N-nitrosodimethylamine (NDMA) induced apoptosis dependent on Fas/FasL complex in human leukocytes.

OBJECTIVE: To investigate the mechanism of apoptosis dependent on the Fas/FasL (Fas ligand) complex in the presence of N-nitrosodimethylamine (NDMA) in human leukocytes. METHODS: Polymorphonuclear neutrophils (PMNs) and peripheral blood mononuclear cells (PBMCs) were isolated form whole blood by density centrifugation. The concentration of NDMA was assessed by cellular toxicity assay. Apoptotic cells were assessed with flow cytometry and the expression of pro- and antiapoptotic proteins was investigated by Western blotting in PMNs and PBMCs treated with NDMA and/or FasL. RESULTS: PMNs showed a higher ratio of apoptotic cells than PBMCs after exposure to NDMA and/or FasL. Enhanced apoptosis was related to the increased expression of proapoptotic proteins in neutrophils following exposure to either NDMA or FasL. In PBMCs, the relation was observed after exposure to FasL only. PMNs and PBMCs incubated with NDMA and FasL simultaneously demonstrated the highest increase in protein expression. CONCLUSIONS: NDMA shows a stronger proapoptotic effect with PMNs than with PBMCs. The Fas/FasL complex, along with other proapoptotic proteins of the receptor (Fas, FADD) and mitochondrial pathway (Noxa, Puma, Bim), plays a key role in the induction of neutrophil apoptosis. Synergic effects of NDMA and FasL which lead to higher induction of apoptosis in PMNs than in PBMCs indicates a multistage and varied regulation of apoptosis in different populations of leukocytes.

Short-term in vitro effects of bisphenol A activity on phenotype and function of peripheral blood immune system cells.

Bisphenol A (BPA) is a phenolic compound being constituent of numerous everyday-used products. Additionally, good absorption through skin and gastrointestinal tract is an unquestionable evidence for high risk of BPA exposure and its possible influence on health. Effects of BPA action on cells and tissues are associated with its structural and functional similarities to steroid hormones. Here we investigated whether BPA could possibly influence immune cells through steroid receptors present on majority of these cells. In in vitro experiments with 200 nM and 1000 nM BPA concentrations we found that high levels affect activation of lymphocytes through increased CD25 expression, with no changes in functional response based on IFN-gamma production. We demonstrated that BPA influence not only phenotype of monocytes with increased frequency of CD14++CD16- subtypes, but also activation inhibition with decline of HLADR expression within monocytes. Similarly to lymphocytes, no changes were observed in context of monocytes function in BPA-exposed cells. Our study revealed that BPA actions are also associated with its direct role in modulation of immune system cells. We presume that further experiments would allow establishing connection between presented data and increased risk of cancer and metabolic diseases in subject exposed to bisphenol A.

Cytokine Network & NETs.

The process of forming and releasing neutrophil extracellular traps (NETs) can be regulated by exogenous and endogenous factors, including cytokines. The study aimed to assess the impact of proinflammatory cytokines, IL-15, IL-17, IL-18, and anti-inflammatory IL-10 on the formation of NETs, all in comparison to IL-8 and pathogenic factors: LPS, fMLP. Also, the expression of myeloperoxidase (MPO), one of the main elements of neutrophil traps, was evaluated. After isolating the neutrophils with Polymorphprep™, the cells were sorted using CD16 MACS® microbeads and incubated with selected factors. The formation of NETs was registered using a BD Pathway 855 microscope system and the expression of MPO was evaluated using flow cytometry. The amounts of circulating DNA in cell supernatants was fluorescently quantified. Microscopic photographs indicated that rhIL-15, rhIL-17, rhIL-18 and fMLP induce formation and release of NETs at a similar timespan, while in the presence of rhIL-10, the formation of the traps was delayed. The presence of the studied cytokines indicated two populations of neutrophils displaying differing MPO expression (MPOlow and MPOhigh). Moreover, stimulation of neutrophils with LPS and fMLP revealed two populations of these cells that differed not only in the expression of MPO, but also in size.

Glucocorticoids upregulate decreased IL-7 receptor expression in asthmatic patients and simian immunodeficiency virus-infected non-human primates.

Signaling through interleukin-7 receptor (IL-7R) is essential for regulation of T-cell homeostasis and survival. Previously, we and others have found diminished IL-7R levels in simian immunodeficiency virus (SIV) - infected non-human primates and human immunodeficiency virus (HIV) - infected patients. To date, it remains unknown whether changes in IL-7R expression could also be linked to non-infectious inflammatory diseases such as asthma or anti-inflammatory drug use. Here, we investigated through flow cytometry the levels of IL-7R expression on CD4+ and CD4- T-cells in asthmatic patients in relation to disease severity, immune status and glucocorticoid (GC) use. In addition, we sought to evaluate the effects of in vivo and in vitro GC treatment on IL-7R expression in both asthmatic patients and SIV-infected non-human primates. We demonstrated that expression of IL-7R on peripheral blood CD4+ T-cells was significantly decreased in clinically stable GC-naive mild and moderate asthmatic patients. Accordingly, the development of asthmatic reaction following bronchial allergen challenge performed in sensitized subjects was associated with a significant drop in levels of IL-7R on circulating CD4+ T-cells. In contrast, CD4+ T-cells from both, mild and moderate, but not severe asthmatics, treated with inhaled GC displayed levels of IL-7R similar to that seen in healthy controls. We did not find significant differences with serum or sputum interleukin-7 levels among healthy controls and GC-naïve and GC-treated asthmatic patients. Furthermore, both in vitro GC treatment and short-term oral GC administration to asthmatic patients resulted in a significant enhancement of IL-7R. Similarly, we demonstrated that GC-stimulated T-cells from SIV-infected non-human primates up-regulated IL-7R expression. Accordingly, experimental short-term systemic in vivo administration of GC to SIV-infected macaques led to enhancement of IL-7R expression on circulating T-cells. Our data indicate that GC bear potential to recover diminished IL-7R expression, as well in asthma as in lentiviral infection.