Jakub Jablonski

Toll-like receptors types 2 and 6 and the apoptotic process in human neutrophils

Abstract.Introduction: Neutrophils (PMN) apoptosis plays an important role in limiting the last phase of inflammatory processes. It is unknown whether Toll-like receptor (TLR)2 acts independently or together with TLR6 in this process. Materials and Methods: The aim of this study was to estimate the relationship between the expressions of TLR2 and TLR6 and the apoptosis of human neutrophils in physiological conditions. We investigated the influence of recombinant human interleukin (IL)-18 and N-formyl-metionyl-leucyl-phenylalanine (fMLP) on the relationships between these receptors and neutrophil apoptosis. Results: Our results showed that after 4-h incubation, the percentage of apoptotic PMNs significantly increased compared with PMN counts before incubation. The stronger expression of TLR2 on the neutrophils suggests that this receptor contributes more significantly to the induction of PMN apoptosis than does TLR6. We also demonstrated an influence of recombinant human IL-18 (rhIL-18) on the expression of TLR6, whereas this effect was not observed in the expression of TLR2. We observed that both rhIL-18 and fMLP inhibited the apoptosis of PMNs and that rhIL-18 had a stronger effect than fMLP. Conclusions: The obtained results suggest that not only TLR2, but also TLR6 plays an important role in the regulation of the apoptosis of PMNs. Changes in the expression of TLR6 and inhibition of apoptosis of PMNs by rhIL-18 seem to confirm the vital role this receptor and of rhIL-18 in regulating the survival of these cells. These data can be useful in developing methods to regulate PMN apoptosis in conditions associated with their excessive and unfavorable activation.

The release of soluble forms of TRAIL and DR5 by neutrophils of oral cavity cancer patients

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.

The influence of very low doses of N-nitrosodimethylamine (NDMA) on the apoptosis of rat neutrophils in vivo. The role of reactive oxygen species.

N-nitrosodimethylamine (NDMA) causes the apoptosis of neutrophils in vitro experiments. This compound also has the ability to stimulate neutrophils for the production of reactive oxygen species. It has been decided to examine more closely whether the apoptosis of neutrophils by NDMA is caused by the influence of the radicals produced by these cells and whether the stimulation to undergo apoptosis of neutrophils is caused by NDMA in either the original form or by its metabolites. The experiment was conducted on rats. The animals were administered a one-time dose of NDMA intragastrically, 1.5 mg/kg. The research was conducted 1,2,4,12 h consecutively following NDMA administration. The concentration of NDMA in blood was evaluated by means of the gas chromatography method. The neutrophils were isolated from blood by means of differential centrifugation. Respiratory burst was assessed in cells, by means of the cytochrome c reduction method. The percentage of cells revealing morphological properties of apoptosis was determined under the fluorescent microscope. It has been observed that the activation of the respiratory burst is caused mainly by non-metabolised NDMA. Probably the non-metabolised molecules of this compound also have a decisive role in the initiation of apoptosis of neutrophils. It can be assumed that the main factor responsible for the apoptosis of neutrophil rats following a one-time NDMA administration is the induction of respiratory burst in neutrophils by this compound.

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.

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.

Role of AP-1 family proteins in regulation of inducible nitric oxide synthase (iNOS) in human neutrophils

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.

ROLE OF IL-18 IN THE SECRETION OF IL-1β, sIL-1RII, AND IL-1Ra BY HUMAN NEUTROPHILS

In the present study we investigated the effect of IL-18 on the production of IL-1β, IL-1Ra and sIL-1RII by human neutrophils. Our observations indicate that rhIL-18 induces IL-1β and, to a lesser extend, IL-1Ra and sIL-1RII production by human neutrophils isolated form peripheral blood. However, this effect was less important in comparison with LPS-stimulation. Moreover, the results obtained suggest that IL-18 can induce priming of neutrophils for IL-1β and, to a lesser extend, IL-1Ra and sIL-1RII production by LPS-stimulated cells. The capacity of IL-18 to serve as an effective modulator for IL-1β and its regulatory proteins may have significance in the inflammatory and immune reactions mediated by IL-1β.

