The Effect of N-Nitrosodimethylamine on TRAIL and DR5 Expression in Human Neutrophils—Preliminary Study

The intracellular mechanisms of NDMA-induced apoptosis of neutrophils have not yet been fully understood. The aim of this study was to explain whether the TRAIL/DR5 system is implicated in NDMA-induced apoptosis of human neutrophils. The expression of TRAIL and DR5 was examined, as well as the secretion of sTRAIL and sDR5 by human neutrophils treated with NDMA confronted with intensity apoptosis of these cells. For comparative purposes similar examinations in autologous peripheral blood mononuclear cells (PBMC) were performed. Decreased expression and secretion of TRAIL and increased expression and secretion of DR5 associated with increased intensity of apoptosis of polymorphonuclear leukocytes (PMNs) suggest that NDMA-induced apoptosis in these cells may be depend on TRAIL/DR5 system. Autologous PBMCs no exerted that changes in the expression and secretion of TRAIL as well as in the intensity of apoptosis. However, the expression and secretion of DR5 by PBMCs were similar to those by PMNs. Differences above suggest that PMNs are more sensitive to unfavorable action of NDMA than PBMCs.     

iNOS expression and NO production by neutrophils in cancer patients

INTRODUCTION: The tumor-polymorphonuclear neutrophil (PMN) relationship can be altered by the release of toxic molecules, such as nitric oxide (NO). The aim of the present study was to examine the expression of the inducible synthase of NO (iNOS)and NO production by human neutrophils of patients with oral cavity cancer. For comparison we performed similar examinations in autologous peripheral blood mononuclear cells (PBMCs). MATERIAL/METHODS: PMNs and PBMCs were isolated from the whole blood of 27 patients with squamous cell carcinoma of the oral cavity. iNOS protein expression in these cells was detected by Western blot. Total nitrite as an indicator of NO concentrations in the culture supernatants and the serum of patients was measured using a colorimetric assay. RESULTS: The PMNs of oral cavity cancer patients showed a significantly lower intensity of iNOS expression than those of healthy controls. The PBMCs of patients showed a more intensive expression of iNOS than the PMNs, but a lower intensity than the PBMCs of the controls. The expression of iNOS in rhIL-6 and rhIL-15-stimulated PMNs and PBMCs of patients increased in comparison with unstimulated cells. We observed lower productions of NO by PMNs and PBMCs of patients than those of the control group. CONCLUSIONS: The results revealed that altered iNOS expression and NO production are more characteristic of PMNs than of PBMCs of patients with oral cavity cancer. Additionally, this study provided new information about IL-6 and IL-15 activity in a tumor-bearing host.     

重组人可溶性TRAIL分子诱导白血病细胞株凋亡的研究

比较重组人可溶性TRAIL(rhsTRAIL)诱导Jurkat细胞株、K562细胞株以及HL-60细胞株凋亡之间的差异,探讨这些差异与细胞表面TRAIL受体(DR4、DR5、DcR1和DcR2)表达量的关系。不同浓度的rhsTRAIL分别处理Jurkat细胞、K562细胞和HL-60细胞12 h、24 h和48 h后,用流式细胞仪检测经碘化丙啶(PI)染色后的细胞凋亡情况;用RT-PCR方法检测细胞表面受体DR4、DR5、DcR1、DcR2的表达。培养12 h、24 h、48 h后,不同浓度rhsTRAIL诱导Jurkat细胞株的凋亡率均明显高于对照组,且具有剂量依赖性和时间依赖性;但K562细胞株和HL-60细胞株未见明显的凋亡发生。RT-PCR结果显示,培养12 h、24 h、48 h后,Jurkat细胞株表面DR4的表达随时间的延长和rhsTRAIL浓度的升高而升高,而DR5、DcR1和DcR2的表达未检出;K562和HL-60细胞株表面DR4的表达没有明显变化,而且DR5、DcR1和DcR2的表达也未检出。rhsTRAIL诱导Jurkat细胞株的凋亡具有剂量依赖性和时间依赖性,且与其细胞表面DR4的表达呈正相关;在一定的浓度条件下,rhsTRAIL未能诱导K 562和HL-60细胞株发生明显凋亡,且其细胞表面DR4的表达也未见明显变化。这些结果提示,应用TRAIL治疗不同种类白血病时,应注意它的使用剂量和适应范围。     

