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Objective To investigate the effect of neutrophil extracellular traps (NETs) on inflammation of rheumatoid arthritis (RA), especially angiogenesis. Methods The presence of NETs in synovial tissues of RA and osteoarthritis (OA) was observed by immunofluorescence assay. Neutrophils were isolated from peripheral blood of health volunteers. Neutrophils were cultured in vitro, the formation of NETs was observed. NETs were extracted as a stimulating agent. The effects of NETs on the proliferation of human umbilical vein endothelial cells (HUVECs) and synovial fibroblasts (RAFLS) were evaluated by MTT, and which were classified into two groups: HUVECs group and RAFLS group, with the following treatment: control and NETs (0.28 mg/L). Wound repair assay was employed to evaluate the effect on the cell migration stimulated with NETs. The experiment was divided into three groups:control, VEGF (40μg/L VEGF) and NETs (0.28 mg/L NETs). Results (1) Compared with OA, NETs were found more in the synovial tissue of RA. (2) NETs formation was induced by stimulator in vitro. The concentration of extracted NETs-DNA was 27.8 mg/L. (3) MTT assay showed that compared with the control groups, low concentration of NETs (0.28 mg/L) promoted the proliferation of HUVECs (0.499 ± 0.011 vs. 0.393 ± 0.009, P<0.05) and RAFLS (0.266 ± 0.007 vs. 0.192 ± 0.007, P<0.05). (4) It was showed that a significant wound closure induced by low concentration of NETs (0.28 mg/L) was found compared with control. Conclusion Our present study suggests that NETs are found more in the synovial tissue of RA, and low concentration of NETs can promote angiogenesis in RA.
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Objective To explore whether serum amyloid A (SAA) can induce the formation of neutrophil extracellular traps(NETs)in neutrophils in vitro. Methods A stable method for inducing NETs formation in vitro was established, in-cluding isolation of peripheral blood neutrophils, cell culture, and NETs formation and observation. The neutrophils were iso-lated from peripheral blood of healthy volunteers. And cells were cultured in vitro and classified into three groups:negative control (NC) group, SAA group and lipopolysaccharide (LPS) group. Following the distinct stimulation in three groups, NETs formation was observed and its percentage was calculated. The concentration of hinstone (h) 3 in supernatant was detected by ELISA. Results The purification and vitality of isolated neutrophils were both more than 95%. The nuclei of neutrophils lost their shape and spread, NETs formation was found. More NETs formation was found in SAA group than that in NC group (P < 0.05). Moreover, the concentration of h3 in supernatant was significantly higher in SAA group than that in NC group (P<0.05). Conclusion SAA can induce the formation of NETs in vitro.
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Objective Calreticulin (CRT)is a multifunctional protein of endoplasmic reticulum implicated in the pathogenesis of rheumatoid arthritis(RA). The present study was undertaken to determine whether CRT was involved in an?giogenesis events in RA. Methods Serum CRT levels were measured by enzyme-linked immnuosorbent assay(ELISA)in 106 patients with established RA, 75 osteoarthritis(OA)and 80 healthy controls(HC). CRT levels in synovial fluid were al?so measured in 25 RA and 22 OA patients. The expression of CRT in synovial tissue was examined by immunohistology. In order to investigate the role of CRT on angiogenesis, human umbilical vein endothelial cells(HUVECs)were isolated and cultured for in vitro experiments. The proliferation, migration and tube formation of HUVECs following CRT stimulation were examined in vitro by MTT assay, scratch wound healing assay and tube formation assay. Results Our results showed a sig?nificantly higher concentration of CRT in serum [(6.4±3.1) μg/L] of RA patients compared to that of OA [(3.7±0.9) μg/L, P<0.01] and HC [(3.4±1.0) μg/L, P<0.01];and significantly higher CRT in synovial fluid [(6.9±3.4) μg/L] of RA vs OA [(3.9± 0.7) μg/L, P<0.01]. Increased CRT expression predominantly localized to vascular endothelial cells, inflammatory cells and perivascular areas in both the lining and sublining layers of RA synovial tissue. Furthermore, CRT significantly promoted the proliferation, migration and tube formation of HUVECs, as showed by MTT assay, scratch wound healing assay and tube for?mation assay. Conclusion These findings suggested that CRT may be involved in synovitis and pannus formation events via promoting angiogenesis in RA.
