Monocyte apoptosis in dialysis patients is Fas ligand-mediated

BACKGROUND: The mononuclear phagocyte system plays an important role in host defense. Since dialysis patients have been reported to show enhanced leukocytes apoptosis, we evaluated the mechanism of increased apoptosis of monocytes in dialysis patients. METHODS: Apoptotic studies were carried out on monocytes isolated from dialysis patients as well as healthy subjects. The effect of dialysis sera and membranes was evaluated on monocyte apoptosis as well as monocyte expression of proapoptotic proteins such as Fas and FasL. To confirm the role of FasL, we evaluated the effect of activated secretory products on T cell apoptosis. In addition, we studied FasL content of dialysis sera and supernatants of activated monocytes. RESULTS: Monocytes isolated from dialysis patients (MDP) showed a greater magnitude of apoptosis when compared to monocytes isolated from healthy subjects (MHS) (MHS, 3.6 +/- 1.1% vs. MDP, 24.3 +/-1.4%). Sera of hemodialysis patients (SHD) promoted (p < 0.001) apoptosis of MHS when compared to pooled control sera (HPS) (HPS, 0.8 +/- 0.5% vs. SHD, 11.5 +/- 0.5% apoptotic cells/field). Dialysis membranes, cellulose acetate membranes in particular, promoted monocyte apoptosis. Interestingly, anti-FasL antibodies partly inhibited dialysis sera-induced monocyte apoptosis. Dialysis membranes also modulated monocyte expression of both Fas and FasL. Secretory products of activated monocytes also promoted T cell apoptosis. Dialysis sera and activated monocyte secretory products showed increased FasL content. CONCLUSIONS: These results suggest that dialysis patients have an increased rate of monocyte apoptosis, which is mediated through a uremic milieu (serum factors). One of these serum factors seems to be FasL. In addition, dialysis membranes seem to promote apoptosis independent of the uremic milieu. The present study provides a mechanistical insight into the enhanced apoptosis of monocytes in dialysis patients.     

Dialysis membrane-induced neutrophil apoptosis is mediated through free radicals

Patients on hemodialysis are prone to infection. Neutrophils are the host's first line of defense against certain invading pathogenic microorganisms. Since apoptotic neutrophils are functionally compromised we examined the effect of dialysis membranes on neutrophil apoptosis. Dialysis patients showed greater (p < 0.001) neutrophil apoptosis when compared with control subjects. Cellulose acetate membranes directly promoted (p < 0.001) neutrophil apoptosis. Cellulose acetate membrane-treated neutrophils exhibited greater apoptosis (p < 0.01) when compared with polysulfone membrane-treated neutrophils. Superoxide dismutase (SOD) partly inhibited the cellulose acetate membrane-induced neutrophil apoptosis, whereas both catalase and dimethylthiourea (DMTU) inhibited the polysulfone membrane-induced neutrophil apoptosis. Similarly, L-NAME, a nitric oxide synthase inhibitor, attenuated both the cellulose acetate and the polysulfone membrane-induced neutrophil apoptosis. In addition, cellulose acetate and monocyte interaction products promoted (p < 0.001) neutrophil apoptosis. These results suggest that dialysis membranes can promote neutrophil apoptosis directly as well as through their interaction with monocytes. The direct effect of dialysis membranes seems to be mediated partly through the generation of reactive oxygen species.     

Apoptotic markers on lymphocytes and monocytes are unchanged during single hemodialysis sessions using either regenerated cellulose or polysulfone membranes

