[Show abstract][Hide abstract] ABSTRACT: The aim of this work was to assess the role of ethanol-derived acetate and acetate-mediated histone acetylation in arachidonic acid-induced stress in HepG2 cells and cells overexpressing CYP2E1. Cells were grown for 7 days with 1 mM sodium acetate or 100 mM ethanol; their acetylated histone proteins and histone deacetylase 2 expression was quantified using Western blot. Ethanol- or acetate-pretreated cells were also treated for 24 h with 60 μM arachidonic acid to induce oxidative stress. Cytotoxicity was estimated by lactate dehydrogenase release, 3-[4,5-dimethylthiazolyl-2] 2,5-diphenyltetrazolium bromide test, and by DNA damage, while oxidative stress was quantified using dichlorofluorescein diacetate. Cells grown with ethanol or acetate had increased acetylated histone H3 levels in both cell types and elevated acetylated histone H4 levels in cells overexpressing CYP2E1 but not in naïve cells. In cells overexpressing CYP2E1 grown with ethanol, expression of histone deacetylase 2 was reduced by about 40 %. Arachidonic acid altered cell proliferation and was cytotoxic mostly to cells engineered to overexpress CYP2E1 but both effects were significantly lower in cells pretreated with ethanol or acetate. Cytotoxicity was also significantly decreased by 4-methylpyrazole-a CYP2E1 inhibitor and by trichostatin-an inhibitor of histone deacetylases. In cells pretreated with acetate or ethanol, the oxidative stress induced by arachidonic acid was also significantly lower. Our data indicate that histone hyperacetylation may in some extent protect the cells against oxidative stress. It is possible that acetate may act as an antioxidant at histone level. This mechanism may be relevant to alcohol-induced liver injury.
Full-text · Article · Nov 2013 · Archiv für Experimentelle Pathologie und Pharmakologie
[Show abstract][Hide abstract] ABSTRACT: Abstract Neutrophils (PMN) play diverse regulatory and effector functions in the immune system through the release of reactive nitrogen species, including nitric oxide (NO). The enzyme responsible for NO synthesis in PMN is inducible nitric oxide synthase (iNOS) that is regulated by various signaling pathways, e.g. PI3K-Akt/PKB, and transcription factors. N-Nitrosodimethylamine (NDMA), a xenobiotic widespread in the human environment, affects immune cells. The study objective here was to examine the role of the PI3K-Akt/PKB pathway in induction of NO synthesis (with involvement of iNOS) in human PMN, as well as in autologous mononuclear cells (PBMC), exposed to NDMA. Isolated cells were incubated for 2 h with a sub-lethal dose of NDMA and then the expression of several select proteins in the cell cytoplasmic and nuclear fractions were determined by Western blot analyses. The results indicated that NDMA enhanced expression of iNOS, phospho-PI3K, and phospho-IκBα in the cytoplasmic fraction of the PMN and PBMC. The nuclear fraction of these cells also had a higher NF-κB expression. Moreover, in PMN, NDMA caused an increased expression of phospho-Akt (T308), phospho-Akt (S473), and phospho-IKKαβ in the cytoplasm, and c-Jun and FosB in the nuclear fraction. Blocking of PI3K caused a decrease in expression of all these proteins in NDMA-exposed PMN. However, inhibition of PI3K led to a drop in expression of iNOS, phospho-PI3K, and phospho-IκBα in the cytoplasm, and in NF-κB in the nuclear fraction, of PBMC. The results of these studies indicated to us that NDMA activates the PI3K-Akt/PKB pathway in human PMN and that this, in turn, contributes to the activation of transcription factors NF-κB, c-Jun, and FosB involved in NO production (through modulation of iNOS expression).
Full-text · Article · Aug 2013 · Journal of Immunotoxicology
[Show abstract][Hide abstract] ABSTRACT: Purpose:
The role of MAP kinases in the activation of AP-1 (c-Jun, c-Fos) and NF-κB p65 engaged in the regulation of iNOS expression in human neutrophils (PMNs) exposed to N-nitrosodimethylamine (NDMA) was analyzed in the study.
Material and methods:
The study included a group of 20 healthy individuals. Isolated human PMN were incubated in the presence of NDMA. Selective MAP kinases inhibitors were used. The expression of proteins in the cytoplasmic and nuclear fractions was assessed using Western blot method.
