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ABSTRACT: To investigate whether monocytes activated with lipopolysaccharide(LPS) have an effect on Th17 cell differentiation in humans, CD4(+) T cell and CD14(+) monocytes activated with LPS were treated in the absence or presence of anti-CD3 mAb with various concentrations at different time points.
Purification of CD4(+) T cell and CD14(+) monocytes were performed by magnetic cell sorting and cultured together. Cultures were stimulated with LPS alone or anti-CD3 mAb alone or LPS plus anti-CD3 mAb for 3 days. In the anti-CD3 mAb stimulation cells were added different concentrations of LPS. Cells were activated under LPS/anti-CD3 costimulation for 3, 6, or 10 days. The percentage of IL-17(+) T cells and INF-γ(+) T was determined by flow cytometry.
LPS or anti-CD3 mAb alone induced only very low levels of IL-17(+) T cells, (1.30 ± 0.19)%, (1.10 ± 0.21)%, respectively. The percentage was substantially higher in the LPS and anti-CD3 mAb costimulationa as much as(2.01 ± 0.46)%. In the presence of 0.1 μg/mL, 1 μg/mL, 10 μg/mL LPS, the proportion of Th17 reached to (1.92 ± 0.21)%, (1.30 ± 0.37)%, (1.01 ± 0.25)%. Low-concentration LPS (0.1 μg/mL) stimulation favored Th17 differentiation. The highest proportion of IL-17(+) T cells was found at day 3(2.13 ± 0.32)%, with levels declining at day 6 and day 10, while, Th1 at day 6(17.45 ± 3.04)%, declining at day 10.
Low-concentration LPS stimulation plus anti-CD3 mAb in short term support optimal Th17 generation. Nevertheless, this model closely mimics the environment of rheumatoid arthritis in vivo and proposes an effective model for the generation of human Th17 cells.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 04/2012; 28(4):377-80.
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ABSTRACT: To observe the therapeutic effect of cyclosporine A (CsA) on bleomycin (BLM) induced pulmonary fibrosis and to investigate its mechanism.
One hundred and twenty C57BL/6 female mice were divided randomly into five groups: BLM model group, control saline group, CsA30 mg treatment group, CsA50 mg treatment group and control treatment group. Treatment groups and model groups were administrated BLM intratracheally to induce interstitial pulmonary disease model, with control saline group administrated with equal volume of normal saline instead. Mice in treatment groups were intraperitoneal injected with CsA, while control treatment group were injected with equal volume of normal saline instead. On the 4th, 7th and 14th day after administration, 8 mice of each group were sacrificed, and the peripheral blood was obtained to count total leucocytes with counting chamber and quantify CD4(+); T cells, CD14(+); monocytes and CD19(+); B cells by flow cytometry (FCM). Bronchoalveolar levage fluid was harvested for cell counting and Giemsa staining. Lung tissues were harvested for immunohistochemical staining and pathological examination.
The quantity of total leucocyte was higher in BLM model group than those in control saline group.The proportion of CD14(+); T cells and CD19(+);B cells in BLM model group were increased markedly than those in control saline group on the 4th, 7th and 14th day post BLM. With CsA treatment, The proportion of CD14(+); T cells was lower than BLM model group at the same time point, especially on the 4th day. The proportion of CD19(+); B cells were significantly lower than those of BLM model group at the same time point(7 d, 14 d). The total and classification of cells of BLM model group were increased markedly than those in control saline group, and decreased obviously in the treatment groups at the same time point. Examination of lung tissues: With the prolonged time of BLM administration, it showed wider alveolar septum, more collagen deposition, as well as more infiltrating inflammatory cells which consisted of generous lymphocyte and few mononuclear macrophages than those in saline control group. With the prolonged time of CsA injection, the interstitial pulmonary inflammation was remissive, and there was less fibroblast infiltration and collagen deposition in pulmonary interstitium and periphery of bronchiole. Alveolar epithelial cells, bronchiolar epithelial cells, mononuclear macrophages, neutrophils and lymphocytes were demonstrated to express CD147, there was higher CD147 expression in BLM model group than those in CsA treatment groups.
CsA may heal BLM induced interstitial pulmonary disease by blocking CD147-CypA interaction, then decreasing chemotaxis for the immunocyte, and reducing migration of immunocytes to the lung and collagen deposition in the lung.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 03/2012; 28(3):232-6.
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ABSTRACT: To observe whether cyclophilin A (CypA)has an effect on macrophage-derived foam cells, and to investigate the involvement of CypA in the development of atherosclerosis.
The foam cell model was established through incubating the human monocyte line (THP-1 cells) with oxidized low density lipoproteins (ox-LDL). The cells were stained with fresh oil red O to study the morphology of the macrophage-derived foam cells. The cell adhesion, invasion and the production of matrix metalloproteinase (MMPs) of the macrophage-derived foam cells were detected by adhesion assay, invasion assay and gelatin zymography respectively both in the absence or presence of different concentrations of purified CypA (50, 100, 200 μg/L). Then the foam cells were respectively pre-treated with CsA, c7b8f10, HAb18 mAb, and dual treatment of c7b8f10 and HAb18 mAb respectively, to investigate the inhibitory effect on macrophage-derived foam cells.
