.     

Low representation of Fc receptor-like 1–5 molecules in leukemic cells from Iranian patients with acute lymphoblastic leukemia

Recent studies have demonstrated expression of Fc receptor-like (FCRL) molecules, a newly identified family with preferential B-cell lineage expression, in some chronic B-cell leukemias with possible implication for classification and/or targeted immunotherapy. In this study, the expression pattern of FCRL1-5 genes was studied in 73 Iranian ALL patients and 35 normal subjects using semi-quantitative RT-PCR method. FCRL protein expression was also investigated by flow cytometry. Our results indicate significant down-regulation of all FCRL genes in ALL compared to normal subjects. Although, FCRL mRNA expression was almost exclusively confined to normal isolated B-cells compared to T-cells, but these genes were similarly expressed in B-ALL, T-ALL and different B-ALL immunophenotypic subtypes. Surface protein expression of FCRL1, 2, 4, and 5 molecules in 10 ALL and 5 normal samples confirmed the PCR results. Expression profile of FCRL molecules in different subtypes of ALL argues against their potential implication as suitable targets for classification and/or immunotherapy of ALL. T. Kazemi, H. Asgarian-Omran and A. Memarian have contributed equally to this study.     

Interleukin-28A promotes IFN-γ production by peripheral blood mononuclear cells from patients with Behçet’s disease

Behçet’s disease (BD) is an autoimmune disease of unknown etiology. Interleukin-28A (IL-28A) promotes immune responses and may participate in the pathogenesis of autoimmune diseases. To examine the role of IL-28A in the pathogenesis of BD, we measured the expression of IFN-γ and IL-17 by IL-28A-stimulated peripheral blood mononuclear cells (PBMCs) from 19 patients with BD and 16 healthy individuals. We found that IFN-γ and IL-17 were undetectable in the sera from BD patients and control subjects. The mRNA expression and protein production of IFN-γ by IL-28A-stimulated PBMCs from BD patients were significantly increased compared to healthy individuals. No significant difference was observed in the mRNA expression and protein production of IL-17 by IL-28A-stimulated PBMCs between BD patients and normal individuals.     

Aberrant Circulating Th17 Cells in Patients with B-Cell Non-Hodgkin’s Lymphoma

Non-Hodgkin’s lymphomas (NHLs) are a heterogeneous group of neoplasm in which 90% are B-cell lymphomas and 10% T-cell lymphomas. Although T-helper 17 (Th17) cells have been implicated to be essential in the pathogenesis of autoimmune and inflammatory diseases, its role in B-cell non-Hodgkin’s lymphoma (B-NHL) remains unknown. In this study, we observed a significantly decreased frequency of Th17 cells in peripheral blood from B-NHL patients compared with healthy individuals, accompanied with increased Th1 cells. IL-17AF plasma levels were remarkably decreased in B-NHL patients, accompanied with undetectable IL-17FF and unchangeable IL-17AA. Moreover, Th17 and Th1 cells became normalized after one or two cycles of chemotherapy. Interestingly, in B-NHL, circulating Th17 cells frequencies were significantly higher in relapsed patients than those in untreated patients or normal individuals. Meanwhile, there was no statistical difference regarding the frequencies of Th1 cells between relapsed and untreated patients. Taken these data together, circulating Th17 subset immune response may be associated with the response of patients to treatment and with different stages of disease.     

Primary B-cell deficiencies reveal a link between human IL-17-producing CD4 T-cell homeostasis and B-cell differentiation

IL-17 is a pro-inflammatory cytokine implicated in autoimmune and inflammatory conditions. The development/survival of IL-17-producing CD4 T cells (Th17) share critical cues with B-cell differentiation and the circulating follicular T helper subset was recently shown to be enriched in Th17 cells able to help B-cell differentiation. We investigated a putative link between Th17-cell homeostasis and B cells by studying the Th17-cell compartment in primary B-cell immunodeficiencies. Common Variable Immunodeficiency Disorders (CVID), defined by defects in B-cell differentiation into plasma and memory B cells, are frequently associated with autoimmune and inflammatory manifestations but we found no relationship between these and Th17-cell frequency. In fact, CVID patients showed a decrease in Th17-cell frequency in parallel with the expansion of activated non-differentiated B cells (CD21(low)CD38(low)). Moreover, Congenital Agammaglobulinemia patients, lacking B cells due to impaired early B-cell development, had a severe reduction of circulating Th17 cells. Finally, we found a direct correlation in healthy individuals between circulating Th17-cell frequency and both switched-memory B cells and serum BAFF levels, a crucial cytokine for B-cell survival. Overall, our data support a relationship between Th17-cell homeostasis and B-cell maturation, with implications for the understanding of the pathogenesis of inflammatory/autoimmune diseases and the physiology of B-cell depleting therapies.     

