Chrong-Reen Wang

Decreased CD4+CD25+ T cells in peripheral blood of patients with systemic lupus erythematosus.

Recent animal studies have shown that CD4+CD25+ T cells play a crucial role in the suppression of the immune response and that depletion of this subset of T cells might lead to development of autoimmune diseases. The aim of the present study was to investigate the levels of CD4+CD25+ T cells in the peripheral blood of patients with systemic lupus erythematosus (SLE). Ninety‐four SLE patients, 52 patients with rheumatoid arthritis (RA) and 50 age‐ and gender‐matched healthy individuals were enrolled in the study. A flowcytometric method was applied in the measurement of CD4+CD25+ T cells. The results showed that patients with SLE had statistically lower levels of CD4+CD25+ T cells than did normal controls, when expressed as either percentages of peripheral blood mononuclear cells (PBMCs) (mean ± SD, 8.49 ± 6.36 versus 11.11 ± 4.58%, P < 0.05) or absolute cell numbers (98.77 ± 97.52 versus 213.93 ± 104.52 cells/mm3, P < 0.05). In terms of CD25brightCD4+ T cells, defined as having a fluorescence intensity of CD25 expression exceeding 100, SLE patients still had significantly lower levels than did normal controls expressed as percentages of PBMCs (1.76 ± 1.32 versus 3.73 ± 1.30%, P < 0.05). No significant differences could be found between RA patients and normal controls. The overwhelming majority of CD4+CD25+ T cells belonged to CD45RO+ cells and most did not express the CD69 molecule. Although decreased CD4+CD25+ T cells were found in SLE patients, we failed to find a significant correlation between the levels of CD4+CD25+ T cells and disease activities of SLE. To the best of our knowledge, this is the first study to demonstrate that patients with SLE had decreased CD4+CD25+ T cells. However, the exact role of the decreased CD4+CD25+ T cells in the pathogenesis of SLE remains to be elucidated.

src homology 2 domain–containing tyrosine phosphatase SHP-1 controls the development of allergic airway inflammation

Th2 cells are generated from naive CD4 T cells upon T cell receptor (TCR) recognition of antigen and IL-4 stimulation and play crucial roles in humoral immunity against infectious microorganisms and the pathogenesis of allergic and autoimmune diseases. A tyrosine phosphatase, SHP-1, that contains src homology 2 (SH2) domains is recognized as a negative regulator for various intracellular signaling molecules, including those downstream of the TCR and the IL-4 receptor. Here we assessed the role of SHP-1 in Th1/Th2 cell differentiation and in the development of Th2-dependent allergic airway inflammation by using a natural SHP-1 mutant, the motheaten mouse. CD4 T cells appear to develop normally in the heterozygous motheaten (me/+) thymus even though they express decreased amounts of SHP-1 (about one-third the level of wild-type thymus). The me/+ naive splenic CD4 T cells showed enhanced activation by IL-4 receptor-mediated signaling but only marginal enhancement of TCR-mediated signaling. Interestingly, the generation of Th2 cells was increased and specific cytokine production of mast cells was enhanced in me/+ mice. In an OVA-induced allergic airway inflammation model, eosinophilic inflammation, mucus hyperproduction, and airway hyperresponsiveness were enhanced in me/+ mice. Thus, SHP-1 may have a role as a negative regulator in the development of allergic responses, such as allergic asthma.

Increased expression of soluble cytotoxic T-lymphocyte-associated antigen-4 molecule in patients with systemic lupus erythematosus.

A soluble form of cytotoxic T‐lymphocyte‐associated antigen‐4 (sCTLA‐4) was recently found and shown to possess a downregulatory function as a membrane‐bound CTLA‐4 molecule. The purpose of the study was to investigate the expression of sCTLA‐4 molecule in patients with systemic lupus erythematosus (SLE). One hundred patients with SLE and 40 age‐ and sex‐matched healthy individuals were enrolled in the study. The results showed that patients with SLE have significantly higher levels of sCTLA‐4 in sera than healthy controls (21.6 ± 12.3 ng/ml versus 5.9 ± 5.4 ng/ml, P < 0.001). Increased expression of sCTLA‐4 mRNA in peripheral blood mononuclear cells (PBMCs) was also found in SLE patients. However, we could not find a statistically significant correlation between the serum levels of sCTLA‐4 and lupus disease activities. The reported CTLA‐4 gene polymorphism in promoter region at position −318 did not affect the levels of sCTLA‐4. To the best of our knowledge, this is the first report showing that patients with SLE have increased sCTLA‐4 expression. However, the mechanism and role of increased sCTLA‐4 in the pathogenesis of SLE remains elucidated.