TNF-相关诱导凋亡因子及其受体在人胎盘组织中的免疫组织化学定位

目的 探讨TNF相关诱导凋亡因子(TRAIL)及其DR4、DR5受体在人胎盘组织的表达及其意义。方法 免疫组织化学结合图像分析定量方法观察TRAIL及其DR4、DR5受体在人胎盘组织的表达及其含量的周龄变化。结果 人胎盘滋养层细胞、绒毛基质细胞及毛细血管的内皮细胞均呈TRAIL及DR4、DR5受体免疫反应阳性，阳性反应物分布于胞膜及胞质，胞核阴性。在人胎盘绒毛的不同发育阶段中，TRAIL的含量相对稳定，而其受体DR4、DR5的含量随着周龄的增加而升高。结论 结果显示胎盘不仅能产生TRAIL，而且也是TRAIL的靶器官，TRAIL及其DR4、DR5受体系统可能参与胎盘的特免调节。     

Application of flow cytometry to molecular medicine: detection of tumor necrosis factor-related apoptosis-inducing ligand receptors in acute myeloid leukaemia blasts

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), a cytokine belonging to the TNF (tumor necrosis factor) family, is currently regarded as a potential anti-cancer agent. Nevertheless, several types of cancer cells display a low sensitivity to TRAIL or are completely resistant to this pro-apoptotic cytokine. TRAIL signalling is dependent on four receptors. Two of them, death receptors 4 and 5 (DR4 and DR5), induce apoptosis, whereas decoy receptors 1 and 2 (DcR1 and DcR2) are unable to evoke cell death upon TRAIL binding. TRAIL resistance may be related to the expression of TRAIL decoy receptors. TRAIL has been proposed as a novel therapeutic agent for the treatment of haematological disorders, including acute myeloid leukaemia (AML). Surprisingly, however, very limited information is available concerning the expression of TRAIL receptors in AML blasts. Here, we have evaluated, using flow cytometry, TRAIL receptor surface expression and sensitivity to TRAIL-dependent apoptosis of AML blasts from 30 patients. We observed frequent expression of TRAIL DcR1 and DcR2, while expression of DR4 and DR5 was less frequent. Nevertheless, the expression of DR4 or DR5 in leukaemic cells was always matched by a similar expression of one of the decoy receptors. Leukaemic blasts were invariably resistant, even to a high concentration (1000 ng/ml) of TRAIL. We suggest that AML blasts are resistant to TRAIL apoptosis in vitro. Therefore, it is unlikely that TRAIL alone might be used in the future as an innovative pharmacological agent for the treatment of AML.     

Evaluation of the influence of N-nitrosodimethyloamine (NDMA) on the apoptosis of neutrophils of peripheral blood (PMN) and the expression of the IL-6R membrane receptor--in vitro research.

Evaluation of the influence of N-nitrosodimethyloamine (NDMA) on the apoptosis of neutrophils of peripheral blood (PMN) and the expression of the IL-6R membrane receptor - in vitro research. The aim of the present work was the evaluation of N-nitrosodimethyloamine (NDMA) on the induction of apoptosis in the neutrophils of peripheral blood as well as the evaluation of the surface receptors for IL-6. The isolated neutrophils were incubated for 1 and 3 hours with NDMA of a concentration of 2.5, 5, 7.5, and 10 mg/ml. In the samples incubated for 1 hour a significant, dose- dependent increase of apoptosis in the examined cells was observed. In the cells incubated for 3 hours, the increase of apoptosis was observed only at concentration of NDMA of 2.5 and 5 mg/ml. In case of higher concentration used, probably necrotic processes dominated in the cells. No influence of NDMA on the expression of IL-6R was observed.     

重组人可溶性TRAIL的制备及其联合硼替佐米诱导肿瘤细胞的凋亡作用

目的:构建重组人肿瘤坏死因子相关的凋亡诱导配体(tumor necrosis factor-related apoptosis-inducing ligand,TRAIL)原核表达质粒p ET-28a(+)-TRAIL114-281,优化蛋白表达和纯化条件,制备重组人可溶性TRAIL并鉴定其活性。方法:使用CCK-8初步验证TRAIL是否具有抑制肿瘤细胞生长的生物活性;将制备的TRAIL单独或联合50 nmol/L硼替佐米应用于H460细胞(对TRAIL敏感)和K562细胞(对TRAIL抵抗)24 h,流式细胞术检测细胞凋亡率,比色法检测caspase-8、-9、-3的活化程度,Western blot分析细胞中Bax、Bcl-2和c FLIP蛋白的表达。流式细胞术检测硼替佐米处理H460细胞和K562细胞24 h后DR4和DR5的表达量变化。结果:制备了具有生物学活性且性质稳定的重组人可溶性TRAIL,且成功诱导H460和K562细胞凋亡。不同浓度TRAIL处理H460细胞后其凋亡率随着TRAIL浓度升高而显著升高(P0.05),但K562细胞凋亡率并未随着TRAIL浓度明显升高。联合用药组的H460和K562细胞凋亡率均显著高于单独用药组(P0.05),凋亡过程中caspase-8、-9、-3均被活化,药物处理组的Bcl-2和c FLIP表达量均比对照组下降,尤其联合用药组表达量下降最为显著(P0.05),而Bax表达量无明显变化。硼替佐米处理H460和K562细胞后DR4和DR5表达量均上调(P0.05)。结论:硼替佐米能协同TRAIL启动内源性凋亡途径诱导H460和K562细胞凋亡,其可能机制是通过上调死亡受体DR4和DR5的表达量、下调抗凋亡蛋白Bcl-2和c FLIP的表达量来实现的。     