The results show that NDMA intensifies iNOS, c-Jun, NF-κB p65 and IκB-α expression in the analyzed PMNs. The blocking of the p38 pathway led to lower iNOS expression, and higher expression of c-Jun and c-Fos in the cytoplasmic fraction, and also lower c-Jun expression in the nuclear fraction of PMNs exposed to NDMA. A decrease in iNOS expression in the cytoplasmic fraction, and also c-Jun in both fractions of the examined cells, was observed as a result of JNK pathway inhibition. The blocking of the ERK5 pathway led to higher iNOS, c-Jun and c-Fos expression in the cytoplasmic fraction, and higher c-Jun expression in the nuclear fraction of PMNs exposed to NDMA. The study also demonstrated that blocking of the p38 and JNK pathways resulted in higher expression of NF-κB p65 and IκB-α in the cytoplasmic fraction and their lower expression in the nuclear fraction of these cells.
Our data indicate the role of MAP kinases p38 and JNK in the activation of c-Jun and NF-κB p65 transcription factors engaged in the regulation of iNOS expression in human neutrophils exposed to NDMA. However ERK5 kinase is not involved in the regulation of iNOS and NO production by those cells.
No preview · Article · Aug 2013 · Advances in Medical Sciences
[Show abstract][Hide abstract] ABSTRACT: Potential role of ERK1/2 kinase in conjunction with p38 in the regulation of inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production, and superoxide anion generation by human neutrophils (PMNs) exposed to N-nitrosodimethylamine (NDMA) was determined. Increased synthesis of NO due to the involvement of iNOS in neutrophils exposed to NDMA was observed. In addition, intensified activation of ERK1/2 and p38 kinases was determined in these cells. Inhibition of kinase regulated by extracellular signals (ERK1/2) pathway, in contrast to p38 pathway, led to an increased production of NO and expression of iNOS in PMNs. Moreover, as a result of inhibition of ERK1/2 pathway, a decreased activation of p38 kinase was observed in neutrophils, while inhibition of p38 kinase did not affect activation of ERK1/2 pathway in these cells. An increased ability to release superoxide anion by the studied PMNs was observed, which decreased after ERK1/2 pathway inhibition. In conclusion, in human neutrophils, ERK1/2 kinase is not directly involved in the regulation of iNOS and NO production induced by NDMA; however, the kinase participates in superoxide anion production in these cells.
No preview · Article · Feb 2013 · Indian journal of experimental biology
[Show abstract][Hide abstract] ABSTRACT: The study objective was to assess the role of the JAK/STAT pathway in the induction of nitric oxide (NO) synthesis with the involvement of inducible nitric oxide synthase (iNOS) and in the generation of superoxide anion radical by human neutrophils (PMNs) exposed to N-nitrosodimethylamine (NDMA). Isolated PMNs were incubated in the presence of NDMA. The expression of the study proteins in the cytoplasmic and nuclear fraction was determined by western blotting. Total NO was assayed in cell cultures by Griess reaction. The ability of PMNs to generate superoxide anion radical was assessed using the cytochrome-c reduction test. The current results indicate that NDMA potentiates the expression of iNOS and phospho-JAK2 in the cytoplasmic fraction of human PMNs. Moreover, it increases the expression of phospho-STAT1 and phospho-STAT3 in both fractions of the cells studied. PMNs treated with NDMA showed greater capability to release NO and superoxide anion radical, which was reduced after administration of the JAK/ STAT pathway inhibitor. The current findings indicate that NDMA in PMNs activates the JAK/STAT pathway, which plays a role in the production of NO with the involvement of iNOS and in the generation of superoxide anion radical by these cells.
