Activation of TAK1 by Chemotactic and Growth Factors, and Its Impact on Human Neutrophil Signaling and Functional Responses.

The MAP3 kinase, TAK1, is known to act upstream of IKK and MAPK cascades in several cell types, and is typically activated in response to cytokines (e.g., TNF, IL-1) and TLR ligands. In this article, we report that in human neutrophils, TAK1 can also be activated by different classes of inflammatory stimuli, namely, chemoattractants and growth factors. After stimulation with such agents, TAK1 becomes rapidly and transiently activated. Blocking TAK1 kinase activity with a highly selective inhibitor (5z-7-oxozeaenol) attenuated the inducible phosphorylation of ERK occurring in response to these stimuli but had little or no effect on that of p38 MAPK or PI3K. Inhibition of TAK1 also impaired MEKK3 (but not MEKK1) activation by fMLF. Moreover, both TAK1 and the MEK/ERK module were found to influence inflammatory cytokine expression and release in fMLF- and GM-CSF-activated neutrophils, whereas the PI3K pathway influenced this response independently of TAK1. Besides cytokine production, other responses were found to be under TAK1 control in neutrophils stimulated with chemoattractants and/or GM-CSF, namely, delayed apoptosis and leukotriene biosynthesis. Our data further emphasize the central role of TAK1 in controlling signaling cascades and functional responses in primary neutrophils, making it a promising target for therapeutic intervention in view of the foremost role of neutrophils in several chronic inflammatory conditions.

Translational control of human neutrophil responses by MNK1.

A growing number of inflammatory and immune processes in vivo have been shown to be influenced by neutrophil-derived cytokines. Whereas the underlying transcriptional mechanisms are increasingly well understood, the translational regulation of this neutrophil response remains largely unexplored. Here, we show that the MNK1, which participates in translational control in several cell types, is activated in response to physiological neutrophil agonists (LPS, TNF-α) in the cytoplasmic and nuclear compartments. With the use of various pharmacological inhibitors, we found that MNK1 activation takes place downstream of the TAK1-p38 MAPK axis in neutrophils, whereas the MEK/ERK, JNK, PI3K, and PKC pathways are not involved. Pharmacological blockade of MNK1, as well as overexpression experiments, established that cytokine protein synthesis (but not gene expression) is under the control of MNK1 in neutrophils. Likewise, MNK1 inhibition reversed the antiapoptotic effect of LPS and TNF-α in neutrophils, and this was accompanied by a decreased expression of the antiapoptotic protein Mcl-1. Thus, MNK1 appears to be an important regulator of neutrophil responses. Although MNK1 inhibition did not affect protein recruitment to mRNA caps, it decreased the phosphorylation of molecules implicated in translation initiation control, such as S6K, S6, and hyperphosphorylated 4E-BP1. These molecular targets of MNK1 are shared with those of PI3K in neutrophils, and accordingly, MNK1 inhibition partially impaired the belated PI3K/Akt activation elicited by LPS or TNF in these cells. Given the importance of neutrophils and their products in numerous chronic inflammatory disorders, MNK1 could represent an attractive therapeutic target.

A class IA PI3K controls inflammatory cytokine production in human neutrophils.

Neutrophils are generally the first leukocytes to arrive at sites of inflammation or injury, where they release a variety of inflammatory mediators, which contribute to shaping the ensuing immune response. Here, we show that in neutrophils exposed to physiological stimuli (i.e. LPS and TNF-α), inhibition of the PI3K signaling pathway impairs the synthesis and secretion of IL-8, Mip-1α, and Mip-1ß. Further investigation showed that Mip-1α and Mip-1ß gene transcription was similarly decreased, whereas IL-8 transcription and steady-state mRNA levels were unaffected. Accordingly, PI3K inhibition had no impact on NF-κB or C/EBP activation, which are essential for IL-8 transcription, but the basis for this selective inhibition of chemokine transcription remains elusive. We nevertheless identified translational targets of the PI3K pathway (S6, S6 kinase, 4E-BP1). Inhibitor studies and overexpression experiments further established that the various effects of PI3K on chemokine production can be ascribed to p85α and p110δ subunits. Finally, we show that in LPS- and TNF-activated neutrophils, PI3K acts downstream of the kinases p38 MAPK and TAK1. Given the importance of neutrophils and their products in numerous chronic inflammatory disorders, the PI3K pathway could represent an attractive therapeutic target.

Potential role of immunosenescence in cancer development.

