Apoptosis In Human Leukocytes Research Articles

Apoptosis and cellular stress induction in human leukocytes by dental X-rays

In clinical practice, the use of ionizing radiation, such as X-rays, is of importance in diagnostic procedures. The X-ray equipment is operated by the dentist himself before, during, and after an oral cavity procedure in dental offices. The occupationally exposed personnel (POES) receive an exposure dose, causing stochastic damage that is not commonly considered but is cumulative. Due to the above, a relationship between leukopenia and exposure to X-rays has been reported. Analysing the expression of bioindicators that allow determination of the degree of homeostatic tolerance to different exposure doses is one way to diagnose cell damage caused by this. This study aimed to evaluate cell viability, the expression of Hsp70, p53, caspase 8 and 3, and morphological changes related to apoptosis in human leukocytes exposed to X-rays from dental equipment. Whole blood from healthy subjects was used and exposed to different doses of X-rays from dental diagnostic equipment (2.28 ± 0.05, 4.56 ± 0.09, 9.12 ± 0.18, and 13.69 ± 0.27 mGy). Following this, morphology was assessed by Wright's stained blood smear of whole blood leukocytes. Subsequently, leukocytes were isolated by Ficoll Histopaque gradients from whole blood, and cell viability was measured. Finally, the Western blot technique identified the leukocyte proteins Hsp70, p53, and caspases. In the observed results, a decrease in cell viability from 96.6% to 45.4% was noticed; viability decreased as the dose of X-ray exposure increased. The expression of Hsp70, p53, Caspase 8, and 3 gradually increased up to the third dose and decreased in the last dose. Likewise, morphological changes characteristic of apoptotic cell death were observed from the first dose of exposure and became more significant at higher doses. In conclusion, there is cell damage in leukocytes exposed to low doses of X-rays; this information is relevant to implementing protection mechanisms for POES that regularly use this equipment for diagnosis in the field of dentistry.

Expression of selected proteins of the extrinsic and intrinsic pathways of apoptosis in human leukocytes exposed to N-nitrosodimethylamine.

N-nitrosodimethylamine (NDMA) is a xenobiotic widespread in human environment capable of regulating the lifespan of immune cells. In this study, we examined the roles of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/death receptor 5 (DR5) complex and the Fas molecule in the induction of the extrinsic apoptosis pathway in human neutrophils (polymorphonuclear neutrophils (PMNs)) and peripheral blood mononuclear cells (PBMCs) exposed to NDMA. Also we assessed these proteins ability to trigger the intrinsic apoptosis pathway in those cells. For this purpose, we examined the expression of Fas-associated protein with death domain, truncated Bid (tBid) proteins, and apoptogenic factors such as apoptosis-inducing factor, Smac/Diablo, Omi/HtrA2, and caspase-3 as an indication of accomplished apoptosis phenomenon. PMNs and PBMCs were isolated from whole blood by density gradient centrifugation using Polymorphrep. Apoptotic cells were assessed with flow cytometry using a ready-made kit. The expression of proapoptotic molecules was investigated by Western blot analysis of PMNs and PBMCs treated with NDMA and/or rhTRAIL. The obtained results confirm the proapoptotic effects of NDMA on the examined human leukocytes and indicate an active participation of the TRAIL/DR5 complex and Fas protein in the process of apoptosis. Moreover, the research revealed distinct mechanisms of intrinsic apoptosis pathway activation between PMNs and PBMCs exposed to NDMA, as confirmed by the different levels of tBid, Smac/Diablo, Omi/HtrA2, and caspase-3 expression in those cells.

Gene-Specific Repression of Proinflammatory Cytokines in Stimulated Human Macrophages by Nuclear IκBα

We have previously shown that increased nuclear accumulation of IkappaBalpha inhibits NF-kappaB activity and induces apoptosis in human leukocytes. In this study, we wanted to explore the possibility that the nucleocytoplasmic distribution of IkappaBalpha can be used as a therapeutic target for the regulation of NF-kappaB-dependent cytokine synthesis. Treatment of LPS-stimulated human U937 macrophages with an inhibitor of chromosome region maintenance 1-dependent nuclear export, leptomycin B, resulted in the increased nuclear accumulation of IkappaBalpha and inhibition of NF-kappaB DNA binding activity, caused by the nuclear IkappaBalpha-p65 NF-kappaB interaction. Surprisingly, however, whereas mRNA expression and cellular release of TNF-alpha, the beta form of pro-IL-1 (IL-1beta), and IL-6 were inhibited by the leptomycin B-induced nuclear IkappaBalpha, IL-8 mRNA expression and cellular release were not significantly affected. Analysis of in vivo recruitment of p65 NF-kappaB to NF-kappaB-regulated promoters by chromatin immunoprecipitation in U937 cells and human PBMCs indicated that although the p65 recruitment to TNF-alpha, IL-1beta, and IL-6 promoters was inhibited by the nuclear IkappaBalpha, p65 recruitment to IL-8 promoter was not repressed. Chromatin immunoprecipitation analyses using IkappaBalpha and S536 phosphospecific p65 NF-kappaB Abs demonstrated that although the newly synthesized IkappaBalpha induced by postinduction repression is recruited to TNF-alpha, IL-1beta, and IL-6 promoters but not to the IL-8 promoter, S536-phosphorylated p65 is recruited to IL-8 promoter, but not to TNF-alpha, IL-1beta, or IL-6 promoters. Together, these data indicate that the inhibition of NF-kappaB-dependent transcription by nuclear IkappaBalpha in LPS-stimulated macrophages is gene specific and depends on the S536 phosphorylation status of the recruited p65 NF-kappaB.

