Study on Nitric Oxide Mediated Signaling in Neutrophils Free Radical Generation and Extracellular Traps Formation

Abstract

Neutrophils/polymorphonuclear leukocytes (PMNs), the terminally differentiated cells, are critical component of innate immune system with shortest life span (4 hours -1 day), which reach first to the inflammatory / infection site. Neutrophils engulf invading microbes by forming a phagocytic vacuole, a phagosome. Subsequently, intracellular granules fuse with the phagosome and discharge their contents to form a phagolysosome, and microbes are killed by the combination of both non-oxidative and oxidative mechanisms (Klebanoff 2005; Segal 2005; Nathan 2006). Neutrophil mediated inflammatory response can be regarded a multistep process initiated by adhesion of circulating neutrophils to activated endothelium, extravasations and migration towards invaders and terminated through phagocytosis, generation of reactive oxygen and nitrogen species (ROS and RNS) and release of microbicidal substances to execute the microorganisms (Faurschou et al. 2003). PMNs are generated in the bone marrow under the influence of cytokines, such as granulocyte colony-stimulating factor (G-CSF) and granulocyte/ macrophage colonystimulating factor (GM-CSF), from hematopoietic stem cells (Cluitmans et al. 1995). PMNs differentiation and formation in the bone marrow has classically been divided into six stages myeloblasts (MBs), promyelocytes (PMs), myelocytes (MCs), metamyelocytes (MMs), band cells (BCs), and segmented neutrophils on the basis of cell size, nuclear morphology, and granular content (Bainton et al. 1971; Borregaard et al. 1997). Transit time for generation of neutrophils in the bone marrow is approximately 10-14 days and marrow maintain a five day supply of mature neutrophils (Bainton et al. 1971). The turnover of neutrophil production is 1010-1011 per day in human. PMNs have shortest life span of only 4-10 hrs in circulation and 1-2 days in tissue. Brinkmann et al. (2004), reported a novel mechanism of microbial killing known as neutrophil extracellular traps (NETs) to eliminate invading pathogens. NETs are large DNA-associated molecule complexes carrying nucleic and cytoplasmic proteins such as histones, elastase, myeloperoxidase (MPO), pentraxin, lactoferrin, and bactericidal/permeability-increasing (BPI) protein, each with strong antimicrobial and/or immunomodulating properties (Brinkmann et al. 2004; Jaillon et al. 2007). These structures bind Gram-positive and -negative bacteria, as well as fungi (Urban et al. 2006). Formation of NETs is an active process, which is distinct from apoptosis and necrosis and is dependent on the reactive oxygen species generation from activated NADPH oxidase and myeloperoxidase (Fuchs et al. 2007; Metzler et al. 2010; Patel et al. 2010). Neutrophil elastase (NE) is essential to initiate NET formation and it synergizes with MPO to drive chromatin decondensation (Papayannopoulos et al. 2010). High circulating levels of DNA have been reported in malaria and sepsis patients (Swarup et al. 2007; Baker et al. 2008; Margraf et al. 2008). Increase in NETs content in the plasma may predict multiorgan failure and sepsis after multiple traumas (Margraf et al. 2008). In vivo NETs contents are expectedly abundant at the site of infection and acute inflammation (Brinkmann et al. 2004; Beiter et al. 2006; Buchanan et al. 2006). Recent report from this lab demonstrated that treatment of PMNs with nitric oxide (NO) donors such as SNP and SNAP released NETs, which was mediated by the activation of NADPH oxidase and myeloperoxidase (Patel et al. 2010). Identification of mechanisms involved in NETs release is thus an area of intense research and have immense implications so as to identify new therapeutic targets. NO, a ubiquitous signaling molecule, plays essential bioregulatory roles in a wide range of processes, including vasodilation, cell proliferation, nerve transmission, tumor surveillance, antimicrobial defense, and regulation of inflammatory responses (Ignarro 1990; Schmidt et al. 1994). Nitric oxide synthase (NOS, EC1.14.13.39) is a family of heme-containing monooxygenases that catalyzes the production of NO from L-arginine. In mammals, there are three distinct isozymes of NOS, each of which is regulated by a different gene (Marsden et al. 1993). The neuronal NOS (nNOS or NOS I) and endothelial NOS (eNOS or NOS III) isoforms are constitutively expressed and Ca++ dependent, while inducible form (iNOS or NOS II) is regulated at the translational level by bacterial endotoxins and various cytokines and its