The incidence and prevalence of most cancers increase with age. The reasons for this may include tumor escape mechanisms and decreased immunosurveillance, but most are caused by the time required for carcinogenesis, according to most scientists. The immune system is a unique mechanism of defense against pathogens and possibly cancers; however, there is a body of evidence that the immune system of the aged is eroded, a phenomenon termed immunosenescence. There is a growing interest in immunosenescence and how it may contribute to the increased number of cancers with aging. Each arm of the immune system, innate and adaptive, is altered with aging, contributing to increased tumorigenesis. Understanding the contribution of immunosenescence to cancer development and progression may lead to better interventions for the elderly.

MT6-MMP is present in lipid rafts and faces inward in living human PMNs but translocates to the cell surface during neutrophil apoptosis.

Polymorphonuclear neutrophils (PMNs) are the first line of defense against invading organisms in humans; in addition, PMNs contribute to the linking of innate and adaptive immunity. To fulfill their biological behavior, PMNs utilize an arsenal of proteolytic enzymes, including members of the matrix metalloproteinase family of zinc-dependent endopeptidases. PMNs express high levels of MT6-MMP (MMP-25), a glycosyl-phosphatidylinositol-anchored MMP, that belongs to the subfamily of membrane-anchored matrix metalloproteinases. Due to the paucity of information on MT6-MMP in primary cells, we set to investigate the localization and potential function of MT6-MMP in human PMNs. We found that MT6-MMP is present in the membrane, granules and nuclear/endoplasmic reticulum/Golgi fractions of PMNs where it is displayed as a disulfide-linked homodimer of 120 kDa. Stimulation of PMNs resulted in secretion of active MT6-MMP into the supernatants. Membrane-bound MT6-MMP, conversely, is located in the lipid rafts of resting PMNs and stimulation does not alter this location. In addition, TIMP-2, a natural inhibitor of MT6-MMP, does not co-localize with it in the lipid rafts. Interestingly, living PMNs do not display MT6-MMP on the cell surface. However, induction of apoptosis induces MT6-MMP relocation on PMNs' cell surface. Our studies suggest that metalloproteinases may play a role in respiratory burst and IL-8 secretion, but not chemotaxis or granulocyte macrophage colony-stimulating factor-induced survival. Collectively, these results provide new insights on the role of MT6-MMP in the physiology of human PMNs.

Constitutive association of TGF-beta-activated kinase 1 with the IkappaB kinase complex in the nucleus and cytoplasm of human neutrophils and its impact on downstream processes.

Neutrophils influence innate and adaptative immunity by generating numerous mediators whose regulation largely depends on the IkappaB kinase (IKK)/IkappaB/NF-kappaB signaling cascade. A singular feature of neutrophils is that they express several components of this pathway (namely, NF-kappaB/Rel proteins and IkappaB-alpha) in both the nucleus and cytoplasm. We recently reported that the IKK complex of neutrophils is similarly expressed and activated in both cellular compartments. However, the upstream IKK kinase has not yet been identified. In this study, we report that neutrophils express the mitogen-activated protein 3 kinase, TGF-beta-activated kinase 1 (TAK1), as well as its associated partners, TAK1-binding protein (TAB) 1, TAB2, and TAB4, in both the cytoplasm and nucleus. Following cell stimulation by TNF-alpha or LPS, TAK1 becomes rapidly and transiently activated. Blocking TAK1 kinase activity with a highly selective inhibitor (5z-7-oxozeaenol) attenuated the phosphorylation of nuclear and cytoplasmic IKKalpha/beta, IkappaB-alpha, and RelA, and also impaired IkappaB-alpha degradation and NF-kappaB DNA binding in activated neutrophils. Moreover, TAK1 was found to be involved in the activation of p38 MAPK and ERK, which also influence cytokine generation in neutrophils. As a result, inflammatory cytokine expression and release were profoundly impaired following TAK1 inhibition. Similarly, the delayed apoptosis observed in response to LPS or TNF-alpha was reversed by TAK1 inhibition. By contrast, IKKgamma phosphorylation and STAT1 activation were unaffected by TAK1 inhibition. Our data establish the central role of TAK1 in controlling nuclear and cytoplasmic signaling cascades in primary neutrophils, making it a promising target for therapeutic intervention in view of the foremost role of neutrophils in several chronic inflammatory conditions.

Sepsis, leukocytes, and nitric oxide (NO): an intricate affair.

