Jin Ding

Function of HAb18G/CD147 in Invasion of Host Cells by Severe Acute Respiratory Syndrome Coronavirus

Abstract To identify the function of HAb18G/CD147 in invasion of host cells by severe acute respiratory syndrome (SARS) coronavirus (CoV), we analyzed the protein-protein interaction among HAb18G/CD147, cyclophilin A (CyPA), and SARS-CoV structural proteins by coimmunoprecipitation and surface plasmon resonance analysis. Although none of the SARS-CoV proteins was found to be directly bound to HAb18G/CD147, the nucleocapsid (N) protein of SARS-CoV was bound to CyPA, which interacted with HAb18G/CD147. Further research showed that HAb18G/CD147, a transmembrane molecule, was highly expressed on 293 cells and that CyPA was integrated with SARS-CoV. HAb18G/CD147–antagonistic peptide (AP)–9, an AP of HAb18G/CD147, had a high rate of binding to 293 cells and an inhibitory effect on SARS-CoV. These results show that HAb18G/CD147, mediated by CyPA bound to SARS-CoV N protein, plays a functional role in facilitating invasion of host cells by SARS-CoV. Our findings provide some evidence for the cytologic mechanism of invasion by SARS-CoV and provide a molecular basis for screening anti-SARS drugs

Expression of CD147 (EMMPRIN) on neutrophils in rheumatoid arthritis enhances chemotaxis, matrix metalloproteinase production and invasiveness of synoviocytes

The occurrence of neutrophils at the pannus‐cartilage border is an important phenomenon for understanding the pathogenesis of rheumatoid arthritis (RA). Matrix metalloproteinases (MMPs) are predominant enzymes responsible for the cartilage degradation. The present article studied the expression of CD147 on neutrophils and its potential role in neutrophil chemotaxis, MMPs production and the invasiveness of fibroblast‐like synoviocytes (FLS). The results of flow cytometry revealed that the mean fluorescence intensity of CD147 expression on neutrophils of peripheral blood from RA patients was higher than that in healthy individual. The potential role of CD147 in cyclophilin A (CyPA)‐mediated cell migration was studied using chemotaxis assay and it was found that the addition of anti‐CD147 antibody significantly decreased the chemotactic index of the neutrophils. Significantly elevated release and activation of MMPs were seen in the co‐culture of neutrophil and FLS compared with cultures of the cells alone. An increased number of cells invading through the filters in the invasion assays were also observed in the co‐cultured cells. The addition of anti‐CD147 antibody had some inhibitory effect, not only on MMP production but also on cell invasion in the co‐culture model. Our study demonstrates that the increased expression of CD147 on neutrophils in RA may be responsible for CyPA‐mediated neutrophil migration into the joints, elevated MMPs secretion and cell invasion of synoviocytes, all of which may contribute to the cartilage invasion and bone destruction of RA. Better knowledge of these findings will hopefully provide a new insight into the pathogenesis of RA.

Interferon-γ Contributes to HLA-B27-associated Unfolded Protein Response in Spondyloarthropathies

Objective. HLA-B27 positivity strongly influences the susceptibility to and phenotype of spondyloarthropathies (SpA). This study was designed to screen factors that activate the promoter of HLA-B27 in U937 cells, and to assess whether these promoter-activating factors induce the unfolded protein response (UPR) in HLA-B27-expressing cells. Methods. Cytometric Bead Array, flow cytometry, and real-time polymerase chain reaction were used to detect the expression of cytokines and UPR-associated proteins in peripheral blood and synovial fluid of patients with SpA. The HLA-B27 promotor transfectant was incubated separately with cytokines and Toll-like receptor ligands. After interferon-γ (IFN-γ) stimulation, expressions of GRP78, CHOP, and XBP-1 were tested in HLA-B27-expressing U937 cells and peripheral blood mononuclear cell (PBMC) of patients with ankylosing spondylitis (AS). (Clinical trial registration no. ChiCTR-OCC-11001565) Results. Expressions of GRP78, CHOP, and XBP-1 in monocytes/macrophages of SpA peripheral blood and synovial fluid were higher than those in healthy controls and patients with osteoarthritis (OA) (p < 0.05). Tumor necrosis factor-α (TNF-α) and IFN-α, IFN-ß, and IFN-γ were found to have activated the HLA-B27 promoter in the U937 cell line (p < 0.05). Following stimulation with IFN-γ, the expressions of GRP78, CHOP and XBP-1 in HLA-B27-transfected U937 cells and PBMC of HLA-B27-positive AS patients were more intense than those in A2-U937 cells, HLA-B27-negative AS patients, or healthy controls (p < 0.05). Conclusion. Expressions of GRP78, CHOP, and XBP-1 were higher in monocytes/macrophages of patients with SpA than those in both OA patients and healthy controls, suggesting that UPR may participate in the pathogenesis of SpA. TNF-α and IFN-α, IFN-ß, and IFN-γ significantly activated HLA-B27 promoter in the U937 cell line, and IFN-γ, the strongest activating factor, may induce the UPR in HLA-B27-expressing cells.