BACKGROUND: There is an increased rate of apoptosis of peripheral blood mononuclear cells (PBMCs) in patients undergoing hemodialysis (HD), but little is known about how different dialysis membranes may contribute to the process. We, therefore, studied the influence of two different dialysis membranes on apoptotic markers during HD. METHODS: 8 healthy controls and 8 patients on regular HD 3 times per week were enrolled in this cross-controlled study. Patients received HD using polysulfone and then regenerated cellulose dialysis membranes for one week each, sequentially. Serum was collected for C-reactive protein (CRP) detection; flow cytometry with dual antibody staining was used to measure the apoptotic markers Fas (CD95), FasL (CD 178) and TNF-R2 (CD120b) in T cells (CD3+), B cells (CD19+), and monocytes (CD14+) at 0, 15, 120 and 240 min after starting HD. We also measured total leukocyte numbers and differential white cell counts. RESULTS: Hemodialysis patients revealed lymphocytopenia, monocytopenia, higher CRP levels and higher Fas and TNF-R2 expression on lymphocytes and monocytes at baseline when compared with normal controls. Leukocyte numbers, including neutrophils, lymphocytes and monocytes, dropped significantly after 15 min of dialysis. There were no significant differences in Fas levels during hemodialysis on T and B lymphocytes or on monocytes. T lymphocyte FasL (CD 178) levels remained unchanged throughout the process. There was a significantly lower overall level of CD120b at 15 min of HD, whereas this marker was higher on monocytes after dialysis. There were no significant differences in the levels of apoptotic markers between the two membranes. CONCLUSION: Our results suggest that uremia itself contributes to PBMC apoptosis. The two different dialysis membranes used in this study did not influence apoptotic markers on PBMCs significantly, but increased TNF-R2 expression on monocytes during a single dialysis session.     

Effect of uremia and dialysis modality on mononuclear cell apoptosis

The aim of this study was to evaluate the effect of both uremia itself and hemodialysis (HD) membranes on the induction of apoptosis. Four groups of subjects were evaluated: 21 nondialyzed (Non-D) patients, 10 continuous ambulatory peritoneal dialysis (CAPD) patients, and 53 HD patients who were on hemophan, cuprophan, cellulose acetate, AN69, and polysulfone; control subjects were nine healthy volunteers. Circulating mononuclear cells were obtained before dialysis and cultured for 48 h. Mean percentage of apoptosis was analyzed by a FACScan flow cytometer using Annexin V-FITC. Cell apoptosis was increased in Non-D patients (11.5 +/- 5.5%) compared with control subjects (2.1 +/- 0.7%, P < 0.001) and CAPD patients (7. 0 +/- 5.8%, P < 0.05). In patients on HD with cuprophan, apoptosis was higher than in control subjects and Non-D and CAPD patients. In Non-D patients, apoptosis was inversely correlated with renal creatinine clearance (r = -0.62, P = 0.003). Cell apoptosis was higher in hemophan than the other HD membranes. In seven patients on hemophan, switching to polysulfone resulted in decreased apoptosis (P < 0.01). Mononuclear cell circulation through mini-dialyzers made of different types of membranes (cuprophan, hemophan, cellulose acetate, AN69, and polysulfone) prouced a significant increase in apoptosis. However, there was a marked difference in the percentage of apoptosis induced by these five membranes, being significantly increased in hemophan and cuprohan compared with the other three membranes. Similar results were obtained when whole blood from healthy donors was circulated through the mini-dialyzers, showing that mononuclear cell apoptosis was increased in hemophan and cuprophan compared with polysulfone. In conclusion, uremia and membrane characteristics may independently affect the mononuclear cell apoptosis.     

Normal T lymphocyte function in patients with end-stage renal disease hemodialyzed with 'high-flux' polysulfone membranes

T lymphocyte function was analyzed in patients hemodialyzed with 'high-flux' polysulfone membranes, which have been reported to improve the patients' overall clinical condition and well-being. For comparison purposes, patients treated by the use of 'low-flux' cuprophane membranes were also studied. Peripheral blood white cell counts, numbers of lymphocytes as well as the numbers of T cells and their CD4 and CD8 subsets were within normal range in both patient groups. The absolute number of B cells was slightly decreased in cuprophane-membrane- but not polysulfone-membrane-treated patients. The proliferative response of T lymphocytes after stimulation with optimal concentration of phytohemagglutinin (PHA) was normal in patients treated with 'high-flux' membrane dialysis but significantly reduced in those treated with cuprophane membranes. The generation of interleukin-2 (IL-2) receptor on T lymphocytes after PHA stimulation was normal in the polysulfone-membrane-treated group and slightly impaired in the cuprophane-membrane-dialyzed patients. Production of both IL-2 and interleukin-1, as well as the natural killer cell activity, in patients treated by 'high-flux' membrane dialysis were also comparable to controls. The levels of serum beta 2-microglobulin were significantly elevated in patients-maintained on 'high-flux' dialysis membranes but did not reach the levels seen in patients dialyzed by cuprophane membranes. The beta 2-microglobulin at levels seen in patients on cuprophane dialysis had no effects on activation and proliferation of control lymphocytes in vitro. These results suggest that impaired functional responses of T lymphocytes seen in end-stage disease patients on prolonged hemodialysis with cuprophane membranes are not seen in similar patients hemodialyzed with polysulfone membranes.     