Sepsis is exceedingly burdensome for hospital intensive care unit caregivers, and its incidence, as well as sepsis-related deaths, is increasing steadily. Sepsis is characterized by a robust increase in NO production throughout the organism that is driven by iNOS. Moreover, NO is an important factor in the development of septic shock and is synthesized by NOS, an enzyme expressed by a variety of cells, including vascular endothelium, macrophages, and neutrophils. However, the effects of NO on leukocyte functions, and the underlying mechanisms, are relatively unknown. Thus, the present review focuses on the effects of NO and its derivatives on cells of the immune system. Experimental evidences discussed herein show that NO induces posttranslational modifications of key proteins in targeted processes with the potential of deterring cellular physiology. Consequently, the manipulation of NO distribution in septic patients, used in conjunction with conventional treatments aimed at restoring normal immune functions, may represent a valuable therapeutic strategy.

Aging and innate immunity in the mouse: impact of intrinsic and extrinsic factors.

Aging affects every innate immune cell, including changes in cell numbers and function. Defects in the function of some cells are intrinsic, whereas for other cells, defects are extrinsic and possibly the consequence of the complex interactions with other cell types or the environmental milieu that is altered with aging. Abnormal function contributes to worsened outcomes after injury or infection and leads to diseases observed in the elderly. Knowing the mechanisms responsible for the aberrant function of innate immune cells might lead to the development of therapeutic strategies designed to improve innate immunity in aged individuals. Herein, advances in the field of innate immunity and aging with a focus on neutrophils, macrophages and dendritic cells in laboratory animals are discussed.

Autocrine role of endogenous interleukin-18 on inflammatory cytokine generation by human neutrophils.

Neutrophils are key players of innate immunity and influence inflammatory and immune reactions through the production of numerous cytokines. Interleukin-18 (IL-18) is known to stimulate several neutrophil responses, and recent evidence suggests that neutrophils might represent a source of IL-18. Here, we show that neutrophils constitutively produce both IL-18 and its antagonist, IL-18BP. Cell activation does not affect IL-18BP release but leads to an increased gene expression and secretion of IL-18, a process that depends on NF-kappaB activation. Moreover, endogenous IL-18 feeds back on the neutrophils to augment cytokine generation in lipopolysaccharide-treated cells. Accordingly, exogenous IL-18 can induce the gene expression and release of several inflammatory cytokines in neutrophils, including its own expression. We finally report that IL-18 activates the p38 MAPK, MEK/ERK, and PI3K/Akt pathways in neutrophils. The IKK cascade is also activated by IL-18, resulting in IkappaB-alpha degradation, NF-kappaB activation, and RelA phosphorylation. Accordingly, these pathways contribute to the generation of inflammatory cytokines in IL-18-stimulated neutrophils. By contrast, the phosphorylation and DNA-binding activity of various STAT proteins were not induced by IL-18. Collectively, our results unveil new interactions between IL-18 and neutrophils and further support a role for these cells in influencing both innate and adaptive immunity.

Aging and neutrophils: there is still much to do.

Human neutrophils are activated by a wide array of compounds through their receptors. This elicits their classical functions, such as chemotaxis, phagocytosis, and the production of reactive oxygen species (ROS). Upon stimulation, neutrophils also produce lipid and immune mediators and can present antigen through the major histocompatibility complex I (MHC-I). The age-related impairment of the classical functions of neutrophils is well described, but experimental evidence showing alterations in the production of mediators and antigen presentation with aging are lacking. This review highlights the role of neutrophils in age-related pathologies such as Alzheimer's disease, atherosclerosis, cancer, and autoimmune diseases. Furthermore, we discuss how aging potentially affects the production and release of mediators by human neutrophils in ways that may contribute to the development of these pathologies.

Effects of aging on triggering receptor expressed on myeloid cells (TREM)-1-induced PMN functions.

Triggering receptor expressed on myeloid cell-1 (TREM-1) is a recently described receptor that has many effects on polymorphonuclear neutrophil (PMN), as the engagement of this receptor on PMN can induce phagocytosis, respiratory burst and degranulation. We studied the effects of aging on TREM-1 engagement in human PMN. PMN from elderly were found to have impaired response following TREM-1 engagement. Notably they were not able to modulate the TREM-1-induced respiratory burst as PMN from young did. TREM-1 engagement could not reverse PMN survival following incubation with LPS or GM-CSF in the elderly whereas it did in the young. The phosphorylation of TREM-1 signal transduction molecules was altered with aging. Finally, TREM-1 engagement could not drive the recruitment of TREM-1 in the lipid-rafts of the elderly explaining in part the altered response. The observed alterations in TREM-1 response are possibly an important contributing factor in the higher incidence of sepsis-related deaths in the elderly population.

Effects of TREM-1 activation in human neutrophils: activation of signaling pathways, recruitment into lipid rafts and association with TLR4.