Full-text · Article · Jan 2013 · Turkish Journal of Biology
[Show abstract][Hide abstract] ABSTRACT: The aim of the study was to assess the activity of AP-1 family proteins, e.g. Fra-1, Fra-2, JunB, JunD, and FosB, engaged in the regulation of inducible nitric oxide synthase (iNOS) expression and the production of NO by neutrophils (PMN) exposed to N-nitrosodimethylamine (NDMA) xenobiotic. Isolated human PMN were incubated in the presence of NDMA. iNOS mRNA expression was then analyzed using Northern blot and the expression of other proteins in the cytoplasmic and nuclear fractions were assessed using Western blot. The obtained results indicate that NDMA increased iNOS mRNA and protein expression in human PMN. Furthermore, it increased the expression of Fra-1, Fra-2, JunB, and JunD in the cytoplasmic fraction, and FosB expression in the fractions of analyzed cells. As a consequence of inhibiting p38 pathway and JNK, reduced iNOS expression and NO production was noted in PMN exposed to NDMA. Inhibition of the p38 pathway resulted in reduced expression of all analyzed proteins in the cytoplasmic fraction of PMN exposed to NDMA. Furthermore, increased Fra-2 expression and reduced FosB expression were found in the nuclear fraction of those cells. Inhibiting ERK5 pathway resulted in increased JunB expression in both fractions of the analyzed cells. Therefore, no changes in the expression of analyzed proteins in the presence of NDMA were observed in PMN pre-incubated with JNK pathway inhibitor. In conclusion, the results here indicate a role of Fra-1, Fra-2, JunB, JunD, and FosB transcription factors in the regulation of iNOS expression and NO production by human neutrophils exposed to NDMA.
No preview · Article · Jun 2012 · Journal of Immunotoxicology
[Show abstract][Hide abstract] ABSTRACT: In the present study we examined a role of pro-apoptotic Bax and anti-apoptotic Mcl-1 proteins, participating in the regulation of intrinsic apoptosis pathway in human neutrophils (PMNs) exposed to N-nitrosodimethylamine (NDMA), the environmental xenobiotic. For the purpose comparison, the same studies were conducted in autologous peripheral blood mononuclear cells (PBMCs). The production of cytochrome c by PMNs was also determined. A deficit of anti-apoptotic Mcl-1 and overexpression of the pro-apoptotic protein Bax suggest that the apoptosis process in human neutrophils exposed to NDMA is dependent on changes in the expression of these proteins. PMNs were more sensitive to NDMA than PBMCs.
Full-text · Article · Sep 2011 · Bulletin of Environmental Contamination and Toxicology
[Show abstract][Hide abstract] ABSTRACT: Ratajczak-Wrona W, Jablonska E, Garley M, Jablonski J, Radziwon P. Effect of N-nitrosodimethylamine on inducible nitric oxide synthase expression and production of nitric oxide by neutrophils and mononuclear cells: the role of JNK signalling pathway, APMIS 2011; 119: 431–41.
In neutrophils (PMN) and mononuclear cells (PBMC), one of the enzymes responsible for nitric oxide (NO) synthesis is inducible nitric oxide synthase (iNOS). Changes in iNOS expression result from various signalling pathways including the mitogen-activated protein kinase (MAPK) pathway activated by endogenic and exogenic factors. N-nitrosodimethylamine (NDMA) is a xenobiotic with widespread occurrence in human environment and has an effect on cells of the immune system. The aim of this study was to determine the effect of NDMA on iNOS expression and NO production by human PMN and PBMC cells in the light of superoxide anion production by PMN cells. Moreover, the role of JNK and p38 pathways in NO production with involvement of iNOS was studied. Additionally, the function of JNK pathway in generation of superoxide anion was determined. Moreover, nitrotyrosine expression was studied in PMN and PBMC cells in the presence of NDMA. This work shows that NDMA increases iNOS expression and NO production by PMN and PBMC cells. In addition, elevated expression of phospho-JNK and phospho-p38, which are markers of JNK and p38 MAPK pathways activation, were observed. Lower iNOS expression and NO production by neutrophils exposed to extended action of NDMA were observed after application of inhibitor of JNK and p38 pathways. Lower phospho-p38 expression in PMN stimulated by NDMA was found as a result of arresting JNK pathway, whereas, application of inhibitor of p38 pathway resulted in enhanced phospho-JNK expression in PMN and PBMC cells. Increased ability to release superoxide anion by NDMA-stimulated PMN cells was observed. This ability was reduced after the application of inhibitor of JNK pathway. In PMN and PBMC cells exposed to NDMA, an increased expression of nitrotyrosine, which is dependant on JNK and p38 pathways that are activated by this particular xenobiotic, was observed. Generally, increased induction of iNOS related to elevated production of NO by PMN and PBMC cells exposed to NDMA may result in dysfunction of regulation of immunity responses controlled by this molecule in various conditions. Increased expression of nitrotyrosine in PMN and PBMC cells exposed to NDMA may affect their functions in an auto- and/or a paracrine way. Mutual interactions of JNK and p38 MAPK during the induction of iNOS expression in cells exposed to NDMA indicate complex mechanism of induction of iNOS synthase.