Increased levels of (class switched) memory B cells in peripheral blood of current smokers

There is increasing evidence that a specific immune response contributes to the pathogenesis of COPD. B-cell follicles are present in lung tissue and increased anti-elastin titers have been found in plasma of COPD patients. Additionally, regulatory T cells (Tregs) have been implicated in its pathogenesis as they control immunological reactions. We hypothesize that the specific immune response in COPD is smoke induced, either by a direct effect of smoking or as a result of smoke-induced lung tissue destruction (i.e. formation of neo-epitopes or auto antigens). Furthermore, we propose that Tregs are involved in the suppression of this smoke-induced specific immune response.The presence of B cells, memory B cells and Tregs was assessed by flow cytometry in peripheral blood of 20 COPD patients and 29 healthy individuals and related to their current smoking status.COPD patients had lower (memory) B-cell percentages and higher Treg percentages in peripheral blood than healthy individuals, with a significant negative correlation between these cells. Interestingly, current smokers had higher percentages of (class-switched) memory B cells than ex-smokers and never smokers, irrespective of COPD.This increase in (class-switched) memory B cells in current smokers is intriguing and suggests that smoke-induced neo-antigens may be constantly induced in the lung. The negative correlation between B cells and Tregs in blood is in line with previously published observations that Tregs can suppress B cells. Future studies focusing on the presence of these (class switched) memory B cells in the lung, their antigen specificity and their interaction with Tregs are necessary to further elucidate the specific B-cell response in COPD.     

FCRL3 Gene Polymorphisms Confer Autoimmunity Risk for Allergic Rhinitis in a Chinese Han Population

Background

Heredity and environmental exposures may contribute to a predisposition to allergic rhinitis (AR). Autoimmunity may also involve into this pathologic process. FCRL3 (Fc receptor-like 3 gene), a novel immunoregulatory gene, has recently been reported to play a role in autoimmune diseases.

Objective

This study was performed to evaluate the potential association of FCRL3 polymorphisms with AR in a Chinese Han population.

Methods

Five single-nucleotide polymorphisms of FCRL3, rs945635, rs3761959, rs7522061, rs10489678 and rs7528684 were genotyped in 540 AR patients and 600 healthy controls using a PCR-restriction fragment length polymorphism assay. Allele, genotype and haplotype frequencies were compared between patients and controls using the χ2 test. The online software platform SHEsis was used to analyze their haplotypes.

Results

This study identified three strong risk SNPs rs7528684, rs10489678, rs7522061 and one weak risk SNP rs945635 of FCRL3 in Chinese Han AR patients. For rs7528684, a significantly increased prevalence of the AA genotype and A allele in AR patients was recorded. The frequency of the GG genotype and G allele of rs10489678 was markedly higher in AR patients than those in controls. For rs7522061, a higher frequency of the TT genotype, and a lower frequency of the CT genotype were found in AR patients. Concerning rs945635, a lower frequency of the CC genotype, and a higher frequency of G allele were observed in AR patients. According to the analysis of the three strong positive SNPs, the haplotype of AGT increased significantly in AR cases (AR = 38.8%, Controls = 24.3%, P = 8.29×10-14, OR [95% CI] 1.978 [1.652~2.368]).

Conclusions

This study found a significant association between the SNPs in FCRL3 gene and AR in Chinese Han patients. The results suggest these gene polymorphisms might be the autoimmunity risk for AR.     

Identification of the gene variations in human CD22

 CD22, a member of the immunoglobulin superfamily, is a B-cell transmembrane glycoprotein that acts as an accessory-signaling component of the B-cell antigen receptor (BCR). Recent evidence indicating the role of CD22 as a negative regulator of BCR signal transduction prompted us to test the possibility that genetic variations of human CD22 may be associated with autoimmune diseases. In this study, variation screening of the entire CD22 coding region was performed, and possible association with rheumatic diseases was tested, using the genomic DNA from 207 healthy Japanese individuals, 68 patients with systemic lupus erythematosus (SLE), and 119 patients with rheumatoid arthritis (RA). Through the variation screening, seven non-synonymous and four synonymous substitutions were identified. In addition, single base substitutions were found in two introns flanking exon-intron junctions. Among these variations, Q152E substitution within the second extracellular domain was observed with a marginally higher frequency in the patients with SLE (3/68, 4.4%) than that in healthy individuals (1/207, 0.5%) (P=0.048. SLE vs healthy individuals), although this difference was no longer significant after correction for the number of comparisons (Pc=0.62). No significant association was observed between any of the variations and RA. These findings indicate that a number of genetic variants are present in CD22, and suggest that CD22 could be considered a candidate for the susceptibility genes to autoimmune diseases. Received: 14 July 1998 / Revised: 7 September 1998     

Proteomic analysis of peripheral blood mononuclear cells: selective protein processing observed in patients with rheumatoid arthritis

In a comparative proteome analysis of peripheral blood mononuclear cells (PBMCs), we analyzed 130 two-dimensional gels obtained from 33 healthy control individuals and 32 patients diagnosed with rheumatoid arthritis (RA). We found 16 protein spots that are deregulated in patients with RA and, using peptide mass fingerprinting and Western blot analyses, identified these spots as belonging to 9 distinct proteins. A hierarchical clustering procedure organizes the study subjects into two main clusters based on the expression of these 16 protein spots, one that contains mostly healthy control individuals and the other mostly RA patients. The majority of the proteins differentially expressed in RA patients when compared with healthy controls can be detected as protein fragments in PBMCs obtained from RA patients. This set of deregulated proteins includes several factors that have been shown to be autoantigens in autoimmune diseases.     

