| Tumor necrosis factor-α is a pleiotropic cytokine with a variety of biological activities produced chiefly by activated monocytes/macrophages, and lymphocytes. TNF-α has a wide range of biologieal effects such as tumoricidal activity, promotion of inflammation, regulation of immune response etc. TNF-α exerts biological effects with two forms, namely a 26kD transmembrane TNF-α (mTNF-α) and a 17kD secreted TNF-α ( sTNF-α) . mTNF-α is anchored on the plasma membrane with the hydrophobic region of its signal peptide. The extracellular segment of mTNF-α can be proteolytically cleaved by TNF-α converted enzyme with the release of sTNF. The two forms of TNF-α elicit a particularly broad spectrum of biological responses by binding two receptors, TNFR1 and TNFR2.The activation-induced cell death (AICD) of mature T lymphocytes represents a major mechanism of the body in the elimination excessive effector cells, modulation of the immune response, maintenance of immunological homeostasis and induction of peripheral immunological tolerance. It has been demonstrated that AICD is mediated mainly but not exclusively by the expression of the death molecule, Fas/FasL. No final conclusion has been reached so far about the role of TNF-α in AICD. Besides, the relevant studies carried out are focused only on sTNF-α, little has been reported concerning the role of mTNF-α in AICD.It was shown by recent studies that the binding of mTNF-α to TNF receptor. resulted in not only the transduction of a signal from TNFR to target cell (positivesignaling) but also the transmission of a signal from mTNF-α, like a receptor, toeffector cell (reverse signaling). It was reported that Infliximab treatment inducesapoptosis of lamina propria T lymphocytes and activated peripheral blood Tlymphocytes in Crohn's disease.The aim of the present study is to compare the role of mTNF-a and sTNF-a in AICD of CD4~+ leukemia Jurkat T cells induced by PHA in an attempt to clarify the biological characteristics and regularities of TNF-a, particularly of positive and reverse signaling mTNF-a, so as to provide experimental basis for further study of the molecular mechanisms of AICD, and provide clues for treatment of autoimmune diseases.1. The experimental model of AICD(1) Dose dependent of AICD induced by PHA in Jurkat cellsTo induce AICD, we stimulated Jurkat cells with various concentrations of PHA.Jurkat cells can be induced to be apoptosis by PHA in a dose dependent manner, the apoptosis rate are proportional to the concentrations of PHA. The apoptosis are 50%, 63% and 80% at PHA 1μg/ml, 5μg/ml, 20μg/ml separately, suggesting that an experimental model of AICD was successfully established.(2) Time-course of AICD induced by PHA in Jurkat cellsTo observe the apoptosis development of AICD, the apoptosis rates were determined at PHA 5μg/ml. The apoptosis activity was already detectable (22%) at 2h, and increased rapidly to 56% at 6h, after that it increased slowly at 24h(63%), 72h(83%). Jurkat cells were incubated for 24h with 5μg/ml of PHA as a model of AICD in the present work to induce the apoptosis effectively and also moderately in order to observe the promoting and inhibitory effect.(3) TNF-α was involved in AICD of Jurkat cells induced by PHAAICD could be partially blocked by anti-TNF-α mAb which cannot exhibit the reverse signaling to neutralize the endogenous TNF-α, (inhibitory rate 14.01%, p<0.05), indicating that TNF-α was involved in AICD of Jurkat cells induced by PHA.2. Positive signal of mTNF-α and sTNF-α in AICD of Jurkat cells (1) Positive signal of mTNF-α in AICDA. Expression of endogenous mTNF-α in AICDThe mTNF-α expression level on the surface of Jurkat cells was analyzed by flow cytometry before and after PHA stimulation. The mTNF-α expression was significantly increased in the PHA-activated Jurkat T cells compared with the control Jurkat cells (4.5% versus 45.8% at 24h). The expression rate of mTNF-α increased gradually in a time dependent manner.B. Cytotoxicyty of mTNF-α against non-activated Jurkat cellsJurkat cells were stimulated with PHA for 24h, then washed and fixed with 1% polyformaldehyde, washed with acidified buffer (glycine HC1 buffer), were used as effectors which express mTNF-α. The effector cells were mixed with non-activated Jurkat cells as targets at different ratios and the extent of cell death was determined by the WST-1 assay. Prestimulated Jurkat cells induced death of their non-activated counterparts. The