[Show abstract][Hide abstract] ABSTRACT: NDMA is a toxic compound strongly interfering with the regulatory mechanisms of the immune system. The objective of the current study was to assess the effect of this nitrosamine on the expressions of IL-17A, IL-17E and their specific receptors in human neutrophils. Isolated from the peripheral blood of healthy subjects, neutrophils were incubated in the presence of NDMA at a concentration of 5 mg/ml. Protein expression was evaluated using Western blot method. Elevated expressions of IL-17A and IL-17R and reduced expressions of IL-17E and IL-17BR were observed in the cells exposed to the action of NDMA. The findings indicate that NDMA may enhance proinflammatory properties of human neutrophils through a change in the expression of IL-17 family proteins.
No preview · Article · Jan 2011 · International Review of Allergology and Clinical Immunology
[Show abstract][Hide abstract] ABSTRACT: Effective elimination of neutrophils (PMNs) via the apoptotic pathway is an essential mechanism regulating the number of these cells in blood circulation. Disorders in neutrophil apoptosis are a likely cause of inflammatory states responsible for tissue damage. Numerous reports show that survival of PMNs depends on different molecules, including Toll-like receptor type 2 (TLR2). According to many data, its expression on neutrophils is regulated by various cytokines, such as IL-1, IL-2, IL-3, IL-6, GM-CSF, TNF-α. The last findings seem to confirm a significant role of TLR2 in the regulation of PMN apoptosis. TLR2 is a new "death receptor" that enhanced apoptosis machinery - TLR2 signals for apoptosis through MyD88 and then FADD and caspase-8. The aim of the current study was to estimate the influence of rhIL-17 on neutrophil TLR2 expression and neutrophil apoptosis in physiological conditions. Results. The findings indicate enhanced apoptosis and a simultaneous increase in TLR2 expression on the rhIL-17-stimulated PMNs. Conclusions. The study suggests a potential role of rhIL-17 in the regulation of PMN survival via a TLR2-dependent pathway.
No preview · Article · Jan 2010 · International Review of Allergology and Clinical Immunology
[Show abstract][Hide abstract] ABSTRACT: The biological activities of IL-18 include its ability to induce the production of inflammatory cytokines such as IL-1 or IL-8 by immunocompetent cells. Our previous study demonstrated that rhIL-18 induces IL-1beta and, to a lesser exted, the secretion of IL-1beta regulatory proteins involving interleukin-1 receptor antagonist (IL-IRa) and soluble interleukin-1 receptor II (sIL-1RII) by neutrophils (PMN), suggesting a significant role of IL-18 in the reactions mediated by IL-1beta. In this study, we estimated the effect of rhIL-18 on the induction of IL-6 and its soluble receptors - sIL-6Ralpha and sgp130 by these cells. Results obtained indicate that IL-18 is a promising candidate for the enhanced secretion of IL-6 by human neutrophils. In contrast, we have not found a significant effect of IL-18 on the release of both soluble receptors of IL-6. The influence of IL-18 on the IL-6 production by PMN appears to indicate a potential role of IL-18 in the early steps of the inflammatory cascade and other immune reactions mediated by IL-6.
No preview · Article · Jul 2009 · Immunological investigations
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Jul 2009 · Immunological investigations
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to assess the influence of N-nitrosodimethylamine (NDMA) on expression of inducible nitric oxide synthase (iNOS), as well as production of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) by human neutrophils (PMN) and peripheral blood mononuclear cells (PBMC), and the participation of the p38 MAPK kinase in this process. Furthermore, the ability of neutrophils to release superoxide anion was determined. The influence of N-nitrosodimethylamine on iNOS expression was determined in isolated PMN and PBMC cells from peripheral blood of healthy individuals. The mononuclear cells showed higher sensitivity to NDMA. Moreover, cytotoxic effect of NDMA can be influenced in some way by the impact of this xenobiotic on nitric oxide and superoxide anion release from human leukocytes. Furthermore, increased generation of these radicals by human leukocytes suggest that neutrophils and mononuclear cells that are exposed to NDMA activity can play a key role in endogenous NDMA generation. However the relationship between iNOS expression and phospho-p38 MAPK in neutrophils and mononuclear cells shows that p38 MAPK pathway participates in induction of iNOS expression in the presence of NDMA.
