Detection of peripheral blood Treg cells and secreted IL-35 level in patients with rheumatoid arthritis and its clinical significance / 重庆医学

Objective To detect the number of peripheral blood Treg cells and secreted IL-35 expression level in the patients with rheumatoid arthritis(RA) and to explore their correlation with RA occurrence.Methods Peripheral blood was collected from 45 cases of RA,22 cases of osteoarthritis(OA) and 26 persons undergoing healthy physical examination(control group).The number of CD4+ CD25+ foxp3+ regulatory T cells was determined by flow cytometry,while plasma IL-35 level was determined by ELISA.Then the relationship between Treg and expressed IL-35 with clinical indicators was analyzed.Results The percentage of peripheral blood Treg ceils to total CD4+T cells in the RA group was (5.65 ± 2.33)％,which was significantly increased compared with (4.12 ± 1.75) ％ in the control group(P＜0.05);the difference between the OA group and RA group was not statistically significant(P=0.086).The average fluorescence intensity of foxp3 had no statistical difference among 3 groups(P＞0.05).The higher the DAS28 score,the lower the peripheral blood Treg cells number and foxp3 fluorescence intensity.The plasma IL-35 level in the RA group [(34.22± 14.35)ng/L] was significantly lower than that in the OA group[(78.63± 24.58)ng/L] and control group [(67.56±25.43)ng/L],the difference was statistically significant(P＜0.05).The Treg number in RA patients was negatively correlated with ESR and DAS28 score(r=-0.223,-0.343,P=0.023,0.011),but had no correlation with rheumatoid factor,C-reactive protein and anti-CCP antibody.Conclusion The peripheral blood Treg cells number in RA patients is elevated,while the IL-35 level is decreased,the negative regulation ability in the patients with Treg cell function deficit is attenuated.

Apolipoprotein C3 enhanced THP-1 cell adhesion to aortas of mice / 中华微生物学和免疫学杂志

Objective To investigate the enhancing effect of apolipoprotein C3 (APOC3) on THP-1 cell adhesion to aortas of mice.Methods Microsurgery was performed to separate the aorta of C57BL/6 mice in sterile condition.After stimulated by APOC3 (100 △g/ml) in vitro for 16 h,the aorta was allowed to adhere for 1 h with CFSE labeled THP-1 cells (1 ×106/ml).Then the adhesion effect was observed,and the expressions of vascular adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) were detected by immunohistochemical method.Results Adhesion effect of the mice aorta with THP-1 cells in the APOC3 stimulated group was stronger than the control group.Both the expressions of VCAM-1 and ICAM-1 in aortas were increased by APOC3,but the former was significantly up-regulated than the latter.Conclusion Apolipoprotein C3 could enhance THP-1 cell adhesion to aortas of mice.

The expression of sialic acid-binding immunoglobulin-like lectin-1 on peripheral blood mononuclear cells and its relationship with disease activity in rheumatoid arthritis / 中华风湿病学杂志

Objective To investigate the expression of sialic acid-binding immunoglobulin-like lectin-1 (Siglec-1) in the peripheral blood mononuclear cells (PBMCs) in patients with rheumatoid arthritis (RA),osteoarthritis (OA) and healthy controls and to explore the relationship between Siglec-1 expression and disease activity in RA.Methods Siglec-1 protein and mRNA levels were measured by flow cytometry and real-time quantitative reversetranscription-polymerase chain reaction (qRT-PCR) in 42 RA patients,28 OA patients and 26 healthy controls,respectively.The correlation studies between Siglec-1 and disease activity score 28 (DAS28) or C-reactive protein were performed.T-test was used for comparisons between groups and Pearson's correlation test was used for correlation analysis.Results The percentage of Siglec-1 positive cells of PBMCs in RA group [(15.2±7.6)％] was significantly higher than that in the OA group [(2.3 ±2.6)％] or healthy controls [(2.1±1.6)％,t=8.615,8.661; all P＜0.01].And the major cell type in PBMCs that expressed Siglec-1 was monocytes.The relative Siglec-1 mRNA expression in PBMCs in the RA group (3.4±1.5) was also significantly higher than that in the OA group (1.2±0.4) or healthy controls [(1.0± 0.4),t=3.446,3.966; all P＜0.05].But no significant differences of Siglec-1 protein and mRNA between the OA group and healthy controls were found.Furthermore,positive correlations between Siglec-1 protein and DAS28 or hs-CRP were found in RA patients (r=0.89,P＜0.01; r=0.48,P＜0.01).Conclusion PBMCs are activated which are characterized by elevated expression of Siglec-1 in RA patients.Circulating Siglec-1 may be considered as a potential noninvasive biomarker for monitoring disease activity in RA.