Brief Report: Amelioration of collagen-induced arthritis in mice by lentivirus-mediated silencing of microRNA-223

OBJECTIVE MicroRNA (miRNA) plays a role in autoimmune diseases. MiRNA-223 (miR-223) is up-regulated in patients with rheumatoid arthritis (RA) and is involved in osteoclastogenesis, which contributes to erosive disease. The aim of this study was to test the feasibility of using lentiviral vectors expressing the miR-223 target sequence (miR-223T) to suppress miR-223 activity as a therapeutic strategy in a mouse model of collagen-induced arthritis (CIA). METHODS Levels of miR-223 in the synovial tissue of patients with RA or osteoarthritis (OA), as well as in the ankle joints of mice with CIA, were determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Lentiviral vectors expressing miR-223T (LVmiR-223T) or luciferase short hairpin RNA (LVshLuc) as a control vector were injected intraperitoneally into mice with CIA. Treatment responses and disease-related bone mineral density were monitored. Levels of nuclear factor 1A (NF-1A), a direct target of miR-223, and macrophage colony-stimulating factor receptor (M-CSFR), which is critical for osteoclastogenesis, were measured by immunohistochemistry and quantitative RT-PCR. Osteoclasts were assessed by tartrate-resistant acid phosphatase staining. RESULTS MiR-223 expression was significantly higher in the synovium of RA patients and in the ankle joints of mice with CIA as compared to OA patients and normal mice. LVmiR-223T treatment reduced the arthritis score, histologic score, miR-223 expression, osteoclastogenesis, and bone erosion in mice with CIA. Down-regulation of miR-223 with concomitant increases in NF-1A levels and decreases in M-CSFR levels was detected in the synovium of LVmiR-223T-treated mice. CONCLUSION This study is the first to demonstrate that lentivirus-mediated silencing of miR-223 can reduce disease severity of experimental arthritis. Furthermore, our results indicate that inhibition of miR-223 activity should be further explored as a therapeutic strategy in RA.

The Presence of Cytokine‐Suppressive CD4+CD25+ T Cells in the Peripheral Blood and Synovial Fluid of Patients with Rheumatoid Arthritis

CD4+CD25+ T cells have been shown to play a regulatory or suppressive role in the immune response and are possibly relevant to the pathogenesis of autoimmune diseases. In the present study, we attempted to investigate the levels of CD4+CD25+ T cells in the peripheral blood (PB) and synovial fluid (SF) of patients with rheumatoid arthritis (RA) and the effects of CD4+CD25+ T cells on the in vitro cytokine production by stimulated SF mononuclear cells (SFMC). The results showed that RA patients had similar frequencies of CD4+CD25+ T cells in PB, expressed as a percentages of the lymphocyte population, as did healthy subjects (mean ± SD: 10.52 ± 5.87% versus 11.11 ± 4.58%., respectively). But in contrast to PB, the SF of RA patients contained significantly higher levels of CD4+CD25+ T cells (17.77 ± 7.92% versus 10.52 ± 5.87%, respectively. P < 0.001). When cocultured in vitro with SFMC, CD4+CD25+ T cells purified from either PB or SF were found to exert a considerable suppressive effect on the production of cytokines including TNF‐α, IFN‐γ and interleukin‐10 (IL‐10). The percentages of inhibition of each cytokines ranged from 41.8 to 98.4% (mean, 80.0%) for TNF‐α, 42.8 to 98.9% (mean, 83.2%) for IFN‐γ and 59.3 to 96.6% (mean, 80.0%) for IL‐10. Because both pro‐inflammatory and anti‐inflammatory cytokines were suppressed by CD4+CD25+ T cells, whether CD4+CD25+ T cells might play a beneficial role in the suppression of sustained inflammation in rheumatoid synovium remains to be elucidated.