Ex vivo flow cytometry determination of intracytoplasmic expression of IL-2, IL-6, IFN-gamma, and TNF-alpha in monocytes and T lymphocytes, in chronic hemodialysis patients

AIMS: To determine the intracytoplasmic expression of TNF-alpha, IL-2, IL-6 and IFN-gamma, ex vivo and in vitro, in both monocytes and T lymphocytes by flow cytometry after appropriate stimulation using phorbol myristate acetate (PMA)/ionomycin or lipopolysaccharides (LPS) in the presence of monensin, in order to assess the bio(in)compatibility of different dialysis membranes. METHODS: We examined monocytes and T lymphocytes taken from chronic hemodialysis patients (using either cuprophane (CUP), n = 6; polyacrylonitrile (AN 69), n = 6; or polysulfone (PS), n = 6 membranes), before and after a dialysis session. We compared the results with those obtained from end-stage chronic renal failure patients (n = 3) and healthy volunteers (n = 11). RESULTS: Before any stimulation there was a statistically significant difference in the percentages of TNF-alpha, IL-6, and IFN-gamma- expressing monocytes with respect to the dialysis membrane used. The highest percentages were observed for CUP and AN69 patients with figures of around 30% for each cytokine; the lowest percentages were found in PS patients and healthy volunteers. One hour after LPS stimulation the patterns remained unchanged for TNF-alpha and IFN-gamma, whereas the percentages of IL-6-expressing cells in PS patients and in healthy volunteers reached the figures obtained in the other groups. When we examined the percentage of IFN-gamma-, TNF-alpha- and IL-6-expressing monocytes in patients before and after a dialysis session, before any stimulation, we found that the results were significantly different for the three membranes (p = 0.01). Thus, a dialysis session with polysulfone membranes had no significant effect on the precentages of IFN-gamma-, TNF-alpha-, and IL-6-expressing monocytes, whereas percentages were significantly lower after the dialysis session when using cuprophane or AN69 membranes, suggesting a release of these cytokines by the monocytes during dialysis. A significant number of IFN-gamma- and IL-2-expressing T lymphocytes were only detected after 18 hours of PMA/ionomycin stimulation. The percentages of IFN-gamma-expressing T cells recorded for the different membranes were not statistically different from those recorded for healthy subjects or pre-dialysis patients, i.e., they were between 11.5 and 20%. However, the percentages of IL-2-expressing T lymphocytes were significantly different between the 5 groups, i.e., 31.3, 30.5, 18.6, 13.9 and 7. 6%, respectively, for CUP patients, pre-dialysis patients, healthy volunteers, PS and AN69 patients. This suggests that pre-dialysis and CUP patients have, at baseline, a stimulation of their T lymphocytes. Finally, a 4-hour dialysis session had no impact on the percentages of IL-2-expressing T lymphocytes, whereas it was associated with a significant decrease in the percentage of IFN-gamma-expressing cells, but only when cuprophane membranes were used. CONCLUSION: Cytokine flow cytometry enables one to study, ex vivo, i.e., without any stimulation of the cells, and in vitro after appropriate stimulation, the bio(in)compatibility of dialysis membranes assessed by the intracytoplasmic cytokine profiles of TNF-alpha, IFN-gamma, IL-6 and IL-2, evaluated at the single cell level.     