The role of monocytes Siglec-1 in stimulating CD4 + and CD8 + T cells proliferation and activation in atherosclerosis / 中华检验医学杂志

Objective To investigate the role of atherosclerotic monocytes Siglec-1 in stimulating CD4 + and CD8 + T lymphocytes proliferation and activation.Methods Experimental research.Cluster of differentiation antigen 14 (CD14) positive monocytes of 18 acute coronary syndrome (ACS),41 stable angina (SA) and 32 healthy volunteers were separated by magnetic-activated cell sorting.Different concentration of interferon-α (IFN-α,0,2,5,10 ng/ml) were used to up-regulate Siglec-1 and small interfering RNA (siRNA) or blocking antibody were used to down-regulate Siglec-1.Then monocytes were cocultured with CD4 + T/CD8 + T cells from a third healthy volunteer for 5 days.The experiment was designed for 11 groups (n=10 for each group),that was (1) normal CD14,(2) normal CD14 + IFN-α 5 ng/ml,(3) normal CD14 + IFN-α 5 ng/ml + anti-Siglec-1 2 μg/ml,(4) ACS CD14,(5) ACS CD14 + control siRNA group (Mock),(6) ACS CD14 + siRNA 679 40 nmol/L,(7) ACS CD14 + anti-Siglec-1 2 μg/ml,(8) SA CD14,(9) SA CD14 + Mock,(10) SA CD14 + siRNA 679 40 nmol/L and (11) SA CD14 + antiSiglec-1 2 μg/ml.Cell Counting Kit-8 (CCK-8) was used to determine T cells proliferation and ELISA was used to detect Interleukin-2 (IL-2),IL-10,IL-12 and IFN-γ of culture supematant.Data of cytokines detection were expressed as medium (quartiles) and analyzed by nonparametric rank sum test.Results By the blockage of Siglec-1 (group 6),the proliferation of CD4 and CD8 were decreased.Secretion of IL-2,IL-12 and IFN-γ by CD4 cells [67.00(62.50-87.30),0.86(0-1.63),and 47.82(37.60-56.67) pg/ml,respectively] were decreased and IL-10 [56.00(46.25-67.40) pg/ml] was increased compared with those in control group [group 4,213.70 (187.50-275.30),6.87 (4.90-8.93),114.90 (89.50-167.40),and 21.08 (15.70-33.20) pg/ml,respectively,with U =8.50,17.00,8.50,and 87.50,respectively.all P ＜ 0.05].When monocytes Siglec-1 from control group was up-regulated by IFN-α (group 2),secretion of IL-2,IL-12 and IFN-γ [220.44(174.30-312.30),7.90(6.540-10.40) and 143.75(78.20-210.00) pg/ml,respectively] were increased and IL-10 [21.95 (16.30-25.00) pg/ml] was decreased (compared with group 1,with U =89.50,98.00,100.00,and 0,respectively.all P ＜ 0.05).Regulation of Siglec-1 had no role in cytokines production in cocultured CD8 + T cells (all P ＞ 0.05).Conclusions IFN-α can upregulate monocytes Siglec-1.Siglec-1 may participate in the pathogenesis of AS via promoting proliferation of CD4 +/CD8 + T cells and Thl cytokines secretion of CD4 + T cells.

Construction of lentiviral vector containing siRNA sequence of Siglec-1 and verification of inhibition efficiency / 中华微生物学和免疫学杂志

Objective To construct lentiviral vectors containing small interfering RNA (siRNA) sequence of Siglec-1 and to screen the effective vector.Methods Three fragments of Siglec-1 siRNA were designed and cloned into pGCSIL-GFP lentiviral plasmid.And then the plasmid was cotransfected into 293T cells with pHelper 1.0 and pHelper 2.0 plasmids.Forty-eight hours later,culture supernatant with virus particles was collected and concentrated.Virus titer was determined by 10-fold serial dilution method and virus was transduced into primary cultured mouse bone marrow-derived macrophages (BMM).Flow cytometry and QRT-PCR were used to screen effective vector with inhibition ability.Results Three vshRNA lentiviral plasmids and a control plasmid were constructed successfully and verified by DNA sequencing.Virus titer was between 1×10s TU/ml and 1×109 TU/ml,which was suitable for in vitro and in vivo experiments.The Lv-1 could inhibit Siglec-1 expression effectively in vitro transduction of BMM.Conclusion Lentiviral vectors containing siRNA sequence of Siglec-1 were constructed successfully and an effective vector was screened,which may lay the foundation for using the vector in gene knockdown experiment in vivo.