Use of HLA-B*58:01 genotyping to prevent allopurinol induced severe cutaneous adverse reactions in Taiwan: national prospective cohort study

Objective To evaluate the use of prospective screening for the HLA-B*58:01 allele to identify Taiwanese individuals at risk of severe cutaneous adverse reactions (SCARs) induced by allopurinol treatment. Design National prospective cohort study. Setting 15 medical centres in different regions of Taiwan, from July 2009 to August 2014. Participants 2926 people who had an indication for allopurinol treatment but had not taken allopurinol previously. Participants were excluded if they had undergone a bone marrow transplant, were not of Han Chinese descent, and had a history of allopurinol induced hypersensitivity. DNA purified from 2910 participants’ peripheral blood was used to assess the presence of HLA-B*58:01. Main outcome measures Incidence of allopurinol induced SCARs with and without screening. Results Participants who tested positive for HLA-B*58:01 (19.6%, n=571) were advised to avoid allopurinol, and were referred to an alternate drug treatment or advised to continue with their prestudy treatment. Participants who tested negative (80.4%, n=2339) were given allopurinol. Participants were interviewed once a week for two months to monitor symptoms. The historical incidence of allopurinol induced SCARs, estimated by the National Health Insurance research database of Taiwan, was used for comparison. Mild, transient rash without blisters developed in 97 (3%) participants during follow-up. None of the participants was admitted to hospital owing to adverse drug reactions. SCARs did not develop in any of the participants receiving allopurinol who screened negative for HLA-B*58:01. By contrast, seven cases of SCARs were expected, based on the estimated historical incidence of allopurinol induced SCARs nationwide (0.30% per year, 95% confidence interval 0.28% to 0.31%; P=0.0026; two side one sample binomial test). Conclusions Prospective screening of the HLA-B*58:01 allele, coupled with an alternative drug treatment for carriers, significantly decreased the incidence of allopurinol induced SCARs in Taiwanese medical centres.

Intraarticular gene transfer of thrombospondin-1 suppresses the disease progression of experimental osteoarthritis

In osteoarthritis, angiogenesis, which occurs in the osteochondral junction and synovium, may accelerate inflammation and contribute to the severity of the disease. We used anterior cruciate ligament‐transection (ACLT) to investigate the therapeutic effect of an angiogenesis inhibitor, thrombospondin‐1 (TSP‐1), in a rat model of osteoarthritis. Osteoarthritis was induced in Wistar rats in the knee of one hind leg. After ACLT, AdTSP‐1 (adenoviral vector encoding mouse TSP‐1) was intraarticularly injected into the knee joints. Transgene expression, angiogenesis, and inflammatory responses in the knee joints were examined. They were also assessed morphologically, radiographically, and histologically for manifestations of disease. The levels of TSP‐1 peaked on day 3 and were substantially maintained for at least 9 days after AdTSP‐1 infection. Adenovirus‐mediated gene expression was detected in the synovial membrane and chondrocytes. TSP‐1 gene transfer induced transforming growth factor‐β (TGF‐β) production, but it reduced microvessel density, macrophage infiltration, and interleukin‐1β (IL‐1β) levels. Gross morphological and histopathological examinations revealed that rats treated with AdTSP‐1 had less severe osteoarthritis than controls. In vivo adenovirus‐mediated TSP‐1 gene transfer significantly reduced microvessel density, inflammation, and suppressed the progression of osteoarthritis. This study provides potential applications of TSP‐1 gene delivery for treating osteoarthritis. Published by Wiley Periodicals, Inc. J Orthop Res 28:1300–1306, 2010

Regulation of CCR5 expression and MIP‐1α production in CD4+ T cells from patients with rheumatoid arthritis