Neutrophilic polymorphonuclears (PMNs) play an important role in the progression of sepsis-related inflammation and become highly activated by a wide array of ligands on the site. The triggering receptor expressed on myeloid cells-1 (TREM-1) is a recently described receptor that has many effects on human PMN. The engagement of TREM-1 on PMN can induce phagocytosis, reactive oxygen species production and release of myeloperoxidase and IL-8. LPS has a priming effect on these functions. We show in this paper that Lyn, AKT, extracellular signal-regulated kinase 1/2 and Jak2 signaling pathways are elicited following TREM-1 engagement and activation by a monoclonal agonist antibody (anti-TREM-1) in human PMN, leading to the phosphorylation of STAT5 and RelA, a subunit of the nuclear factor-kappa B family. We also show that TREM-1 is recruited to ganglioside M1-lipid rafts in PMN upon stimulation with LPS or anti-TREM-1. Moreover, we observed that Toll-like receptor 4 and TREM-1 co-localize upon stimulation and TREM-1 engagement resulted in the phosphorylation of IL-1R-associated kinase 1, but not its stimulant-induced degradation. These data shed a new light on how various receptors implicated in the innate immune response could interact to insure an efficient inflammatory response upon pathogens-associated aggression.

GM-CSF activates the Jak/STAT pathway to rescue polymorphonuclear neutrophils from spontaneous apoptosis in young but not elderly individuals.

Polymorphonuclear neutrophils (PMN) are the first cells to be recruited to the site of tissular aggression. They have a short-life span and die by spontaneous apoptosis. However, their life span and functional activities can be extended in vitro by a number of proinflammatory cytokines, including the granulocyte-macrophage colony stimulating factor (GM-CSF). We have reported that the protective effect of GM-CSF did not occur in PMN of elderly subjects. Data reported here showed that this difference was not due to a change in the expression of the GM-CSF receptor in the PMN of elderly individuals compared to young subjects. Furthermore, we showed here that GM-CSF activated the Janus kinase/signal transducer and activator of transcription (Jak/STAT) pathway and this activation appeared to be maintained for an extended period of time (18 h) playing an important role in the GM-CSF induced delayed PMN apoptosis. In marked contrast, GM-CSF had no effects on Jak2 activation in PMN of elderly individuals. We found that an inhibitor of Jak2 activation (AG490) abolished the protective effect of GM-CSF in PMN from young donors, however had no effect in PMN of elderly subjects. GM-CSF induced a transient activation of STAT3 and STAT5 in PMN of young donors but failed to activate to the same extent these signal transducers in PMN of elderly donors. The levels of proCaspase-3 were reduced in PMN of young donors treated with GM-CSF for 18 h but remained unchanged in PMN of elderly subjects treated under the same conditions compared to the untreated PMN. Our data are consistent with the interpretation that, at least in part (1) the protective effect of GM-CSF against apoptosis results from the activation of the Jak/STAT pathway and (2) decreased rescue from apoptosis in PMN of elderly is related to a failure of GM-CSF to activate this pathway in these cells.

Impairment of SHP-1 down-regulation in the lipid rafts of human neutrophils under GM-CSF stimulation contributes to their age-related, altered functions.

It has been shown that the functions and the rescue from apoptosis by proinflammatory mediators of polymorphonuclear leukocytes (PMN) tend to diminish with aging. Here, we investigated the role of protein tyrosine phosphatases (PTP), especially Src homology domain-containing protein tyrosine phosphatase-1 (SHP-1), in the age-related, altered PMN functions under granulocyte macrophage-colony stimulating factor (GM-CSF) stimulation. The inhibition of PTP suggested a differential effect of GM-CSF on phosphatase activity in modulating PMN functions with aging. The down-regulation of phosphatase activity of immunopurified SHP-1 from lipid rafts of PMN of young donors was found significantly altered at 1 min of stimulation with aging. In young donors, SHP-1 is displaced from lipid rafts at 1 min of stimulation, whereas in the elderly, SHP-1 is constantly present. We assessed in PMN lipid rafts the phosphorylation of tyrosine and serine residues of SHP-1, which regulates its activity. We observed an alteration in the phosphorylation of tyrosine and serine residues of SHP-1 in PMN of elderly subjects, suggesting that GM-CSF was unable to inhibit SHP-1 activity by serine phosphorylation. GM-CSF activates Lyn rapidly, and we found alterations in its activation and translocation to the lipid rafts with aging. We also demonstrate that SHP-1 in the PMN of elderly is constantly recruited to Lyn, which cannot be relieved by GM-CSF. In contrast, in the young, the resting recruitment could be relieved by GM-CSF. Our results suggest an alteration of the SHP-1 modulation by GM-CSF in lipid rafts of PMN with aging. These alterations could contribute to the decreased GM-CSF effects on PMN.