activity is not dependent on the oscillation of intracellular calcium (Cho et al. 1992; Alderton et al. 2001). Blood cells such as neutrophils, monocytes, eosinophils, platelets and red blood cells synthesize NO. Among these PMNs constitute an important proportion and major participant in a number of pathological conditions with suggestive involvement of NO. PMNs are predicted to generate NO at a rate of 10-100nmoles/5min/106 cells, comparable to endothelial cells implicating a potential impact on vascular homeostasis (Salvemini et al. 1989; Wright et al. 1989). PMNs capability to synthesize NO was first discovered by its ability to relax rat aortic rings (Rimele et al. 1988) and by inhibition of platelet aggregation (Dikshit et al. 1993), which was abolished by NOS inhibitors (Faint et al. 1991; Dikshit et al. 1993). Nitrite (NO2 -) content and NOS activity as measured by the conversion of L-arginine to L-citrulline have been correlated with NO production in rat and human PMNs (Miles et al. 1995; Rodenas et al. 1995). Circulating rat or human PMNs do not contain iNOS mRNA, protein, or enzymatic activity (Miles et al. 1995). Exposure of cells to LPS, lipoteichoic acid (LTA), peptidoglycan, bacterial DNA, or to intact bacteria, induces iNOS expression and enhanced NO production (Nathan 1997; Wheeler et al. 1997). nNOS in neutrophils was though demonstrated at mRNA level (Greenberg et al. 1998), but Wallerath et al (1997) failed to observe at protein level. Moreover, Gatto et al. (2000) reported over expression of neutrophil nNOS mRNA and protein in Parkinson’s disease. Constitutive expression of iNOS in human neutrophils has been subsequently documented (Cedergren et al. 2003). A detailed study from this lab by using RT-PCR, Western blotting, and immune electron microscopy demonstrated the presence and intracellular distribution of nNOS and iNOS in rat PMNs (Saini et al. 2006). PMNs iNOS mRNA was augmented in SHR rat neutrophils (Chatterjee et al. 2007), while expression and activity was regulated by ascorbate (Chatterjee et al. 2008). Studies from this lab and others have demonstrated NO mediated modulation of free radical generation from PMNs in various physiological and pathological conditions (Pieper et al. 1994; Sethi et al. 1999; Lee et al. 2000; Sethi et al. 2001; Sharma et al. 2004; Patel et al. 2009). Intracellular and extracellular calcium also have a modulatory effect on NOS activity and free radical generation (Dikshit et al. 2002). Recently effect of NO donors on neutrophil respiratory burst suggests involvement of K+ channels and kinases in NO mediated augmentation of respiratory burst (Patel et al. 2009). Reactive nitrogen species have also been recognised as regulators of ion channels, G proteins like p21ras, protein tyrosine kinases like Fyn and other members of the src family of kinases, protein tyrosine phosphatases, caspases and transcription factors like AP-1 and c-Jun (Bogdan 2001). A wide range of neutrophil function like apoptosis, adhesion and respiratory burst requires the activation of mitogen activated protein kinases (MAPKs). PMA as a receptor-independent stimulus directly activates PKC without G-proteins participation. It also indirectly activates MAPKs and PKTs (Watson et al. 1991; Worthen et al. 1994; Avdi et al. 1996; Krump et al. 1997; Corbit et al. 2003; Moraes et al. 2003). The p38 MAPK pathway is a key regulator of proinflammatory cytokines biosynthesis at the transcriptional and translational levels. p38α and p38 δ isoforms have been found to be expressed in neutrophils (Hale et al. 1999). Several studies have demonstrated that extracellular signal regulated kinases (ERK)1/2 and p38 MAPK, but not c-Jun N-terminal kinase (JNK), are activated in human neutrophils by fMLP, PMA, GM-CSF, and TNF (Avdi et al. 1996; El Benna et al. 1996; Nick et al. 1997; McLeish et al. 1998; Zu et al. 1998). Extracellular stimuli shown previously to inhibit spontaneous neutrophil apoptosis may exploit ERK-dependent antiapoptotic pathways to delay apoptosis at sites of inflammation (Ward et al. 1999). LPS and TNF-α induced activation of JNK was reported in adherent neutrophils (Avdi et al. 2001; Arndt et al. 2004). Impairment of signaling pathway may thus lead to pathological conditions. It was considered worthwhile to undertake the present study to investigate following: 1) Status of NO/NOS and neutrophil extracellular traps (NETs) in systemic inflammatory response syndrome (SIRS) patients. 2) Effect of NO donor (DETA-NONOate) and PMA on free radical generation and NETs release. 3) Signaling pathways involved in NETs formation.