Marzena Garley

NETs in cancer.

Many aspects of neutrophil hyperactivity and its role in numerous immune responses still remain a mystery. A new neutrophil mechanism was discovered recently, i.e., the formation of neutrophil extracellular traps (NETs). These structures, composed of DNA strands and neutrophil granule proteins, are an element of the non-specific immune response and bind pathogens to prevent their spread and ensure increased local concentrations of toxic factors. Research on this phenomenon shows that tumor-associated neutrophils (TANs) also form and release NETs. Reports on the role of NETs in the course of cancer are scarce, and the opinions on the involvement of extracellular traps in the disease are divided, indicating a dual function. There is speculation about the anti-cancer properties of NETs connected with direct killing of cancer cells or stimulation of the immune system. On the other hand, the trap structures might promote migration and immune escape of cancer cells or constitute a physical barrier between cancer cells and immune-competent cells. This article summarizes our knowledge about the proven roles of NETs in the course of cancer with particular focus on the significance of NETs as prognosis biomarkers in the course of the neoplastic process and their potential use in therapy.

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.

New Aspects of the Biology of Neutrophil Extracellular Traps

The formation and release of neutrophil extracellular traps (NETs) discovered in 2004 by Volker Brinkmann and Arturo Zychlinsky cast a new light on the role of neutrophils in the non‐specific immune response of the body. This discovery has resulted in the rapid development of neutrophil studies in different bacterial and autoimmune diseases as well as neoplasms. Research is also being performed on the role of different signalling pathways engaged in the induction of NETosis, a unique form of a programmed cell death leading to the creation of NETs. The literature provides information on the structure and composition of neutrophil extracellular traps. This review presents the latest data on NET formation and the role of their key components, as well as describing the intracellular signalling pathways leading to the generation of NETs that have been discovered.

Heterogeneity Among Neutrophils

Neutrophils (PMNs) play a key role in innate defence mechanisms. Generally, PMNs were considered to have a homogeneous population of mature and diversified cells. It seems, however, that their pleiotropic action results from the existence of different subpopulations in this group of cells. There are data that confirm the involvement of PMNs in the direct activation of other cells in non-specific response, as well as specialised cells in specific response. For example, there have been observations of PMNs with different levels of activity in relation to lymphocytes, and a population was identified which had characteristics similar to those of cells which are capable of presenting antigens. There are also reports of PMNs which demonstrate different survival time or capacity for chemotaxis. Other studies suggest that the neutrophil response to Staphylococcus aureus is diverse (not identical among all neutrophil). There are also reports of PMNs with varying activity during inflammation, which might explain many as yet unknown pathophysiological aspects of their hyperreactivity. The functional dualism of PMNs in the course of neoplastic disorders raises a lot of controversy. This paper presents the current state of knowledge of the heterogeneity of PMNs and their potential roles in different stages of disease.

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 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.

PI3K-Akt/PKB signaling pathway in neutrophils and mononuclear cells exposed to N-nitrosodimethylamine.

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).

TLR4 ligation induces expression of APRIL molecule in human neutrophils — a preliminary study

In the present study we investigate the consequences of TLR4 activation by LPS for the synthesis of a proliferation-inducing ligand (APRIL) by human neutrophils (PMNs), and the possible role of the ERK1/2 kinases signaling pathway. In order to make a comparison, the same examinations were carried out on autologous peripheral blood mononuclear cells (PBMCs). The levels of mRNA for APRIL and TLR4 were measured using the real-time PCR method. Western blot analysis was used to assay the expressions of APRIL and ERK1/2 in cell lysates. We discovered an increased expression of APRIL accompanying the increased expression of TLR4 in the LPS-stimulated PMNs and PBMCs. Furthermore, stimulation with LPS triggered similar changes in phospho-ERK1/2 proteins expression in those cells. The present study suggests that LPS plays a role in TLR4-ligation in APRIL induction through ERK1/2 pathway activation in human neutrophils and mononuclear cells of peripheral blood. The association between TLR4 activation and APRIL expression in examined leukocytes might have important implications for the immune response of the host exposed to TLR4 ligands such as LPS.