ERK MAPK activation mediates the antiapoptotic signaling of melatonin in UVB-stressed U937 cells

The pineal gland hormone melatonin has been recently described to downregulate the intrinsic (or damage-induced) pathway of apoptosis in human leukocytes. These properties appear to depend on a specific mitochondrial signaling of melatonin which is associated with a lower generation of reactive oxygen species and a better control of redox-sensitive components such as the antiapoptotic protein Bcl-2. Other elements upstream in this signaling are expected to contribute regulatory roles that remain unexplored. The aim of this study was to investigate whether the extracellular signal-regulated kinase (ERK), which controls the balance between survival and death-promoting genes throughout the MAPK pathway, is involved in the antiapoptotic signaling of melatonin. Human monocytic U937 cells irradiated with UVB light were used as a model of stress-induced apoptosis. In this model we found that pharmacological concentrations of melatonin (1 mM) were able to decrease superoxide anion production, mitochondrial damage, and caspase-dependent apoptosis by improved Bcl-2 levels and decreased Cyt c release in the cytoplasm. Moreover, melatonin increased the phosphorylative activation of ERK 1/2 independently from the presence of UVB stress, and decreased the UVB-mediated activation of the stress kinases p38 MAPK and JNK. The ERK 1/2 inhibitor PD98059, but not the p38 MAPK inhibitor SB203580, abolished to different extents the effects that melatonin had on the UVB-induced ROS generation, mitochondrial dysfunction, and apoptosis. Using these inhibitors, a cross-talk effect between stress and survival-promoting kinases was tentatively identified, and confirmed the hierarchical role of ERK MAPK phosphorylation in the signaling of melatonin. In conclusion, melatonin sustains the activation of the survival-promoting pathway ERK MAPK which is required to antagonize UVB-induced apoptosis of U937 cells. This kinase mediates also the antioxidant and mitochondrial protection effects of this hormonal substance that may find therapeutic applications in inflammatory and immune diseases associated with leukocyte oxidative stress and accelerated apoptosis.

Unique Role of Junctional Adhesion Molecule-A in Maintaining Mucosal Homeostasis in Inflammatory Bowel Disease

Junctional adhesion molecule-A (JAM-A) is localized at the tight junctions and controls leukocyte migration into the tissues. However, its functional role in inflammatory bowel disease (IBD) is unexplored. Control, Crohn's disease (CD), and ulcerative colitis (UC) tissue specimens were studied for JAM-A expression, as well as the colon of mice given dextran sodium sulfate (DSS). Wild-type and JAM-A(-/-), Tie-2-Cre-JAM-A(-/-) (endothelial/hematopoietic-specific JAM inactivation) mice were studied for susceptibility to DSS. Disease activity and colonic inflammation were assessed using a disease activity index histology and endoscopy, and mucosal cytokines were measured by enzyme-linked immunosorbent assay. JAM-A function was investigated by RNA silencing in epithelial cells, and apoptosis was measured. In both CD and UC, as well as in experimental colitis, there is a loss of epithelial but not endothelial JAM-A expression. Deletion of JAM-A results in a dramatic increase in susceptibility to DSS colitis, as assessed by weight loss, disease activity index, histologic and endoscopic severity, and strikingly high mortality rates. This is not caused by the absence of JAM-A in the endothelial or hematopoietic compartments because Tie-2-Cre-JAM-A(-/-) mice are no more susceptible to DSS colitis than wild-type animals. JAM-A(-/-) mice displayed increased intestinal permeability and inflammatory cytokine production, and marked epithelial apoptosis. Silencing of JAM-A in intestinal epithelial cells resulted in increased permeability in vitro. Our results show a nonredundant and novel role of JAM-A in controlling mucosal homeostasis by regulating the integrity and permeability of epithelial barrier function.