Characterization of Regulatory B Cells in Graves’ Disease and Hashimoto’s Thyroiditis

A hallmark of regulatory B cells is IL-10 production, hence their designation as IL-10⁺ B cells. Little is known about the ability of self-antigens to induce IL-10⁺ B cells in Graves’ disease (GD), Hashimoto’s thyroiditis (HT), or other autoimmune disease. Here we pulsed purified B cells from 12 HT patients, 12 GD patients, and 12 healthy donors with the thyroid self-antigen, thyroglobulin (TG) and added the B cells back to the remaining peripheral blood mononuclear cells (PBMCs). This procedure induced IL-10⁺ B-cell differentiation in GD. A similar tendency was observed in healthy donors, but not in cells from patients with HT. In GD, B cells primed with TG induced IL-10-producing CD4⁺ T cells. To assess the maximal frequency of inducible IL-10⁺ B cells in the three donor groups PBMCs were stimulated with PMA/ionomycin. The resulting IL-10⁺ B-cell frequency was similar in the three groups and correlated with free T₃ levels in GD patients. IL-10⁺ B cells from both patient groups displayed CD25 or TIM-1 more frequently than did those from healthy donors. B-cell expression of two surface marker combinations previously associated with regulatory B-cell functions, CD24^hiCD38^hi and CD27⁺CD43⁺, did not differ between patients and healthy donors. In conclusion, our findings indicate that autoimmune thyroiditis is not associated with reduced frequency of IL-10⁺ B cells. These results do not rule out regulatory B-cell dysfunction, however. The observed phenotypic differences between IL-10⁺ B cells from patients and healthy donors are discussed.     

Profiles of gene expression in human autoimmune disease

Human autoimmune diseases arise from complex interactions between genetic and environmental factors, result from immune attack upon target tissues, and affect 3–5% of the population. We compared gene expression profiles (>4000 genes) in the peripheral blood mononuclear cells of normal individuals after immunization to individuals with four different autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus, insulin-dependent diabetes mellitus, and multiple sclerosis). All autoimmune individuals, including unaffected first-degree relatives, share a common gene expression profile that is completely distinct from the immune profile. Therefore, this expression pattern is not simply a recapitulation of the immune response to nonself, is not a result of the disease process, and results, as least in part, from genetic factors. Surprisingly, these genes are clustered in chromosomal domains suggesting there is some genomewide logic to this unique expression pattern. These data argue that that there is a constant pattern of gene expression in autoimmunity that is independent of the specific autoimmune disease and clinical parameters associated with any individual autoimmune disease.     

Identification of a 48 kDa form of unconventional myosin 1c in blood serum of patients with autoimmune diseases

We searched for protein markers present in blood serum of multiple sclerosis (MS), rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) patients in comparison to healthy human individuals. We used precipitation/extraction methods and MALDI TOF/TOF mass spectrometry, and identified a protein with Mr ~46 kDa as a fragment of human unconventional myosin IC isoform b (Myo1C). Western blotting with specific anti-human Myo1C antibodies confirmed the identity. Screening of blood serum samples from different autoimmune patients for the presence of Myo1c revealed its high level in MS and RA patients, relatively low level in SLE patients, and undetected in healthy donors. These data are suggesting that the level of p46 Myo1C in blood serum is a potential marker for testing of autoimmune diseases.     

No significant association between Fc receptor-like 3 gene polymorphisms and human leukocyte antigen-B27 positive ankylosing spondylitis in Han Chinese population

Ankylosing spondylitis (AS) is a rhematoid arthritis, which is a common autoimmune disease with a complex genetic etiology. Although HLA-B27 has been identified to be associated with AS, a number of other genes may also be involved in the disease. Fc receptor-like 3 (FCRL3) gene has been shown to be associated with rheumatoid arthritis in Japanese population. Here we aim to explore the association FCRL3 gene and susceptibility to human leukocyte antigen (HLA)-B27-positive AS in Han Chinese population. Among 169 AS patients, the frequencies of C and T (rs7522061) in FCRL3 gene were 38.7 and 61.3%, respectively; in 184 controls (HLA-B27-positive), the frequencies of C and T were 38.6 and 61.4%, respectively. The frequencies of alleles and genotype are not of statistically significant difference in two groups (χ² = 0.000, P = 0.983; χ² = 0.099, P = 0.952, respectively),but the distribution of HLA-B27 subtypes are statistically significant difference between cases and controls (χ² = 8.214, P = 0.042). Our data reveal that the FCRL3 gene does not appear associated with susceptibility to HLA-B27-positive AS in Han Chinese population.