No preview · Article · Jun 2009 · Immunopharmacology and Immunotoxicology
[Show abstract][Hide abstract] ABSTRACT: We studied the mechanisms of acetaminophen (APAP) cytotoxicity in HepG2 cells overexpressing cytochrome p4502E1, particularly the role of oxidative/nitrosative stress and ryanodine Ca2+ channel. Cells were grown for 24 h with APAP in the presence or absence of 4-methylpyrazole (4MP), L-arginine methyl ester (L-NAME), superoxide dismutase (SOD), or ruthenium red (RuR). Drug cytotoxicity was also tested in cells pretreated overnight with V-PYRRO/NO. APAP was without effect on empty vector-transfected cells, but damaged CYP2E1-transfected cells and this was abolished by RuR, reduced by 4MP, or V-PYRRO/NO but affected by L-NAME or SOD. APAP increased microsomal [3H]-ryanodine binding, while microsomal Ca2+ uptake was significantly lowered. RuR increased net microsomal Ca2+ uptake and normalized cytosolic Ca2+ levels. We can conclude that neither oxidative nor nitrosative stress is relevant to APAP cytotoxicity in cultured HepG2 cells, but our results point to ryanodine receptors as a potential crucial protein in the early stages of APAP cytotoxicity.
No preview · Article · Sep 2008 · Archiv für Experimentelle Pathologie und Pharmakologie
[Show abstract][Hide abstract] ABSTRACT: In the present study we examined the release of the soluble form of TRAIL by neutrophils (PMN) derived from patients with oral cavity cancer. Simultaneously, we estimated the ability of PMNs of these patients to release the soluble form of DR5 receptor, a natural regulatory protein of TRAIL. The obtained results were confronted with the serum levels of sTRAIL and sDR5. The cells were isolated from 21 patients with squamous cell carcinoma of oral cavity at diagnosis and three weeks after surgery treatment. For comparative purposes we performed similar examinations in autologous peripheral blood mononuclear cells (PBMC). Cytoplasmic protein fractions of the cells were analyzed for the presence of TRAIL and DR5 by western blotting. Soluble TRAIL and soluble DR5 concentrations in the culture supernatants of cells were confronted with their serum levels using ELISA kit. PMN and PBMC of the whole cancer patient group expressed decreased TRAIL protein and unchanged expression of DR5 receptor in comparison with the control group. Unchanged release of sTRAIL by PMNs of patients in Stage II was accompanying the decrease of the ability of PBMC to secrete this protein. In patients in Stage IV the secretion of sTRAIL by PMNs and PBMC was impaired. In contrast to changes in sTRAIL secretion by PMN and PBMC of oral cavity cancer patients, the secretion of sDR5 by these cells was unchanged. The serum levels of sTRAIL were increased in patients in Stage II before treatment and decreased in the same patients after treatment. The altered ability of PMN of PBMC to secrete sTRAIL may have different implications for the immune response of patients with oral cavity cancer cells at different stages of disease.
Preview · Article · Jun 2008 · Folia Histochemica et Cytobiologica
[Show abstract][Hide abstract] ABSTRACT: Inducible nitric oxide synthase (iNOS) is one of the enzymes responsible for NO production in neutrophils (PMN) and in peripheral blood mononuclear cells (PBMC). Several studies have demonstrated that iNOS expression is controlled by a wide group of cytokines which achieve their biological effect through, among others, the activation of the p38 MAPK pathway. The aim of the present study was to define the participation of the p38 MAPK pathway in the induction of iNOS expression and NO production by PMN and PBMC of healthy persons after stimulation of rhIL-15 and rhIL-18. We also estimated the influence of rhIL-15 and rhIL-18 on cGMP production by both population cells and the production of superoxide anion radicals by neutrophils. The results show that rhIL-15 and rhIL-18 induced an increase in the expression ofiNOS and phospho-p38 MAPK in PMN and PBMC. We also found that PMN and PBMC, stimulated by these cytokines, released larger amounts of NO and cGMP in comparison with non-stimulated cells. Additionally, PMN showed a more pronounced ability to produce superoxide anions. The results suggest that iNOS activation in neutrophils and in peripheral blood mononuclear cells stimulated with rhIL-15 and rhIL-18 may be achieved through the assistance of the p38 MAPK pathway.