The role of MAP kinases in the induction of iNOS expression in neutrophils exposed to NDMA: the involvement transcription factors

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. RESULTS 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. CONCLUSION 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.

A proliferation-inducing ligand (APRIL) in neutrophils of patients with oral cavity squamous cell carcinoma

Available data indicating a role for neutrophils in the tumor-host reactions are controversial. In 37 patients with oral cavity squamous cell carcinoma (OSCC), we investigated the expression of a tumor-promoting, proliferation-inducing ligand (APRIL) molecule by peripheral blood neutrophils isolated from blood samples collected at presentation and three weeks after surgery, and the serum levels of TGF-β in the same samples. Additionally, we investigated the consequences of TLR4 activation by LPS for the synthesis of APRIL by those cells. The levels of mRNA for APRIL and TLR4 were measured using a real-time PCR method. Western blot analysis was used to assay the expressions of APRIL and ERK1/2 in cell lysates. The results of the present study revealed the unfavorable features of the detection, in the blood, of neutrophils displaying an enhanced expression of the tumor-promoting APRIL molecule. The increased expression and release of APRIL accompanying advanced stages of disease demonstrated by these cells, combined with the increased number of neutrophils, may be an important marker of disease progression in the patient group examined. Simultaneously, an increased level of circulating TGF-β in the serum of these patients appeared to be associated with the overexpression of APRIL in their neutrophils. In contrast to the healthy controls, TLR4 expression and the ERK1/2 signaling pathway appear to play only minor roles in APRIL induction in the cells of patients with cancer. The changes presented in the current study suggest that modulation of the expression of tumor-promoting APRIL, in addition to TRAIL and BAFF, might be taken into account in the development of new strategies for supportive immunotherapy of OSCC disease and possibly for other types of neoplasm as well.

The Effect of N-nitrosodimethylamine (NDMA) on Bax and Mcl-1 Expression in Human Neutrophils

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.

The expressions of intrinsic and extrinsic apoptotic pathway proteins in neutrophils of oral cavity cancer patients: a preliminary study

Introduction:The biological availability and activity of polymorphonuclear neutrophils (PMNs) are regulated by their short life span, which can be additionally shortened by a malignant process. The signaling pathways leading to apoptotic PMN death are classified in two categories: the intrinsic and the extrinsic. In the present study the expressions of proteins participating in the extrinsic apoptotic pathway (DR5, FADD, caspase-8 activity) and the intensity of apoptosis of PMNs from patients with cancer of the oral cavity were examined. The expression of proteins participating in the intrinsic pathway (Bax and Mcl-1) were also examined in these cells. The results can be helpful in explaining the reasons for the decreased activity of these cells in oral cavity cancer patients.Materials and Methods:The examinations were carried out in patients with squamous cell carcinoma of the oral cavity before and after treatment. The expressions of all the proteins were measured in neutrophils and, for comparison, in autologous peripheral blood mononuclear cells (PBMCs). Western blot analysis was used to assay the expressions of DR5, FADD, Bax, and Mcl-1 in cell lysates. The apoptosis level was determined by flow cytometry and caspase-8 activity by colorimetric assay.Results:A lack of changes in DR5 expression associated with increased FADD protein expression and caspase-8 activity accompanied the accelerated apoptosis rates in the PMNs of the patients before treatment. Decreased expression of anti-apoptotic Mcl-1 protein was associated with an unchanged expression of pro-apoptotic Bax protein. There were no such changes in the patients PBMCs. Increased expression of Mcl-1 in the PMNs of the patients following surgical treatment was found.Conclusion:The acceleration of the apoptosis of PMNs of oral cavity cancer patients before treatment is dependent on both the intrinsic and extrinsic pathways.