Transfection of tubule cells with Fas ligand causes leukocyte apoptosis

BACKGROUND: Since the Fas/Fas Ligand (FasL) interaction is recognized as a major pathway of apoptosis in immune cells, we hypothesized that selective expression of FasL by tubular cells (TC) may promote the resolution of interstitial inflammation by inducing apoptosis of infiltrating immune cells. In this study, the effect of FasL transfection of rat TC on apoptosis of leukocytes was examined. METHODS: Rat tubule cells (NRK52E) were transfected with plasmids constructed using human and rat FasL (hFasL and rFasL). The propensity of activated, transfected TC to undergo apoptosis was examined. Similarly, the effects of FasL transfection on apoptosis of Jurkat cells and activated leukocytes were assessed directly following co-culture for 12 hours and in a cell insert system intended to assess the effects of soluble FasL. Fas and FasL expression was assessed by flow cytometry and apoptosis was examined using Annexin V staining and the TUNEL method. RESULTS: Expression of FasL in TC was increased after FasL transfection. Transfected TC showed no detectable increase in apoptosis following lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha) activation. Jurkat cell apoptosis was increased ninefold and eightfold after co-culture with TC transfected with hFasL and rFasL, respectively (67.0 +/- 12.1% and 60.1 +/- 8.8% vs. 6.7 +/- 1.8% with un-transfected TC, P < 0.01). Similarly, apoptosis of activated leukocytes was increased fourfold by co-culture (26.8 +/- 4.9% vs. 6.7 +/- 2.0% with untransfected TC, P < 0.01). Leukocyte apoptosis also was increased in an insert culture system (18.2 +/- 4.4% vs. 5.8 +/- 2.3% with un- transfected TC, P < 0.01). No increase of TC apoptosis was detected in any of the co-culture experiments. CONCLUSION: Enhanced expression of FasL by TC is capable of inducing apoptosis of activated leukocytes, without evidence for increased susceptibility to apoptosis of the transfected cells themselves. This suggests a potential role for this approach in the limitation and resolution of renal tubulointerstitial inflammation.     

FTY720 mediates apoptosis-independent lymphopenia in human renal allograft recipients: different effects on CD62L+ and CCR5+ T lymphocytes

BACKGROUND: The sphingolipid FTY720 (FTY), a novel immune modulator, induces lymphopenia and prevents allograft rejection. This study was designed to study the effect of FTY on lymphocyte subpopulations and apoptosis in stable renal allograft recipients. METHODS: Stable renal allograft recipients received a single oral dose of 0.25 to 3.5 mg of FTY (n= 13) or placebo (n= 3). Whole blood was drawn immediately before and at 4, 8, 12, 24, 72, and 96 hr after administration. The number of lymphocyte subpopulations, with an emphasis on surface markers involved in lymphocyte migration, was analyzed by flow cytometry. Apoptotic lymphocytes were detected following Annexin V-FITC/PI staining. Lymphocyte mobility was investigated in a modified Boyden chamber. RESULTS: FTY induced a transient lymphopenia by an apoptosis-independent mechanism. In vitro experiments with peripheral blood mononuclear cells (PBMC) confirmed that clinically relevant concentrations of FTY (0.1 microM) increased lymphocyte mobility, whereas only suprapharmacologic concentrations of FTY (10 microM) could induce apoptosis. FTY-treated patients had reversible changes in the composition of peripheral lymphocyte subpopulations. CD62L+ T cells decreased to the greatest extent (-57%). In contrast, CCR5+ T-cell counts declined only marginally (-10%). In vitro, treatment of PBMC with FTY (1 mM-10 microM) did not induce changes in the expression of these surface markers. CONCLUSIONS: The data indicate that FTY mediates apoptosis-independent lymphopenia in human renal allograft recipients. FTY-induced lymphopenia preferentially affects CD62L+ and CCR5- T-lymphocyte subpopulations.     

B lymphopenia in uremia is related to an accelerated in vitro apoptosis and dysregulation of Bcl-2.