Evaluation of the Influence of N-Nitrosodimethyloamine (NDMA) on the Apoptosis of Neutrophils of Peripheral Blood (PMN) and the Expression of the IL-6R Membrane Receptor—in vitro Research

Evaluation of the influence of N-nitrosodimethyloamine (NDMA) on the apoptosis of neutrophils of peripheral blood (PMN) and the expression of the IL-6R membrane receptor - in vitro research.

The aim of the present work was the evaluation of N-nitrosodimethyloamine (NDMA) on the induction of apoptosis in the neutrophils of peripheral blood as well as the evaluation of the surface receptors for IL-6. The isolated neutrophils were incubated for 1 and 3 hours with NDMA of a concentration of 2.5, 5, 7.5, and 10 mg/ml. In the samples incubated for 1 hour a significant, dose- dependent increase of apoptosis in the examined cells was observed. In the cells incubated for 3 hours, the increase of apoptosis was observed only at concentration of NDMA of 2.5 and 5 mg/ml. In case of higher concentration used, probably necrotic processes dominated in the cells. No influence of NDMA on the expression of IL-6R was observed.     

Evaluation of the Influence of N-Nitrosodimethyloamine (NDMA) on the Apoptosis of Neutrophils of Peripheral Blood (PMN) and the Expression of the IL-6R Membrane Receptor—in vitro Research

Evaluation of the influence of N-nitrosodimethyloamine (NDMA) on the apoptosis of neutrophils of peripheral blood (PMN) and the expression of the IL-6R membrane receptor - in vitro research.

The aim of the present work was the evaluation of N-nitrosodimethyloamine (NDMA) on the induction of apoptosis in the neutrophils of peripheral blood as well as the evaluation of the surface receptors for IL-6. The isolated neutrophils were incubated for 1 and 3 hours with NDMA of a concentration of 2.5, 5, 7.5, and 10 mg/ml. In the samples incubated for 1 hour a significant, dose- dependent increase of apoptosis in the examined cells was observed. In the cells incubated for 3 hours, the increase of apoptosis was observed only at concentration of NDMA of 2.5 and 5 mg/ml. In case of higher concentration used, probably necrotic processes dominated in the cells. No influence of NDMA on the expression of IL-6R was observed.     

Expression of TRAIL (TNF-related apoptosis-inducing ligand) and its receptors in normal colonic mucosa,adenomas, and carcinomas

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in tumour cell lines. Four membrane-bound receptors for TRAIL have been identified, two apoptosis-mediating receptors, DR4 and DR5, and two apoptosis-inhibiting receptors, DcR1 and DcR2. The aim of this study was to examine the role of TRAIL and its receptors in colorectal cancer development. The immunohistochemical expression and localization of TRAIL and its receptors were investigated in normal mucosa (n=10), adenomas (n=19), and carcinomas (n=21). Correlations between the expression of TRAIL and its receptors and the degree of apoptosis (assessed by M30 expression) and histopathological characteristics were explored. TRAIL and its receptors were expressed in normal mucosal epithelium. Expression of the receptors was seen in adenomas and carcinomas. TRAIL expression was lost in a subset of colorectal tumours, more frequently in carcinomas than in adenomas (p<0.05). DR4 and DR5 staining was stronger in neoplastic cells than in normal cells and was accompanied by a higher degree of apoptosis. No differences were found between tumour and normal cells regarding DcR1 and DcR2 expression. No correlations were found between TRAIL or TRAIL receptor expression and histopathological characteristics. In conclusion, marked changes were seen in the course of the adenoma-carcinoma sequence with respect to the expression of TRAIL and TRAIL receptors DR4 and DR5. The stronger expression of DR4 and DR5 in neoplastic cells than in normal cells, together with a higher degree of apoptosis, suggests a possible functional role for these receptors in apoptosis induction in neoplastic colorectal cells.     