Kinetic changes of regulatory B10 cells in collagen-induced arthritis could be regulated by cytokines IFN-γ and TGF-β1

ObjectiveThe status of B10 cells in patients with rheumatoid arthritis (RA) has not been consistently reported. In this study, we observed the kinetic changes of the B10 cells in collagen-induced arthritis (CIA) mice and the influence of multiple cytokines on the B10 cells to investigate the potential mechanism underlying the changes of B10 cells.MethodsThe kinetic changes of frequency and function of the CD19+CD1dhiCD5+ cells in splenic cells were observed during the complete progress of CIA mice. The kinetic changes of cytokines IL-4, IL-6, IL-17A, IL-18, TNF-α, IFN-γ and TGF-β1 were also detected. Then influence of these cytokines on the status of B10 cells was investigated both in vitro and in vivo.ResultsThe frequency and suppressive ability of the CD19+CD1dhiCD5+ cells increased to its peak on the 14th day while gradually decreased subsequently. IFN-γ showed a similar tendency with the CD19+CD1dhiCD5+ cells, whereas IL-6, IL-17A, IL-18, TNF-α, and TGF-β1 reached its peak on the 28–35th day. In addition, IFN-γ up-regulated while TGF-β1 down-regulated the frequency and function of the CD19+CD1dhiCD5+ cells both in vitro and in vivo.ConclusionThe B10 cells in CIA mice could be regulated by IFN-γ and TGF-β1, suggesting that the status of B10 cells in RA may be influenced by the balance of pro-inflammatory and anti-inflammatory factors, and the impaired B10 cells could be recovered in vitro by adequate treatment before being used for a therapeutic method in clinical practice.

Metastasis-associated protein 1 (MTA1) signaling in rheumatoid synovium: Regulation of inflammatory response and cytokine-mediated production of prostaglandin E2 (PGE2).

Abnormal perpetual inflammatory response and sequential cytokine-induced prostaglandin E2 (PGE2) play important roles in the pathogenesis of rheumatoid arthritis (RA). The underlying regulatory mechanism, however, remain largely unknown. Here, we discovered that expression level of Metastasis associated protein 1 (MTA1), an important chromatin modifier that plays a critical role in transcriptional regulation by modifying DNA accessibility for cofactors, was upregulated in human rheumatoid synovial tissues. Furthermore, a knockdown of MTA1 by siRNA in the human fibroblast-like synovial cell line MH7A was found to impair the 4-hydroxynonenal (4-HNE)-induced transcriptional expression levels of certain proinflammatory cytokines including IL-1β, TNF-α and IL-6. Moreover, endogenous MTA1 was required for the cytokines-induced PGE2 synthesis by rheumatoid synoviocytes. Collectively, the coordinated existence of MTA1 inside distinct cascade loops points to its indispensable role in the modulation of the integrated cytokine network along the pathogenesis of RA. Further exploration of the functional details of this master transcriptional regulator should be an attractive strategy to identify novel therapeutic target for RA and warrants execution.

Increased expression of human leucocyte antigen class I free heavy chains on monocytes of patients with spondyloarthritis and cells transfected with HLA-B27.

Human leucocyte antigen (HLA)‐B27 expression is correlated with spondyloarthritis (SpA), but its role in disease pathogenesis remains unclear. The aim of the study was to determine whether HLA‐B27 free heavy chain (FHC) contributes to SpA pathogenesis. Flow cytometry was used to analyse the FHC expression on CD3+ and CD14+ cells in the peripheral blood (PB) and synovial fluid (SF) from SpA patients, healthy controls, and rheumatoid arthritis (RA) patients. Human monocytic U937 cell lines stably expressing enhanced green fluorescence protein (EGFP)/HLA‐B27, EGFP/HLA‐A2 or EGFP alone were created to further investigate the relation between HLA‐B27 and FHC expression. The relative FHC level on CD14+ PB cells was significantly higher in SpA patients than in controls, but lower than on the SF cells of SpA patients. No significant correlation was found for relative FHC expression with HLA‐B27 or β2‐microglobulin expression. HLA‐B27‐transfected U937 cells expressed higher FHC levels than either EGFP/HLA‐A2‐ or EGFP‐transfected cells. HLA class I FHC expression was significantly increased on monocytes of SpA patients and HLA‐B27‐transfected cells, implying that FHC, perhaps mostly derived from HLA‐B27, plays an important role in SpA pathogenesis.

Simultaneous detection of DNA synthesis, activation and cytokine secretion in collagen II (250–270)-activated T lymphocytes by flow cytometry

T cell activation and secretion of cytokines from activated peripheral blood mononuclear cells (PBMC) in culture have traditionally been measured by 3H‐thymidine incorporation for assessment of cell proliferation. However, this method has many disadvantages that limit its usage in analyzing antigen‐specific T responses, because of the low specific frequencies of the cells. Collagen II (250–270) may be an important autoantigen involved in the pathology of rheumatoid arthritis (RA). To further study the specific T cells response to CII 250–270, we developed an improved method for measuring lymphocyte proliferation and activation, and intracellular cytokine production, by flow cytometry at the single cell level. BrdU, an analog of thymidine, was incorporated into cellular DNA as a marker of individual cell proliferation. The cells were fixed and permeabilized, and a monoclonal antibody against BrdU conjugated with a fluorescent dye was used to measure BrdU incorporation. A Tris staining technique for the simultaneous determination of cell surface activation markers (CD69 or CD25) and intracellular cytokine production was also used and the parameters were assessed by 3‐color flow cytometry. Optimal conditions were selected to improve the sensitivity and specificity of the assays. This method allowed simultaneous detection of lymphocytic DNA synthesis, phenotype analysis and cytokine production at the single cell level, and thus it may be a useful tool for analyzing immune responses.