The adhesion, invasion and the production of MMP-9 and MMP-2 were enhanced during the differentiation of monocytes into macrophages (P<0.05). CypA, especially in the concentration of 100 ng/mL, significantly promoted the function of macrophage-derived foam cells (P<0.05). CsA, c7b8f10, HAb18 mAb, and c7b8f10- HAb18 mAb combination dramatically inhibited the function of macrophage-derived foam cells both in the absence or presence of CypA (P<0.05). The c7b8f10- HAb18 mAb combination pretreatment had the most obviously suppressive effect on macrophage-derived foam cells (P<0.05).
These findings suggest that CypA up regulates the adhesion, the invasion and the expressions of MMP-2 and MMP-9 in macrophage-derived foam cells. The CypA effect is blocked by the pretreatment of the different antagonists. This research might suggest the correlation between atherosclerosis pathogenesis and the vulnerability of atherosclerotic plaques, and thus give us some good ideas for atherosclerosis therapy in future.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 05/2011; 27(5):515-8.
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ABSTRACT: To ascertain the effect of CyPA and IL-8 in chemotaxis of neutrophil and the level of IL-8 from the CyPA effecting of peripheral blood from RA patients.
12 RA patients matched 4 healthy people were studied. Chemotaxis of IL-8 was measured by Boyden chamber on neutrophil of RA patients and that of 4 normal healthy people controls were studied; the level of IL-8 on neutrophil of RA patients peripheral blood after the effecting of CyPA was assessed by ELISA. Correlations between CyPA and IL-8 were observed in RA.
The chemotaxis of neutrophil which IL-8 mixed with CyPA antibody was lower than IL-8(P<0.05); The secretion of RA peripheral blood in IL-8 was higher than normal people, as well as the secretion of RA peripheral blood in IL-8 after effecting of CyPA was higher than before (P<0.05), but the level of IL-8 after blocking CyPA was not changed.
CyPA could affect the chemotaxis of IL-8 in the neutrophil of RA patients' peripheral blood. The secretion of IL-8 is accelerated by CyPA on neutrophil of RA patients'peripheral blood.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 05/2009; 25(5):423-5.
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ABSTRACT: To observe the correlation between vascular endothelial growth factor (VEGF) and CD147 expressed in the rheumatoid synovium and to investigate the effect of CD147 of cultured rheumatoid fibroblast-like synoviocytes (FLS) on the production of VEGF.
The presence of CD147 and VEGF in the rheumatoid synovium derived from 15 patients with RA and 4 patients with osteoarthritis (OA) was detected by streptavidin/peroxidase (SP) immunostaining. FLS were cultured by enzymatic digestion of synovial tissues and incubated in 24-well plates. Then different concentration of LY294002, PD98059, SP600125, SB203580 and HAb18G mAb was added to each well. VEGF in the culture supernatant was measured by sandwich ELISA.
CD147 and VEGF in synovium from 15 patients with RA showed high expression, while CD147 and VEGF in synovium from 4 patients with OA showed low expression. Macrophages, fibroblast-like synovial cells and lymphocytes were demonstrated to express CD147 while synovial lining cells, fibroblasts surrounding microvessels and vascular smooth muscle cells were demonstrated to express VEGF. Statistic analysis indicates that VEGF production was correlated with the levels of CD147 expression. VEGFproduction was suppressed when CD147 expression was inhibited by LY294002 or HAb18G mAb.
CD147 can regulate the angiogenesis in rheumatoid arthritis by VEGF. The low levels of CD147 expressed by FLS cells decrease the production of VEGF via the PI3K-Akt signaling pathway. These findings further highlight the importance of CD147 in pannus formation and angiogenesis.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 06/2007; 23(5):426-8.
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ABSTRACT: To explore the percentages of CD4(+) CD25(high) regulatory T cells in peripheral blood or synovial fluid from patients with rheumatoid arthritis (RA) on different stages, and to study their correlations with the activity of the disease.
Peripheral blood lymphocytes were obtained from the patients with active RA who had received no previous disease modifying (DMARDs) therapy(n=11), from individuals with stable, well-controlled RA (n=12), from subjects whose disease were poorly improved after DMARDs therapy(n=9), and from healthy controls(n=8). The frequencies of CD4(+) CD25(high) T cells were quantified by using flow cytometry(FCM). Meanwhile, the correlations between the percentage of CD4(+) CD25(high) T cells and the level of Anti-CCP antibody, CRP, ESR and RF were also investigated. Paired blood and synovial fluid was analysed in a small group of RA and other patients.