Regulation of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and TRAIL receptor expression in human neutrophils

Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily, which is capable of inducing apoptosis in many cell types, including tumour and virus-infected cells, but rarely in normal cells. Expression of TRAIL mRNA and TRAIL receptors has previously been detected in neutrophils; however, the expression of TRAIL protein and the regulation of TRAIL and TRAIL receptor expression in these cells remain unknown. Here we report, for the first time, that neutrophils constitutively express TRAIL protein on their cell surface and that the TRAIL protein is shed during culture. TNF-alpha is a down-regulator of TRAIL expression, whereas IFN-gamma up-regulates the expression of TRAIL. Neutrophils did not express a detectable level of TRAIL-R1 or -R4, but constitutively expressed a low, but substantial, level of TRAIL-R2 and a high level of TRAIL-R3. Although the level of TRAIL-R2 was not significantly altered during culture under different experimental conditions, approximately 30% of TNF-alpha-treated cells rapidly lost their high-level TRAIL-R3 expression, whereas the majority of IFN-gamma-treated cells retained a high level of TRAIL-R3 expression. Anti-TRAIL neutralizing antibody significantly inhibited neutrophil apoptosis during cultures in medium alone, or in the presence of TNF-alpha or IFN-gamma. Thus, our study identified human neutrophils as a cellular source of TRAIL and suggests that neutrophil-derived TRAIL may play a role in immune surveillance. Our results also suggest a role for the TRAIL/TRAIL receptor system in neutrophil apoptosis.     

Neisseria gonorrhoeae-mediated inhibition of apoptotic signalling in polymorphonuclear leukocytes

The human pathogen Neisseria gonorrhoeae recruits and interacts extensively with polymorphonuclear leukocytes (PMNs) during infection. N. gonorrhoeae is able to survive the bactericidal activity of these innate immune cells and can actively modulate PMN functions in vitro. PMNs are short-lived cells which readily undergo apoptosis, and thus the effect of N. gonorrhoeae infection on PMN survival has implications for whether PMNs might serve as an important site of bacterial replication during infection. We developed and validated an HL-60 myeloid leukemia cell culture model for PMN infection and used both these cells and primary PMNs to show that N. gonorrhoeae infection alone does not induce apoptosis and furthermore that N. gonorrhoeae can inhibit both spontaneous apoptosis and apoptosis induced by the intrinsic and extrinsic apoptosis inducers staurosporine (STS) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), respectively. N. gonorrhoeae infection also results in the activation of NF-κB signaling in neutrophils and induces secretion of an identical profile of proinflammatory cytokines and chemokines in both HL-60 cells and primary PMNs. Our data show that the HL-60 cell line can be used to effectively model N. gonorrhoeae-PMN interactions and that N. gonorrhoeae actively inhibits apoptosis induced by multiple stimuli to prolong PMN survival and potentially facilitate bacterial survival, replication, and transmission.     

Osteoprotegerin (OPG) acts as an endogenous decoy receptor in tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis of fibroblast-like synovial cells

We examined the role of osteoprotegerin (OPG) on tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in rheumatoid fibroblast-like synovial cells (FLS). OPG protein concentrations in synovial fluid from patients with rheumatoid arthritis (RA) correlated with those of interleukin (IL)-1beta or IL-6. A similar correlation was present between IL-1beta and IL-6 concentrations. Rheumatoid FLS in vitro expressed both death domain-containing receptors [death receptor 4 (DR4) and DR5] and decoy receptors [decoy receptor 1 (DcR1) and DcR2]. DR4 expression on FLS was weak compared with the expression of DR5, DcR1 and DcR2. Recombinant TRAIL (rTRAIL) rapidly induced apoptosis of FLS. DR5 as well as DR4 were functional with regard to TRAIL-mediated apoptosis induction in FLS; however, DR5 appeared be more efficient than DR4. In addition to soluble DR5 (sDR5) and sDR4, OPG administration significantly inhibited TRAIL-induced apoptogenic activity. OPG was identified in the culture supernatants of FLS, and its concentration increased significantly by the addition of IL-1beta in a time-dependent manner. Neither IL-6 nor tumour necrosis factor (TNF)-alpha increased the production of OPG from FLS. TRAIL-induced apoptogenic activity towards FLS was reduced when rTRAIL was added without exchanging the culture media, and this was particularly noticeable in the IL-1beta-stimulated FLS culture; however, the sensitivity of FLS to TRAIL-induced apoptosis itself was not changed by IL-1beta. Interestingly, neutralization of endogenous OPG by adding anti-OPG monoclonal antibody (MoAb) to FLS culture restored TRAIL-mediated apoptosis. Our data demonstrate that OPG is an endogenous decoy receptor for TRAIL-induced apoptosis of FLS. In addition, IL-1beta seems to promote the growth of rheumatoid synovial tissues through stimulation of OPG production, which interferes with TRAIL death signals in a competitive manner.