BACKGROUND: Lymphopenia has been described in patients with chronic renal failure (CRF). It is postulated that the decline in lymphocytes is due to accelerated apoptosis. We investigated whether dysregulation of programmed cell death plays a role in the immunodeficiency described in CRF. METHODS: Peripheral blood lymphocytes (PBL) from pre-dialysis uraemic patients (nHD) and haemodialysed patients (HD) were cultured with no stimulus for 96 h. Apoptosis of lymphocytes was measured by propidium iodide staining and flow cytometry. Expression of Fas and Bcl-2 was also analysed by flow cytometry. RESULTS: Peripheral blood B cells were significantly lower in pre-dialysis and haemodialysis uraemic patients compared to control. Lymphocytes from both groups of patients had a higher rate of apoptosis in vitro than those from healthy controls. This effect was more pronounced in B lymphocytes and a significant correlation between the B lymphopenia and the percentage of apoptotic B cells after 48 h of culture without stimulus was observed. The increased lymphocyte apoptosis in CRF was accompanied by a significantly lower in vitro Bcl-2 expression. However, Fas did not seem to play a role in spontaneous lymphocyte apoptosis in end-stage renal disease. CONCLUSIONS: Our data indicate that B lymphopenia in CRF may be partially attributed to an increased susceptibility to cell death by apoptosis that is associated with a decreased expression of Bcl-2.     

Beta-2-microglobulin in hemodialysis patients. Effects of different dialyzers and different dialysis procedures

beta 2-Microglobulin (beta 2m) has been identified as the major constituent of dialysis-related amyloid. Although there is no clear correlation between absolute beta 2m levels and amyloidosis-related symptoms, elevated serum levels are thought to be the basis for tissue deposition of beta 2m. Besides diminished renal excretion and insufficient removal during hemodialysis, a dialysis-related induction of beta 2m production is discussed as the major cause of elevated serum beta 2m levels. In order to evaluate the influence of hemodialysis membranes and the hemodialysis procedure on beta 2m levels we determined serum beta 2m levels in patients on chronic intermittent hemodialysis. Polymethylmethacrylate 2.0 m2, cuprophane and cellulose acetate dialyzers led to increasing beta 2m levels during dialysis, which was in excess of what could be accounted for by hemoconcentration. The polymethylmethacrylate 1.6 m2 dialyzer did not result in a significant rise of beta 2m levels during dialysis. This indicates that production of beta 2m is not only dependent on the membrane material but also on the surface area of the dialyzer. The use of polysulfone and hemophane low-flux dialyzers did not induce an increase in beta 2m levels during dialysis but a significant clearance of beta 2m was not demonstrable either. Volume-controlled dialysis with high-flux membranes (polysulfone 0.65 m2 and polysulfone 1.25 m2) lowered beta 2m; clearance values, however, were significantly higher when these dialyzers were used in a hemodiafiltration procedure. We conclude from our study that some dialysis membranes appear to induce beta 2m production, whereas others do not.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of membrane composition on cytokine production and clinical symptoms during hemodialysis: a crossover study

BACKGROUND: Intradialytic symptoms including hypotension have been reported during dialysis and it has been suggested that these are related to the release of cytokines like IL-1beta and TNFalpha by blood mononuclear cells when they get activated either due to contact with the dialyzer membrane or by compliment activation. OBJECTIVE: To study the relationship between hemodialysis symptoms, cytokine production, and dialyzer membrane composition. METHOD: In a randomized prospective crossover study, 20 ESRD patients on maintenance hemodialysis were studied over cuprophan (CU) and polysulfone (PS) low flux dialyzer membranes for three weeks each undergoing a biweekly dialysis schedule of 4 h sessions. Serial IL-1beta and TNFalpha levels were measured over 0, 15, 240 min during the first use of the dialyzer for all patients on both membranes. Intradialytic symptoms were monitored in a total of 240 dialysis sessions. RESULTS: IL-1beta levels increased from 16.6 +/- 2.2 to 64.8 +/- 25.1 pg/mL on CU and 21.5 +/- 3.7 to 103.5 +/- 30.7 pg/mL on PS membrane over the 4-h dialysis session. Similarly TNFalpha increased from 42.8 +/- 4.5 to 354.9 +/- 80.4 pg/mL on CU and 117.1+/- 53.7 to 387.0 +/- 78.0 pg/mL on PS membrane. IL-1beta levels increased significantly with PS membrane while TNFalpha rise was significant with both the membranes. Nausea was the most common symptom occurring in 138 dialysis sessions (57.5%). Vomiting, chest pain, fever, chills, and breathlessness occurred significantly more during dialysis with CU membrane as compared with PS membrane (P < 0.01). Nausea, cramps, back pain, itching, restlessness, post dialysis fatigue, and hypotension did not differ between the two membranes. The mean rise in the cytokine levels during the first 15 min of sessions where the symptoms occurred, when compared with the mean rise in sessions where the symptoms did not occur, did not reveal any significant difference. Cytokine release did not correlate with the occurrence of intradialytic symptoms. CONCLUSION: Both CU and PS membranes increase circulating cytokine levels. More intradialytic clinical symptoms are seen in dialysis with CU as compared with PS membrane but the rise in cytokines IL-1beta and TNFalpha does not appear to be responsible for them.     

