Neutrophils (PMNs) and cytokines have a critical role to play in host defense and systemic inflammatory response syndrome (SIRS). Neutrophil extracellular traps (NETs) have been shown to extracellularly kill pathogens, and inflammatory potential of NETs has been shown. Microbial killing inside the phagosomes or by NETs is mediated by reactive oxygen and nitrogen species (ROS/RNS). The present study was undertaken to assess circulating NETs contents and frequency of NETs generation by isolated PMNs from SIRS patients. These patients displayed significant augmentation in the circulating myeloperoxidase (MPO) activity and DNA content, while PMA stimulated PMNs from these patients, generated more free radicals and NETs. Plasma obtained from SIRS patients, if added to the PMNs isolated from healthy subjects, enhanced NETs release and free radical formation. Expressions of inflammatory cytokines (IL-1β, TNFα and IL-8) in the PMNs as well as their circulating levels were significantly augmented in SIRS subjects. Treatment of neutrophils from healthy subjects with TNFα, IL-1β, or IL-8 enhanced free radicals generation and NETs formation, which was mediated through the activation of NADPH oxidase and MPO. Pre-incubation of plasma from SIRS with TNFα, IL-1β, or IL-8 antibodies reduced the NETs release. Role of IL-1β, TNFα and IL-8 thus seems to be involved in the enhanced release of NETs in SIRS subjects.

Neutrophils/polymorphonuclear leukocytes (PMNs), an important component of innate immune system, release extracellular traps (NETs) to eliminate invaded pathogens; however understanding of the role of signaling molecules/proteins need to be elucidated. In the present study role of p38 MAPK and extracellular signal regulated kinase (ERK) against phorbol 12-myristate 13-acetate (PMA) induced reactive oxygen species (ROS) generation and NETs formation has been investigated. Human neutrophils were treated with PMA to induce free radical generation and NETs release, which were monitored by NBT reduction and elastase/DNA release, respectively. PMA treatment led to the time dependent phosphorylation of p38 MAPK and ERK in PMNs. Pretreatment of PMNs with SB202190 or U0126 did not significantly reduce PMA induce free radical generation, but prevented NETs release. Pretreatment of PMNs with NADPH oxidase inhibitor (diphenyleneiodonium chloride) significantly reduced free radical generation, p38 MAPK and ERK phosphorylation as well as NETs release, suggesting that p38 MAPK and ERK activation was downstream to free radical generation. The present study thus demonstrates ROS dependent activation of ERK and p38 MAPK, which mediated PMA induced NETs release from human neutrophils.

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that activates c-jun N-terminal kinase (JNK) and can induce cell death in neurons. By contrast, the activation of phosphatidylinositol 3-kinase and AKT/protein kinase B (PKB) acts to suppress neuronal apoptosis. Here, we report a functional interaction between MLK3 and AKT1/PKB␣. Endogenous MLK3 and AKT1 interact in HepG2 cells, and this interaction is regulated by insulin. The interaction domain maps to the C-terminal half of MLK3 (amino acids 511-847), and this region also contains a putative AKT phosphorylation consensus sequence. Endogenous JNK, MKK7, and MLK3 kinase activities in HepG2 cells are significantly attenuated by insulin treatment, whereas the phosphatidylinositol 3-kinase inhibitors LY294002 and wortmannin reversed the effect. Finally, MLK3-mediated JNK activation is inhibited by AKT1. AKT phosphorylates MLK3 on serine 674 both in vitro and in vivo. Furthermore, the expression of activated AKT1 inhibits MLK3-mediated cell death in a manner dependent on serine 674 phosphorylation. Thus, these data provide the first direct link between MLK3-mediated cell death and its regulation by a cell survival signaling protein, AKT1.The cellular decision to undergo either cell death or cell survival is determined by the integration of multiple survival and death signals. Mixed lineage kinase 3 (MLK3) 1 is a member of a growing family of mixed lineage kinases (1). Recently, it has been shown that overexpression of MLK3 or NGF withdrawal leads to neuronal cell death, which can be prevented by treatment with a small molecule inhibitor of MLKs, CEP-1347 (2). Similarly, CEP-11004, an analog of CEP-1347, has also been shown to prevent neuronal cell death upon NGF withdrawal (3). These results indicate a significant and direct involvement of MLKs in regulating cell death; however, the detailed mechanism by which MLKs are regulated is still unknown.The c-jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK) is stimulated by proinflammatory cytokines, oxidative stress, heat shock, UV, ␥-irradiation, and by other cellular stresses (4, 5). The signals in stress-activated JNK pathway are transmitted through three core modules: MAP3Ks such as members of the mixed lineage kinases or MEKK members, a MAP2K such as SEK1/MKK4 or MKK7, and MAPK such as JNK family members (4, 5). The activated MAP3K phosphorylates and activates MKK7 or SEK1, which in turn phosphorylates and activates JNK. JNKs phosphorylate several nuclear transcription factors that include ELK1, c-Jun, and ATF2 (4, 5). In several cell types, the activation of JNKs is directly linked to cell death (6 -8). Therefore, one mechanism of cell survival could be to block JNK pathway induction. The activation of phosphatidylinositol 3-kinase (PI3K) correlates with increased cell survival, and this effect is largely mediated through the activation of a serine/threonine kinase, AKT (also known as PKB). PI3K agonists such as insulin and insulin-like growth factor-1 (IGF-1) ...

Macrophages are central to the initiation and progression of atherosclerosis and thus can be very appropriate targets for therapy. Cell adhesion molecules mediating monocytes recruitment to the endothelium are attractive therapy targets and their inhibitors are in clinical trials. Macrophage scavenger receptors like SR-A and CD-36 mediate foam cell formation by facilitating the uptake of modified lipids. Peroxisome proliferator-activated receptors (PPAR), liver X receptor (LXR)-mediated signaling, mitogen-activated protein kinase (MAPK) induced phosphorylation events seem to play an important role in this phenomenon. Proteins affecting macrophage cholesterol metabolism and transport, including ATP-binding cassette (ABC) A1, ABCG1, acylCoA:cholesterol acyltransferase (ACAT), apolipoprotein A-1 (ApoA-1), neutral cholesteryl ester hydrolase (NCEH) also regulate foam cell formation and are being developed as therapeutic targets by many pharmaceutical companies. Macrophage proliferation and apoptosis are important events controlling inflammatory response, plaque vulnerability, and destabilization. Free cholesterol (FC) activates the macrophage endoplasmic reticulum (ER) stress pathway and apoptosis. Free radicals and nitric oxide also modulate macrophage foam cell formation and apoptosis. Various antioxidants like AGI-1067 and BO-653 are in clinical trials for atherosclerosis treatment. Macrophage matrix metalloproteinase's (MMP's) play a significant role in weakening and rupture of plaques. Efforts are on to develop isoform specific MMP inhibitor. CD-14, MMP-3, ABCA1, Toll-like receptor-4 (TLR-4), lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), arachidonate lipoxygenase-15 (ALOX-15), and Connexin37 polymorphisms and macrophage dysfunction signify their importance in atherosclerosis. Deciphering the role of macrophages in regulating dyslipidemia and inflammation during atherosclerosis is important for developing them as therapeutic targets. ß 2007 Wiley Periodicals, Inc. Med Res Rev, 28, No. 4, 483-544, 2008

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