percentage of cell death increased with the effector to target ratios, reaching 71% at ratio 5:1. Anti-TNF rabbit pAb and anti-TNF mouse mAb partly block the cytotoxicity (inhibitory rate at 53.86%, 43.37%, respectively, p<0.01). These results indicate that the cytotoxicity induced by activated Jurkat cells were partly mediated by mTNF-α.C. Cytotoxicity of mTNF-α on PHA activated Jurkat cellsThe PHA-activated Jurkat cells (24h) expressing mTNF-α were used as effectors and mixed with PHA-activated cells (2h) as targets for 48h. mTNF-α on PHA activated Jurkat cells induced significant apoptotic cell death (44.37%) against activated Jurkat target cells. Anti-TNF pAb and anti-TNF mAb partly block apoptosis (p<0.01). mTNF-α could therefore account for the AICD of activated Jurkat cells.(2) Positive signal of sTNF-α in AICDA. Secretion of endogenous sTNF-α by PHA activated Jurkat cellsJurkat cells were stimulated with PHA for 24h, and detect the sTNF-α release by the ELISA assay. Endogenous sTNF-α was non-detectable.B. PHA induce TACE on Jurkat cellsWe next investigated the expression of TNF converting enzyme (TACE), FACS analysis showed that TACE expression on Jurkat cells was greatly increased stimulated by PHA (44.39%). It was possible that the TACE-mediated cleavage of mTNF-α generates a large amount of soluble TNFR that affects the detection of sTNF-α.C. Cytotoxicity of exogenous sTNF-α on PHA activated and non-activated Jurkat cellsTo observe the apoptosis effect- induced by sTNF-α, Jurkat cells were treated with exogenous sTNF-α(50U/ml) for 24h. sTNF-α are cytotoxic against both non-activated Jurkat cells and PHA-activated cells, with the apoptosis rate of 23.81% and 78.13%, respectively(p<0.01). 3. mTNF-α reverse signaling in AICD of Jurkat cells(1) Enhancement of AICD via mTNF-α reverse signalingA. mTNF-α reverse signaling triggered by pAb against TNF-α accelerate AICD PHA-activated Jurkat cells were treated with anti-TNF pAb simultaneously. As acontrol, anti-TNF alone did not induce apoptosis of non-activated Jurkat cells, whereas the apoptosis rate of activated Jurkat cells was greatly induced by 20.11%, indicating that mTNF-α reverse signaling could enhance the AICD of Jurkat cells.B. Augment of AICD via mTNF-α reverse signaling triggered by sTNFR1To confirm the direct involvement of mTNF-α reverse signaling, 20μg/ml soluble TNF receptor (sTNFR1) were used to stimulate the reverse signaling of mTNF-α. sTNFR1 was able to increase the apoptosis rate of activated Jurkat cells instead of non-activated cell (86.82%, p<0.01). The apoptosis rate increased in sTNFR1 dose dependent manner.(2) Mechanism mediated by mTNF-α reverse signaling in AICD of Jurkat cells A. mTNF-α reverse signaling upregulate the expression of Fas/FasLAlthough non-activated Jurkat cells expressed almost undectable level of Fas and FasL, the expression of Fas and FasL was significantly induced by PHA. The treatmentof anti-TNF pAb further upregulate the expression of Fas/FasL (33.28% versus 52.19%, 34.98% versus 47.74%, respectively, p<0.01).B. Increase in PFLA induced mTNF-α expression by reverse signaling via mTNF-αUnstimulated Jurkat cells expressed low level of mTNF-α, after stimulated with PHA, the expression of mTNF-α was greatly induced. Anti-TNF pAb was able to significantly enhance the expression of mTNF-α on the surface of Jurkat cells compared to PHA alone (45.51%versus61.15%, p<0.01).C. Effect of mTNF-α reverse signaling on expression of TNFR1 and TNFR2 in PHA induced AICDNon-activated Jurkat cells express basal amount of TNFR1 and TNFR2. PHA predominantly induced the expression of TNFR1 (45.29%), whereas TNFR2 only slightly increased (6.54%), indicating that TNF-α induced the apoptosis in AICD of Jurkat cells mainly through TNFR1 which contains the death domain. The reverse signaling of mTNF-α induced by anti-TNF pAb obviously upregulate the expression of TNFR1 and TNFR2 (59.32% and 55.66%, separately, p<0.01).In summary, the results above suggest that the positive signaling and reverse signaling of mTNF-α might mediate the AICD of Jurkat cells induced by PHA. PHA predominantly induced the expression of TNFR1 compared with TNR2, indicating that TNF-α induced the apoptosis in AICD of Jurkat cells mainly through TNFR1 which contains the death domain. Upregulation of Fas/FasL, mTNF-α and TNFR1/TNFR2 may account for the mechanism of enhancement of AICD mediated by mTNF-α reverse signaling on Jurkat cells. |