Increased expression of Siglec-1 and cytokines by stimulation of oxidized low-density lipoprotein on RAW264.7 cells / 中华微生物学和免疫学杂志

Objective To explore the role of sialic acid-binding immunoglobulin-like lectin-one (Siglec-1) in the process of atherosclerotic inflammation induced by oxidized low-density hpepmtein (ox-LDL). Methods Ox-LDL was synthesized by oxidization of native LDL and different concentration of ox-LDL was added to the culture medium of RAW264.7. Forty-eight hours later, cells and supernatants were collected separately. The expression of Siglec-1 protein and mRNA were measured by flow cytometry(FCM) and real-time quantitative RT-PCR, respectively. The levels of monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1α(MIP-1α) and IL-8 in supernatants were determined by ELISA. Re-sults By the stimulation of ox-LDL, Siglec-1 protein and mRNA on RAW264.7 cells were significantly in-creased, meanwhile, the cytokines levels in culture supematants were significantly higher than that in the control group. And both Siglec-1 expression and cytokine secretion were ox-LDL dose-dependent. Conclu-sion Ox-LDL can increase Siglec-1 protein and mRNA expression and some inflammatory cytokines secre-tion on RAW264.7 cells in a dose-dependent manner. Manifested by enhanced Siglec-1 expression, the acti-vated macmphages may take a part in the development and progression of atherosclerosis.

The role of Siglec-1 in chemokines secretion and phagocytosis of oxidized low-density lipoprotein on macrophages / 中华检验医学杂志

Objective By in vitro culture of mouse macrophage cell line RAW264. 7 and primary mouse bone marrow macrophages, the expression of Siglec-1 when stimulated by ox-LDL was observed. Meanwhile, Siglec-1 was up-regulated by M-CSF and down-regulated by small interference RNA targeting Siglec-1 ( si-RNA-Siglec-1) , and the expression of chemokines and lipid uptake ability by macrophages were observed, to explore the role of Siglec-1 on macrophages in atherosclerosis. Methods LDL was oxidized by copper. According to preliminary experiment results, ox-LDL 100 μg/ml was selected as a stimulus. There were 6 experimental groups:normal control group,ox-LDL 100 μg/ml group, ox-LDL 100 μg/ml + si-RNA 2509 2 ng/ml group,ox-LDL 100 μg/ml + si-RNA 3618 2 ng/ml group,ox-LDL 100 μg/ml + M-CSF 5 ng/ml group and ox-LDL 100 μg/ml + M-CSF 10 ng/ml group. si-RNA-Siglec-1 was transfected into macrophage to inhibit the expression of Siglec-1, whereas M-CSF 10 ng/ml or 5 ng/ml were added into the culture medium to enhance the expression of Siglec-1. Quantitative real-time polymerase chain reaction ( qRT-PCR) was used to determine the interfere efficiency of si-RNA-Siglec-1 or M-CSF. After stimulation with ox-LDL for 48 h, cell culture supernatants were collected to determine MIP-1 alpha, MCP-1 and IL-8 concentration by ELISA (n =3 for each group) to evaluate the activation of macrophages. Internalization of lipid particles by macrophages was analyzed by oil red 0 staining. Results Observed by fluorescence microscope, si-RNA-Siglec-1 could be effectively transfected into macrophages with a transfection efficiency about 90% ;PCR results showed that si-RNA 2509 and si-RNA 3618 in a concentration of 40 pmol/L had an inhibition rate of 0. 54 ±0. 11 or 0. 52 ±0. 16 vs 1. 00 ±0. 24 (control group) , t =5. 227 and 4. 992, respectively, all P < 0.01, while M-CSF 10 ng/ml could increase Siglec-1 mRNA expression approximately 4-fold (4. 16 ± 1. 25 vs 1.00 ±0. 24, t =7. 448, P<0. 01). The secretion of MCP-1, MIP-1 alpha, and MIP-2 in si-RNA3618-Siglec-1 group [(359. 28±47. 80) pg/ml, (33. 76 ± 14. 28) ng/ml and (7.87±1.55) ng/ml for MCP-1,MIP-1 alpha, and MIP-2, respectively] was significantly reduced in compare with ox-LDL 100 μg/ml group [ (577. 89 ± 35. 95 ) pg/ml, (69. 17 ± 11. 82) ng/ml and (12.28 ± 1.19) ng/ml for MCP-1, MIP-1 alpha, and MIP-2, respectively], with P value of 0.01, 0.05 and 0.01. In contrary, ox-LDL 100 μg/ml plus M-CSF 10 ng/ml group could significantly promote macrophage chemokine secretion [ (672. 89 ± 43.80) pg/ml, (101.31 ±24.17) ng/ml and (14.81 ±0.54) ng/ml for MCP-1, MIP-1 alpha, and MIP-2, respectively], with P < 0.05 compared with ox-LDL 100 μg/ml group. Meanwhile, lipid intemalization and foam cell formation was inhibited in si-RNA3618-Siglec-l group while ox-LDL 100 μg/ml plus M-CSF 10 ng/ml group could enhance the phagocytosis of ox-LDL by macrophage. Conclusions Siglec-1 may served as a potential phagocytic receptor for ox-LDL involving in macrophage uptake of lipid and turn into foam cells. Furthermore, it can active macrophages and enhance the secretion of MIP-1 alpha, MCP-1 and IL-8, attracting more macrophages and lymphocytes to the site of inflammatory plaque. Targeted inhibition of Siglec-1 reduces macrophage uptake of lipid and secretion of chemokines. Siglec-1 may possibly serve as a potential target of treatment or delay the development of atherosclerosis.