Production of CCR5 expression and MIP‐1α, a ligand of CCR5, by CD4+ T cells from patients with rheumatoid arthritis (RA) were studied. We analysed further the influence of IL‐15 stimulation, CD40/CD40 ligand (CD40L) interaction and CCR5 promotor polymorphism. One hundred and fifty‐five RA patients and another 155 age‐ and sex‐matched healthy individuals were enrolled. Peripheral CD4+ and double negative (DN) T cells from patients had lower portions of CCR5, whereas synovial CD4+ and DN T cells showed a much higher CCR5 expression. IL‐15 significantly up‐regulated the expression of CCR5 on purified CD4+ T cells. CD40L expression on synovial CD4+ T cells was increased greatly in CCR5+ portions by IL‐15. MIP‐1α production by synovial CD4+ T cells was also enhanced by IL‐15. Co‐culture of CD40 expressing synovial fibroblasts with IL‐15‐activated synovial CD4+ T cells significantly increased MIP‐1α production. Expression of CCR5 on patients’ CD4+ T cells was not influenced by the promotor polymorphism of CCR5 gene. Taken together, these data suggest CCR5+CD4+ T cells infiltrate the inflamed synovium and IL‐15 up‐regulates CCR5 and CD40L expression further and enhance MIP‐1α production in synovial CD4+ T cells. Production of MIP‐1α by synovial fibroblasts is significantly increased by engagement of CD40 with CD40L. Synovial microenvironment plays a potential role in regulation of CCR5+CD4+ T cells in rheumatoid joints.

Expression of CD40 and CD40 Ligand among Cell Populations within Rheumatoid Synovial Compartment

Augmented and prolonged expression of CD40 ligand (CD40L) was recently reported in lymphoid cells from human lupus patients, suggesting that CD40/CD40L pathway was involved in the pathogenesis of systemic autoimmune diseases. This study was thus designed to study the expression of CD40 and CD40L among cell populations within inflammatory joints of patients with rheumatoid arthritis (RA). The result showed that most B cells and monocytes in synovial fluids (SF) expressed CD40. Cultured synovial fibroblasts also stained positive for CD40. Regarding CD40L, we found that T cells as well as B cells could express CD40L. Compared with normal controls, RA patients had higher levels of CD40L+ T cells (8.71 ± 17.69 % vs 1.74 ± 2.30 %, P± 0.05) and CD40L+ B cells (7.71 ± 7.64 % vs 1.12 ± 1.59 % P < 0.05). After in vitro stimulation, T cells from RA patients had higher and longer CD40L expression than T cells from normal peripheral blood. For investigating the effect of CD40 expressed on synovial fibroblasts on TNF-a production in joint compartment, we used anti-CD40 antibody to bind CD40 on fibroblasts for one hour and then co-cultured with synovial fluid mononuclear cells. We found that the levels of TNF-a decreased in the presence of anti-CD40 antibody. We concluded that there was an intrinsic hyperexpression of CD40L on lymphoid cells within rheumatoid joints, and synovial fibroblasts could contribute to articular inflammation through surface CD40 molecule.

CTLA-4 gene polymorphism in promoter and exon-1 regions in Chinese patients with systemic lupus erythematosus.

Cytotoxic T lymphocyte associated antigen 4 (CTLA-4), a structural homologue of CD28, has been reported to be an important negative regulator of autoimmune diseases. Recent studies showed that CTLA-4 gene polymorphism was associated with several kinds of human autoimmune diseases, suggesting that CTLA-4 gene is probably a general susceptibility gene to autoimmune disease. The present study was conducted in Chinese to determine whether there is any association of the CTLA-4 gene polymorphism with the development of systemic lupus erythematosus (SLE). CTLA-4 gene polymorphism in promoter and exon 1 was detected by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method in 81 patients with SLE and 81 normal controls. The results showed that there were no statistically significant differences in both exon 1 and promoter gene polymorphism between SLE patients and normal controls. The preliminary study does not suggest an association of the known polymorphism in exon 1 and promoter of CTLA-4 gene with Chinese SLE. However, SLE is a very heterogeneous syndrome and CTLA-4 gene polymorphism might correlate with some specific clinical features. To exploring this possibility, subgroup analysis in more patients needs to be performed.