硒对淋巴细胞杀伤大肠癌细胞过程中Fas/FasL表达的影响

目的　探讨硒对淋巴细胞抗大肠癌过程中凋亡相关基因Fas/FasL表达的影响。方法　以半胱氨酸硒作为影响因素,人T淋巴细胞为效应细胞及人结肠癌细胞（LoVo细胞株）为靶细胞,采用四甲基偶氮唑蓝(MTT)比色法、凋亡细胞荧光计数检测凋亡细胞的数量,逆转录-聚合酶链(RT-PCR)反应、原位杂交技术检测,硒对效应细胞杀伤肿瘤细胞过程中Fas/FasL表达的影响。结果　不同浓度的半胱氨酸硒（0.5、1.0　mg/L）作用48h后,可增强人T淋巴细胞抗肿瘤活性［分别为（25.12±3.91）%,（46.17±3.68）%,P＜0.05］,且肿瘤细胞的凋亡比例上升［分别为(18.6±4.1)%,（32.7±2.1）%,P＜0.05］;能使免疫效应细胞和肿瘤细胞表达FasL和Fas水平增高。结论　硒可提高淋巴细胞的抗肿瘤作用,可能通过Fas/FasL途径起作用。     

In Vivo Evaluation of Platelet Activation by Different Cellulosic Membranes

Abstract: This study was undertaken to evaluate platelet activation in vivo induced by different cellulosic membranes by measuring the expression of P-selectin on the platelet surface during hemodialysis in 9 uremic patients. Hollow fiber dialyzers of similar surface with different cellulosic membranes (Cuprophan, cellulose acetate, cellulose triacetate, and Hemophan) were evaluated and compared to a synthetic membrane (polysulfone). Blood samples were obtained before hemodialysis and from the efferent and afferent limbs 5 min after the beginning of dialysis. P-select in exposure was evaluated by flow cytometry (FACScan) using a monoclonal antibody (RUU 2.17). The percentage of platelets expressing P-select in before hemodialysis and the percentage from the arterial line during hemodialysis were similar. All membranes evaluated induced platelet activation (estimated as the increase in percentage of platelets expressing P-selectin in samples obtained from the venous line with respect to the arterial line). Cuprophan induced more platelet activation than any other membrane (p < 0.05). The activation induced by cellulose acetate and cellulose triacetate membranes was also higher than that observed with Hemophan (p < 0.05). Hemophan-induced platelet activation was similar to that of polysulfone. These results indicate that all cellulosic membranes induce platelet activation during hemodialysis although there are quantitative differences among them. While Cuprophan induced the highest degree of platelet activation, Hemophan was the only cellulosic membrane that showed a degree of platelet activation similar to the biocompatible membrane polysulfone.     

