ABSTRACT
Objective:To investigate whether autophagy in the inflammatory environment of thrombotic antiphospholipid syndrome (APS) drives polymorphonuclear neutrophils (PMNs) to release neutrophil extracellular traps (NETs) and whether IFN-λ1(IL-29) has a function of inhibiting NETs-related thrombin production.Methods:PMNs isolated from peripheral blood of healthy volunteers were in vitro stimulated by serum samples of patients with active APS. IL-29 and 3-methyladenine (3-MA) were used to regulate NETs release and autophagy. NETs expression was evaluated by immunofluorescence technique (IFT) and flow cytometry (FCM). Autophagy-related proteins were detected by Western blot. The levels of thrombin-antithrombin (TAT) complexes were determined by enzyme-linked immunosorbent assay (ELISA). Results:The percentages of NETs released from PMNs of healthy volunteers after stimulation with serum samples of APS patients were (22.8±3.1)% according to IFT and (10.1±2.7)% according to FCM and used as control group. IL-29 and 3-MA inhibited the release of NETs from PMNs stimulated by serum samples of APS patients and the percentages of NETs were (5.3±2.2)% and (4.9±2.4)% detected by IFT, and (2.1±1.3)% and (2.7±1.4)% by FCM, respectively. There were significant differences with the control group ( q=9.89, 10.67, 11.74, 9.61, all P<0.01). IL-29 inhibited the expression of LC3B, but promoted the expression of p62 on PMNs of healthy volunteers after stimulating with serum samples of APS patients, and the differences with the group without IL-29 pretreatment were statistically significant [LC3B/GAPDH: (0.28±0.03)% vs (0.77±0.06)%, q=8.65; p62/GAPDH: (0.63±0.05)% vs (0.33±0.03)%, q=4.78; both P<0.01]. IL-29 and 3-MA reduced the levels of NETs-related TAT complexes from (7.6±0.6) ng/ml to (3.1±0.5) ng/ml and (4.7±0.4) ng/ml, respectively ( q=6.34 and 5.15, both P<0.01). Conclusions:Autophagy was involved in the formation of NETs and subsequent coagulation cascade activation in PMNs of healthy subjects after stimulation with serum samples of APS patients with thrombosis. IL-29 suppressed the production of NETs and NETs-associated thrombin by inhibiting autophagy.
ABSTRACT
Objective To investigate the roles of a variety of cytokines including transforming growth factor beta (TGF-β),interleukin-6 (IL-6) and interleukin-2 (IL-2) in the differentiation of CD+4 Tlymphocyte cells.Methods T lymphocyte cells either in human peripheral blood or routine spleen were cultured in vitro under different stimulation conditions.Flow cytometry was used to analyze the percentages of CD+4IL-17+ T-helper 17(Th17) cells,CD+8 IL-17+ T cells,CD+4 CD+25 FOXP+3 T regulatory (Treg) cells among activated T cells.Results Differentiation of Treg cells,Th17 cells and CD+8 IL-17+ T lymphocyte cells was enhanced when murine splenic T cells were cultured with TGF-β.The levels of expression were (7.8±2.2)%,(12.6±3.1)%,(10.1±2.6)% ,respectively.Experimental control group was severally same type of T cells without cytokine treatment.The levels of expression were (4.8±0.6) %,(1.7±0.5) %,(1.0±0.4) %,respectively.There were statistically significant differences among them (q=4.09,8.80,9.61.P<0.05 or P<0.01).Under combination treatment with IL-6 and TGF-β,(17.8±5.3) % Th17 cells and (15.0±4.2)% CDCD+8 IL-17CD+ T cells were induced,whereas the levels of Treg cells whose differentiation were restrained were (4.1±1.2) %.The differences were statistically significant compared with the level of same type of T cells in TGF-β group (q=5.03,5.17,5.04,P<0.01).Moreover,combination treatment with IL-2 and TGF-β decreased percentages of Th17 and CDCD+8 IL-17CD+ T cells and increased percentages of Treg cells in T cell population.There was an opposite effect when anti-IL-2 was apphed.The percentages of Th17 and CD+8 IL-17+ T cells were increased and the percentages of Treg cells were reduced The regulation trend of T lymphocyte cells in human peripheral blood was similar with those in routine spleen.Conclusion Various cytokines are of great importance in the regulation of the balance between Th17 and Treg cell.