[Show abstract][Hide abstract] ABSTRACT: The influence of lipopolysaccharide (LPS) and the nitric oxide synthase (iNOS) inhibitor--N-nitro-L-arginine methyl ester (L-NAME)--on the formation of N-nitrosodimethylamine (NDMA) by HepG2 cells, engineered to overexpress CYP2E1, was assessed and compared with data from empty vector-transfected cells. HepG2 cells produced significant amounts of NDMA but its levels in the culture media of cells overexpressing CYP2E1 was significantly lower than in empty-vector transfected cells. LPS increased the formation of NDMA, the expression of the iNOS and the production of the nitric oxide (NO). On the other hand, L-NAME significantly decreased NDMA levels. The results above indicate that the synthesis of NDMA by HepG2 cells depends on NO production. Furthermore, ethanol did not affect iNOS expression but decreased NDMA levels in CYP2E1-transfected cells below the detection limit. It is probably caused by the increased N-nitrosodimethylamine metabolism. In conclusion, HepG2 cells' ability to synthesize NO with simultaneous CYP2E1 activation may lead to an increase of carcinogenic products of the NDMA metabolism.
No preview · Article · Oct 2005 · Human & Experimental Toxicology
[Show abstract][Hide abstract] ABSTRACT: It is known that the relationship between pro-angiogenic and anti-angiogenic factors is responsible for the presence and intensity of neoangiogenesis. The angiogenic factors are produced by tumour cells and/or by tumour-infiltrating inflammatory cells such as macrophages or polymorphonuclear leukocytes (PMN). In the present study we compared VEGF secretion with IL-18 and NO release by PMN derived from oral cavity cancer patients. Knowledge of the relationship between mediators above could help in better understanding the role of PMN in angiogenesis in this patient group. The results from culture supernatants of PMN were confronted with the serum levels of parameters examined. We found an interesting relationship between VEGF and IL-18 concentrations in the culture supernatants of PMN derived from patients with oral cavity cancer. High production of VEGF was associated with low production of IL-18 by PMN derived from patients before treatment. During examinations after treatment we found lower concentrations of VEGF and higher concentrations of IL-18 than those in the study before treatment. In contrast to VEGF and IL-18, the NO production by PMN of cancer patients, before and after treatment, was unchanged. We also demonstrated markedly elevated serum levels of VEGF as well as IL-18 according to the progression of the disease. Results obtained indicate that relations between VEGF and IL-18 released by PMN may promote neoangiogenesis and may be important for benign tumour cells to acquire metastatic phenotype in the early stage of oral cavity cancer. Furthermore, our results suggest that the concentrations of VEGF and IL-18 in the serum are sensitive tumour markers in this patient group before and after treatment.
[Show abstract][Hide abstract] ABSTRACT: In our previous study we found that rhsIL-6R, along with recombinant human interleukin-6, plays a regulatory role in the immune response by modulating the tumour necrosis factor-alpha(TNF-alpha) expression and its production by peripheral blood mononuclear cells (PBMC). We also suggested that sIL-6R with IL-6 secreted by human PMN (neutrophils) influenced the TNF-alpha expression and its production by autologous PBMC.
Since soluble gp130 (sgp130) is a natural inhibitor for sIL-6R/interleukin-6 responses, in the present study we estimated an effect of exogenous recombinant human sgp130 and sgp130 secreted by PMN on the TNF-alpha expression and its production by PBMC.
Cells were isolated from whole blood of healthy persons. The PMN were cultured in 96-well plates for 1 h at 37 degrees C in a humidified incubator with 5% CO2. After the incubation, the culture supernatant of PMN was removed and added to the PBMC. PBMC were incubated for 1 h at 37 degrees C in the same conditions. Cytoplasmic protein fractions of PMN and, for comparative purpose of PBMC, were analysed for presence of sgp130 by western blotting with the use of monoclonal antibody capable of detecting this protein. In the culture supernatants of PMN we examined the concentrations of sgp130 by human enzyme-linked immunosorbent assay. TNF-alpha was measured at the protein levels as well as the mRNA levels.
The present results revealed that exogenous recombinant human sgp130modulates the TNF-alpha expression and production by PBMC. In contrast, we did not find any effect of sgp130 secreted by PMN on the TNF-alpha expression and its production by autologous PBMC.
Preview · Article · Jan 2004 · Mediators of Inflammation