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Objective To investigate the role of scavenger receptor class B type 1 (SR-B1) signaling pathway in serum amyloid A (SAA)-induced angiogenesis in rheumatoid arthritis (RA).Methods The expression and location of SR-B1 in RA and osteoarthritis (OA) synovial tissues were observed by immunohistochemistry.And SR-B1 expression in the resting human umbilical vein endothelial cells (HUVECs) was detected by immunoflourescence.Wound repair assessement and tube formation assessement were employed to evaluate the effect on cell migration and tube formation stimulated by SAA and/or anti-SR-B1 antibody.The t-test and one-way analysis of variance (ANOVA) were used for statistical analysis.Results ① SR-B1 was significantly highly expressed in RA tissue samples (A=6 788±819) when compared to the minimal expression in OA (A =31 849±6 977,t=3.567,P＜0.01).Positive staining of SR-B1 was observed in RA synovial vascular endothelial cells and perivascular areas.② Strong staining for SR-B1 was observed in all HUVECs tested.③ Significant wound healing induced by SAA (MI=2.50±0.17) was found compared with the untreated controls (MI=1.00±0.09,q=14.38,P＜0.01),and the effects were inhibited in the presence of anti-SR-B1 antibody (MI=1.16±0.14,q=13.02,P＜0.01).④ Compared to the untreated group (branch point number:6.6±0.8),there was an enhanced formation of branched and capillary-hke tube structure followed by SAA stimulation (branch point number:19.0±1.1,q=25.04,P＜0.01) after culturing for 72 h,whereas,tube formation decreased markedly upon pre-treated with anti-SR-B1 antibody (branch point number:7.6±1.3,vs SAA,q =23.32,P＜0.01).Conclusion Our present study suggests that serum amyloid A may induce angiogenesis via SR-B1 signaling pathway in RA.
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Objective To investigate the correlation between serum amyloid A (SAA) and disease activity (DAS28) in patients with rheumatoid arthritis (RA). Methods Forty-four patients with RA, 35 patients with systemic lupus erythe-matosus (SLE), 18 patients with osteoarthritis (OA) and 30 healthy controls (HC) were enrolled in this study. The levels of SAA were measured by ELISA. Erythrocyte sedimentation rate (ESR) was measured by the Westergren method. The value of serum C reactive protein (CRP) was examined by immunonephelometry assay. The correlation between SAA and DAS 28, ESR and CRP was assessed, respectively. Results The SAA levels were significantly higher in RA group than those of SLE, OA, and HC groups (P0.05), but there was no significant difference between RA group and SLE group. There was positive correlation between SAA and DAS28, ESR, and CRP levels (rs=0.790, P<0.001;rs=0.674, P<0.001;rs=0.679, P=0.004), respective-ly. Conclusion SAA may be a new serological marker to assess disease activity in RA.
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Objective To investigate the possible mechanism of aggregation and activation of neu-trophils(polymorphonuclear neutrophils,PMN)in mice with chlamydial pneumonitis. Methods C57BL/ 6 mice were inoculated intranasally with 3×103 inclusion-forming units(IFU)of Chlamydia muridarum(Cm) to induce the murine model of chlamydial pneumonitis. Samples of lung tissues collected at different time points after infection were stained by hematoxylin and eosin for histopathological assessment of inflammation. The levels of myelo-peroxidase(MPO)were detected for the evaluation of PMN aggregation. The mononu-clear cells were isolated from lung tissues. The inflammatory cells were counted with Giemsaˊs staining. CD11b+Gr1+ cell population and CD11b expression in lung mononuclear cells were analyzed by flow cytome-try. The expression of chemokines(MIP-2,LIX,KC and MCP-1)in lung tissues at mRNA level was meas-ured by RT-PCR. Results Chlamydial pneumonitis was induced in mice by intranasal inoculation of 3×103 IFU of Cm. Compared with the mice from control group,large amounts of inflammatory cells including PMN, monocytes and lymphocytes were induced in lung tissues of mice with Cm infection. PMN responded earlier than monocytes to the infection. The levels of MPO were significantly increased in mice with Cm infection and reached the highest level on the 7th day after infection. A decline in MPO levels was observed on the 14th day but the levels were still higher than those on day 0. The percentages and total numbers of CD11b+Gr1+ cells were significantly increased after Cm infection. Moreover,an increased expression of PMN CD11b was also detected by flow cytometry. The expression of chemokines(MIP-2,LIX,KC and MCP-1)was in-creased in lung tissues of mice after Cm infection. The results of the study indicated that Cm infection in-duced the expression of PMN chemoattractants,resulting in the recruitment of PMN. Conclusion The infil-tration and activation of PMN in lung tissues of mice were induced by Cm infection through increasing the ex-pression of chemokines. PMN played an important role in immune responses against Cm infection.