Combined detection of uMCP-1 and uTWEAK for rapid discrimination of severe lupus nephritis.

Reliable markers for the rapid discrimination of severe renal damage remain a vital concern for lupus nephritis (LN). To determine a better tool for kidney damage detection, the present study compared the evaluation ability of novel urinary cytokines and chemokines (namely urinary monocyte chemoattractant protein 1 (uMCP-1), tumor necrosis factor-like weak inducer of apoptosis (uTWEAK)) with traditional serum or urinary markers (namely urinary alpha 1-microgrobulin (uα1-MG), beta 2-microglobulin (uβ2-MG) and serum complement C3 (C3), complement C4 (C4), creatinine (Cr), blood urea nitrogen (BUN) and cystatin C (Cys C)) in discriminating LN renal damage. Correlations between markers with Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) renal SLEDAI scores, biopsy activity index (BAI) and biopsy chronicity index (BCI) scores were evaluated. Receiver operating characteristic (ROC) curves were generated to evaluate a single or combined model in discriminating active renal involvement (rSLEDAI scores > 0) and patients with poor pathological outcome (BAI scores ≥ 7). uMCP-1 and uTWEAK possess higher correlation coefficients with renal damage and larger areas under ROC curves (AUCs) than other markers. A combined model of uMCP-1 and uTWEAK showed an AUC of 0.887, sensitivity of 86.67% and specificity of 80.00% to discriminate active LN, and an AUC of 0.778, sensitivity of 75.00% and specificity of 81.82% to discriminate LN with poor outcome, which are better than the utility of any markers individually.

The expression and significance of CD28, Ctla-4, CD80 and CD86 in ankylosing spondylitis were also stimulated

Objective: To explore the expression and significance of CD28, ctla-4, CD80 and CD86 in ankylosing spondylitis. Methods: Flow cytometry was used to test in January 2016-January 2017 in our hospital was 73 cases of ankylosing spondylitis patients and 40 normal controls CD28, CTLA 4, CD80 and CD86 weeks outside expression in lymphocytes. By ELISA method, the lgA serum immunoglobulin IgG, IgM and hypersensitive c-reactive protein (hs CRP) and blood sedimentation (ESR) levels, and to explore, CD80, CD86 and CD28, CTLA - 4, age, duration of the hs CRP, ESR, Bath AS functional index (BASFI) and Bath AS measurement index (BASMI) relevance. Results: The levels of CD28, ctla-4, CD80 and CD86 were significantly higher in patients with ankylosing spondylitis (P<0.05). The levels of IgG and IgA in patients with ankylosing spondylitis were higher than those in the control group, and the difference was statistically significant (P<0.05). The CD28 level of peripheral blood was positively correlated with ESR and BASFI index (P<0.05), which was negatively correlated with hs-crp and BASMI index (P<0.05). The ctla-4 has negative correlation with ESR, hs-crp and BASMI index (P<0.05), and has no relationship with the BASFI index. CD80 was negatively correlated with ESR, BASFI index and BASMI index (P<0.05), and had no relationship with hs-crp. CD80 has positive correlation with hs-crp and BASMI index (P<0.05), which has no relation with ESR and BASFI index. Conclusion: The patients with ankylosing spondylitis is a total stimulus molecular CD28, CTLA 4, higher CD80, CD86 clear expression, the body is in a state of immune activation, has close relationship with immune dysfunction, monitoring peripheral CD28, CTLA 4, CD80, CD86 level is helpful for the early detection of disease, determine the illness treatment strategy.

ACPA mediates the interplay between innate and adaptive immunity in rheumatoid arthritis

The production of anti-citrullinated peptide antibodies (ACPAs) requires the participation of both innate immunity and adaptive immunity. On the one hand, activated innate immunity is able to produce citrullinated auto-antigens that fuel autoimmunity and provide an inflammatory environment that facilitates the breach of self-tolerance, proliferation of self-reactive T/B cells and the production of ACPAs. On the other hand, after their production by plasma B cells, ACPAs are also able to interact with innate immunity to exacerbate the manifestation and chronicity of rheumatoid arthritis (RA). This article discusses the roles of citrullinated peptides and ACPA played in innate immunity and autoimmunity. In addition, we emphasise the relationships between environmental factors and innate immunity, as well as the pathogenic function of ACPAs per se. In doing so, we hope to provide fundamental knowledge of RA pathogenesis and reveal potential therapeutic targets in RA treatment.