不同透析膜对维持性血液透析患者外周血Ｔ细胞凋亡的影响

目的 研究终末期肾病(ESRD)患者外周血T细胞凋亡、Bcl-2和Fas的表达、Th1及Th2类细胞因子特征以及不同透析膜对维持性血液透析患者T细胞凋亡的影响。 方法 研究对象包括ESRD未透析(ND)患者10例；维持性血液透析(HD)的患者45例，分别用醋酸纤维素膜(CA)、低通聚砜膜(PS-LF)、高通聚砜膜(PS-HF)进行透析；以及健康对照(C)8例。上述研究对象的外周血T细胞经植物凝集素(PHA)刺激培养24 h后，应用流式细胞术检测其凋亡情况；免疫组化检测T细胞Bcl-2、Fas的表达；ELISA检测细胞培养上清IFN-γ及IL-4的水平。 结果 ND组及HD组T细胞凋亡率高于C组的(6.82±1.64)%。HD各组T细胞的凋亡率以CA组的(12.26±1.21)%最高，PS-HF组(9.00±0.89)%最低(P < 0.05)。ND组及HD组外周血T细胞Bcl-2的表达低于C组(P < 0.05)，Fas的表达高于C组(P < 0.05)；相关分析显示T细胞凋亡与Fas的表达呈正相关，与Bcl-2呈负相关。ND组及HD组IFN-γ的水平均低于C组(P < 0.05)，与T细胞凋亡呈负相关；IL-4的水平高于C组 (P < 0.05)，与T细胞凋亡呈正相关。 结论 ND组及HD组外周血T细胞凋亡加速，Bcl-2、Fas参与T细胞凋亡的发生。 ND组及HD组患者Th类细胞因子失衡，呈Th2细胞因子优势， Th类细胞因子参与T细胞凋亡的调控。HD患者的T细胞凋亡不仅与透析膜的生物相容性有关还与透析膜的通透性有关。     

Enhancement of susceptibility to Fas-mediated apoptosis of TH1 cells by nonmitogenic anti-CD3epsilon F(ab')2

BACKGROUND: Anti-CD3epsilon F(ab')2 are nonmitogenic for naive T cells but can induce apoptosis of antigen-activated T cells in vitro and in vivo. We studied the mechanisms by which nonmitogenic anti-CD3epsilon F(ab')2 antibodies induce T cell death. METHODS: OVA-responsive T cell lines were generated by immunization in vivo and restimulation in vitro. Fas or Fas ligand (FasL) expression was tested by surface staining with specific mAbs. The apoptotic DNA and cell cycle phase were tested by staining DNA with propidium iodide. Interferon-gamma was measured by ELISA, whereas interleukin (IL)-2 and IL-4 were detected by bioassays. RESULTS: Restimulation with anti-CD3epsilon F(ab')2 induced apoptosis of antigen-activated wild-type T cells, but not Fas or FasL-defective T cells. Anti-CD3epsilon F(ab')2 induced death of cells expressing high levels of Fas and FasL, and preferentially deleted T helper (Th)1 cells but spared Th2 cells. Soluble anti-CD3epsilon F(ab')2 did not regulate Fas or induce FasL expression, indicating that the ability of anti-CD3epsilon F(ab')2 to induce T cell apoptosis depends on a distinct mechanism. T cells in S/G2 were found relatively resistant to Fas-mediated apoptosis, but anti-CD3epsilon F(ab')2 rendered those T cells exquisitely sensitive to Fas-mediated apoptosis. CONCLUSIONS: Anti-CD3epsilon F(ab')2 induces apoptosis of cycling CD4+ T cells through activation of the Fas/FasL pathway. Anti-CD3epsilon F(ab')2 does not regulate Fas or FasL expression but induces susceptibility to Fas-mediated apoptosis of cycling T cells. Anti-CD3epsilon F(ab')2 can induce death of polarized Th1 cells, but not Th2 cells, thus potentially skewing the repertoire of antigen-activated T cells toward the Th2 phenotype. These features predict that nonmitogenic anti-CD3epsilon F(ab')2-like antibodies can be useful to prevent or reverse pathogenic immune responses mediated by Th1 cells.     