There was a smaller proportion of CD4(+) CD25(high) T cells in the peripheral blood of the active RA patients(mean 5.24%) and poorly-improved RA patients(mean 6.43%) than in patients with stable well-controlled RA or in healthy controls(mean 11.79% and 17.17%, respectively, P<0.01 in each case). The frequency of CD4(+) CD25(high) T cells from RA was negatively associated with Anti-CCP antibody(58.0 Ru/mL), ESR(38.8 mm/h) and CRP(2.73 mug/L), (P<0.05 for each). On contrast, The frequency of CD4(+) CD25(high) T cells from healthy controls was not significantly correlated with the level of Anti-CCP antibody(<5.0 Ru/mL), ESR(4.67 mm/h), CRP(0.15 mug/L) and RF(1.37) (all P>0.1). The percentage of CD4(+) CD25(high) regulatory T cells from synovial fluid of RA patients (24.32%) was significantly lower than that of AS patients(30.24%) (P<0.05).
The results demonstrate a smaller CD4(+) CD25(high) regulatory T cell population in peripheral blood of individuals with active RA prior to disease modifying treatment and with poorly-improved RA, and this is negatively associated with the activity of the disease.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 04/2007; 23(4):331-4.
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ABSTRACT: To investigate the expression of CD147 both on cellular membrane and in culture supernatant of in vitro cultured THP-1 cells and monocytes of RA patients before and after stimulation with phorbol myristate acetate (PMA).
Human peripheral blood monocytes (HPBMs) were segregated from RA patients by adherent culture method. After the HPBMs and THP-1 cells were stimulated with PMA, the expression rate and expression intensity of CD147 on THP-1 cells and HPBMs of RA patients were determined by flow cytometry. The soluble CD147 levels in culture supernatant of in vitro cultured THP-1 cells and HPBMs were quantified by double antibody Sandwich ELISA.
The soluble CD147 in the supernatant of cultured THP-1 cells and HPBMs was detectable before PMA stimulation. Both cells showed high CD147 expression. After PMA stimulation, soluble CD147 level in the supernatant of THP-1 cells increased. CD147 expression on THP-1 cells firstly increased then decreased and kept stable for several days. CD147 expression on HPBMs of RA patients decreased, but CD147 level in the culture supernatant of HPBMs of RA patients increased and kept stable for 2 days.
CD147 molecule can be shed from the membrane of THP-1 cells and HPBMs of RA patients, or it can be directly secreted into the culture supernatant from the two kinds of cells. PMA can up-regulate the level of soluble CD147.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 06/2006; 22(3):333-5, 338.
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ABSTRACT: To examine the role of matrix metalloproteinases(MMP) expressed by the tartrate-resistant acid phosphatase (TRAP)-positive mononuclear and multinucleated cells in articular cartilage damage.
C57BL/6 mice were immunized by injection of chicken type II(CII) collagen to construct the collagen-induced rheumatoid arthritis(CIA) model. The presence of TRAP positive cells in the synovial tissue of CIA mice was examined by enzyme histochemistry and expression of MMP-2,9 was assessed in TRAP positive cells by immunohistochemistry.
Expression of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) was detected in TRAP positive mononuclear and multinucleated cell. Quantity of TRAP positive cells and the destruction of articular cartilage had a positive correlation (r(s) =0.903, P<0.01). Expression of MMP-2 and MMP-9 in TRAP positive cells was also correlated significantly with the destruction of articular cartilage (r(s) =0.954, P<0.01).
This study suggests that MMP-2 and MMP-9 expression by TRAP positive mononuclear and multinucleated cells are involved in articular cartilage destruction in CIA.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 05/2005; 21(3):349-52.
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ABSTRACT: To induce bone marrow mesenchymal stem cells or bone marrow stroma cells (MSCs) to differentiate directionally towards chondrocytes in vitro and then identify the differentiated cells.
MSCs were isolated from bone marrows of healthy adult human donors and then the cultured. MSCs at the 3rd passage were induced by TGF-beta1, dexamethasone(Dex) and Vitamin C(Vit C). 14 days later, micro-cell aggregates formed by the induced MSCs were embedded in paraffin, sectioned and stained with HE, toluidine blue or anti-type II collagen mAb (Col II). The expression of Col II in the induced MSCs also were detected by Western blot and RT-PCR.
The induced cells exhibited a chondrocyte-like morphology.The staining of toluidine blue and Col II on extracellular matrix in the micro-cell aggregates was positive. Western blot and RT-PCR showed that Col II was only expressed in the induced cells.
MSCs can differentiate into chondrocytes under the induction of TGF-beta1, Dex and Vit C.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 02/2005; 21(1):79-82.
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ABSTRACT: To observe the positive rate and its clinical significance of serum antibody to ribosomal P protein (anti-P) in patients with systemic lupus erythematosus (SLE).
Anti-P protein antibody was detected by EURO blot with synthetic full-length ribosomal proteins, and then correlation between anti-P antibody, other autoantibodies and clinical characters was analysed .
In 150 SLE patients 36 cases (24%) were positive for anti-P antibody. The incidence of renal and central nervous system lesions in anti-P antibody-positive patients was notably higher than that in anti-P antibody-negative group. The positive rate of anti-P antibody was correlated with that of anti-dsDNA, anti-Sm and anti-RNP antibodies, but had no correlation with the lesions of skin, joint, blood and liver, nor indexes of disease's activity (DAI).
There is correlation between anti-P antibody level with renal and CNS lesions in SLE patients.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 02/2005; 21(1):120-2.