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Objective To investigate the regulation of IFN-γ to Th17 response in Chlamydia muridarum (Cm) lung infection in mice. Methods A murine model of pneumonia induced by intranasal inoculation of Cm was used for this study. Anti-mouse IFN-γ McAbs were used to neutralize endogenous IFN-γfollowing Cm lung infection. Control group received the same dose of isotype antibody (IgG2a). Mice were sacrificed at day 7 postinfection. Chlamydial growth in the lung was assessed by immunoenzyme technique.IL-17 and IL-23 mRNA expression in the lung was assayed by RT-PCR and the proliferation of IL-17 + CD4 +T cells in the spleen was assayed by intracellular cytokine staining. Results IFN-γ-neutralized mice exhibited serious disease course, include greater body weight loss, higher organism growth and much more severe pathological changes in the lung compared with control mice. The mRNA expression of IL-17 and IL-23 in the lung and the proliferation of IL-17 + CD4 + T cells in the spleen significantly decreased in the IL-17- neutralized mice. Conclusion IFN-γ was protective in Cm lung infection through up-regulating the antigen specific Th17 responses.
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Objective: To investigate the effect of human chorionic gonadotrophin (hCG) on the gene expression of migration inhibitory factor (MIF) in human peripheral blood mononuclear cell (PBMC). Methods: The healthy human PBMC was cultured with hCG at 37 ℃, 5%CO_2 for 2 hours. The mRNA of harvested cells was isolated. The MIF mRNA was detected by real-time RT-PCR. Results: In a certain range of doses, the mRNA expression of MIF significantly increased following the increase of hCG in a dose depandent manner, and it reached to a peak 1-2 hours after culture, then returned to the minimum level after 8 hours. Conclusion: In a certain range of doses, hCG can increase the mRNA expression of MIF. This effect is correlated with reacting time. It is suggested that hCG may involve in immune response by up-regulating the production of cytokines by PBMC.
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Objective:To detect the expression of IDO, iNOS, gp91 NADPH ox releated with IFN-? function following Chlamydia trachomatis lung infection in mice and to investigate the immunological defense mechanism of IFN-?. Methods:A murine model of pneumonia induced by intranasal inoculation of Chlamydia trachomatis,mouse pneumonitis (MoPn) biovar,was used for this study. Chlamydial growth in the lung was assessed by inoculating HeLa 229 cell monolayer with lung homogenates followed by HRP conjugate anti-Chlamydial LPS mAb.The mRNA expressions of IDO, iNOS, gp91 NADPH ox and IFN-? in the lung were determined by RT-PCR on day 7 and 14 postinfection.Results:Chlamydial growth in the lung was observed on day 2 postinfection, peaking at day 7 with subsequent decline in quantity. At day 21 following inoculation, the IFU declined to the baseline. Contrast with the uninfected group, Th1-like cytokine IFN-? underwent a significant increase at day 7 and a decrease on day 14 postinfection. mRNA expression for IDO, iNOS, gp91 NADPH ox was significantly increased in the lungs on day 7 and 14 postinfection, IDO and gp91 mRNA expression was significantly highler at day 7(P