Fas配体诱导淋巴细胞凋亡与肾癌免疫攻击作用

目的　探讨Fas配体 (FasL)诱导淋巴细胞凋亡与肾癌免疫攻击作用的关系。　方法　采用免疫组化技术检测 4 4例肾癌组织FasL表达及肿瘤周围浸润淋巴细胞 (TIL)凋亡情况 ,并应用肾癌细胞株 786 0、GRC 1与JurkatT淋巴细胞共培养检测T细胞凋亡率。SP法检测Ki6 7表达 ,评价肾癌预后。　结果　 (1) 4 4例肾癌组织FasL表达阳性率 4 6 .5 % ,高于正常肾组织的 2 3.2 % ,差别有显著性意义 (P <0 .0 1)。随肾癌分期增加 ,FasL表达阳性率增加。肾癌FasL表达率与Ki6 7表达率呈显著正相关 (r =0 .93,P <0 .0 1)。 (2 )肾癌组织TIL凋亡率为 33.9% ,高于正常肾组织的3.5 % ,差别有显著性意义 (P <0 .0 1)。肾癌FasL表达率与TIL凋亡率呈显著正相关 (r =0 .96 ,P <0 .0 1)。 (3) 786 0FasL表达率 18.6 % ,GRC 1表达率 2 .3% ,二者差别有显著性意义 (P <0 .0 1)。Ju rkat细胞与 786 0细胞共培养的凋亡率 14 .9% ,与GRC 1共培养的凋亡率 1.3% ,二者差别有显著性意义 (P <0 .0 1)。中和抗体NOK 2中和 786 0细胞FasL后 ,与之共培养的Jurkat细胞凋亡率显著减少 (P <0 .0 1)。　结论　肾癌组织FasL表达增高 ,以此诱导淋巴细胞凋亡 ,实现对宿主的免疫攻击。     

Enhanced apoptosis of peripheral blood mononuclear cells in cardiac transplanted patients undergoing chronic immunosuppressive treatment

Apoptosis plays a major role in tissue transplantation because intact T-cell-apoptosis pathways are required for the induction of tolerance to allografts. Moreover, immunosuppressive agents commonly used in clinical transplantation medicine promote lymphocyte apoptosis inhibiting the expression and production of cytokines involved in lymphocyte survival. The aim of our study was to evaluate peripheral blood mononuclear cells (PBMC) spontaneous apoptosis in patients undergoing chronic immunosuppressive treatment after cardiac transplantation. PBMC obtained from patients (n = 31) and controls matched for age and sex (n = 25) were cultured for 72 h and apoptosis was evaluated by quantification of fragmented DNA, staining with Hoechst 33258 dye and annexin V binding. We also investigated Fas expression and FasL mRNA expression as well as the ability of an IgM anti-Fas antibody to induce apoptosis. Finally, we evaluated IL2 production induced by PHA and the ability of IL2 to prevent apoptosis. In patients, PBMC underwent enhanced spontaneous apoptosis in comparison with controls. However, we could not find any difference between patients and normals as regards the expression of Fas and of FasL mRNA, even if the cross-linking of the Fas molecule induced apoptosis in PBMC from patients, whereas it failed to induce cell death in normals. We also found that IL2 production was significantly decreased in patients and that the addition of IL2 to the culture medium reduced PBMC spontaneous apoptosis. Our findings suggest that in cardiac transplanted patients PBMC undergo enhanced spontaneous apoptosis, which may contribute to prevent allograft rejection.     

FasL-transfected endothelial cells decrease the proliferative response of allogeneic PBL

Graft endothelium has a key role in organ transplantation because it regulates graft infiltration by allogeneic activated T cells. Overexpression of death molecules that could induce apoptosis of alloreactive T cells might be an alternative to the immunosuppressive treatment currently used in graft transplantation. Several studies have shown that immune-privileged sites express Fas ligand (FasL) and induce apoptosis of activated T-cells. We propose that endothelial cells engineered to express FasL could inhibit alloreactive T cell-proliferation by inducing apoptosis. An expression vector was constructed with human FasL cDNA and used to transfect an endothelial cell line (ECV304 cells). We demonstrated that FasL-transfected ECV304 cells were effective in inducing apoptosis of Jurkat T cell lymphoma as an agonist anti-Fas antibody. Using a mixed lymphocyte-endothelial cell culture model we observed that FasL-transfected ECV304 cells which conserved their two principal costimulatory pathways inhibited alloreactive T cell-proliferation by inducing activated T-cell apoptosis. These results suggest that endothelial cells could be interesting candidates to convey a death signal and induce hyporesponsiveness of alloreactive T cells during organ transplantation.