The expression of sialic acid-binding immunoglobulin-like lectin 1 on peripheral mononuclear cells in patients with coronary heart disease and its clinical significance / 中华检验医学杂志

Objective To investigate the expression of sialic acid-binding immunoglobulin-like lectin-one (Siglec-1, also called CD169) in lymphocytes, monocytes and neutrophils in peripheral blood in patients with coronary heart disease(CHD), and explore the relationship between Siglec-1 expression and atheresclerosis. Methods CD145 CD169 positive cell proportion and CD169 mRNA levels were respectively measured by flow cytometry and real-time quantitative reverse transcription-polymerase chain reaction (FQ-RT-PCR) in 57 CHD patients and 38 healthy controls. And the levels of serum hpids were determined by automatic biochemistry analyzer. Results The flow cytometry analysis showed that CD169 protein was not found in lymphocytes and neutrophils in both CHD patients and healthy controls. The rate of CD14 CD169 double positive ceils in monocytes in CHD group was significandy higher than that in healthy controls [(12.7±2.4)% vs (1.0±0.3)% ,t =23.2,P<0.01]. And FQ-RT-PCR analysis showed that the mean CD± mRNA copy number in PBMCs in CHD group was significantly higher(3.2 fold) than that in healthy controls [t = 6. 59, P < 0.01]. However, neither differences of CD169 protein positivities [[(12. 2 ± 2. 3) %vs (13.4±2.5)% ,t = 1.87,P >0.05] nor mRNA levels [3.64 fold vs 2.79 fold when compared with healthy controls,t =0. 98, P > 0. 05] were found between CHD patients with normal and abnormal levels of serum Lipids. Conclusions CD169 is mainly expressed in human tissue-resident macrophages but not expressed in peripheral blood monecytes. And when the monocytes is stimulated by inflammation, the expression of CD169 is increased. In patients with CHD, the increased expression of CD169 protein and mRNA level has demonstrated the activation of monocytes in peripheral blood. CD169 and CD169-mediated monocytes activation may play an important role in the development and progression of atherosclerosis.

Non-elevated cardiovascular risk in patients with primary biliary cirrhosis and hypercholesterolemia / 第二军医大学学报

Primary biliary cirrhosis(PBC) is an autoimmune disease with unknown causes.Most PBC patients have abnormal lipid metabolism characterized by hypercholesterolemia.Paradoxically,clinical observations and pre-experimental studies showed that the risk of hyperlipidemia associated cardiovascular event and the mortality of PBC patients were not increased.In this review we summarize the possible reasons and the underlying mechanisms.