Patrick P. McDonald

Selective activation and functional significance of p38α mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils

Activation of leukocytes by proinflammatory stimuli selectively initiates intracellular signal transduction via sequential phosphorylation of kinases. Lipopolysaccharide (LPS) stimulation of human neutrophils is known to result in activation of p38 mitogen-activated protein kinase (MAPk); however, the upstream activator(s) of p38 MAPk is unknown, and consequences of p38 MAPk activation remain largely undefined. We investigated the MAPk kinase (MKK) that activates p38 MAPk in response to LPS, the p38 MAPk isoforms that are activated as part of this pathway, and the functional responses affected by p38 MAPk activation. Although MKK3, MKK4, and MKK6 all activated p38 MAPk in experimental models, only MKK3 was found to activate recombinant p38 MAPk in LPS-treated neutrophils. Of p38 MAPk isoforms studied, only p38alpha and p38delta were detected in neutrophils. LPS stimulation selectively activated p38alpha. Specific inhibitors of p38alpha MAPk blocked LPS-induced adhesion, nuclear factor-kappa B (NF-kappaB) activation, and synthesis of tumor necrosis factor-alpha (TNF-alpha). Inhibition of p38alpha MAPk resulted in a transient decrease in TNF-alpha mRNA accumulation but persistent loss of TNF-alpha synthesis. These findings support a pathway by which LPS stimulation of neutrophils results in activation of MKK3, which in turn activates p38alpha MAPk, ultimately regulating adhesion, NF-kappaB activation, enhanced gene expression of TNF-alpha, and regulation of TNF-alpha synthesis.

Hypoxia-enhanced Expression of the Proprotein Convertase Furin Is Mediated by Hypoxia-inducible Factor-1 IMPACT ON THE BIOACTIVATION OF PROPROTEINS

Hypoxia is a common tumorigenesis enhancer, mostly owing to its impact on gene expression of many angiogenic and invasion-related mediators, some of which are natural substrates for the proprotein convertase furin. Analysis of furin promoters revealed the presence of putative binding sites for hypoxia-inducible factor-1 (HIF-1), a transcription complex that plays a pivotal role in cellular adaptation to hypoxia. In fact, we demonstrate herein that the levels of fur mRNA, encoding furin, are remarkably increased upon hypoxic challenge. Cotransfection of a HIF-1α dominant negative form in wild-type (WT) cells or transfection of a furin promoter-reporter gene in HIF-1-deficient cells indicated the requirement of HIF-1 for furin promoter activation by hypoxia. Direct HIF-1 action on the furin promoter was identified as a canonical hypoxia-responsive element site with enhancer capability. The hypoxic/HIF-1 regulation of furin correlated with an increased proteolytic activation of the substrates membrane-type 1 matrix metalloproteinase and transforming growth factor-β1. Our findings unveil a new facet of the physiological consequences of hypoxia/HIF-1, through enhanced furin-induced proteolytic processing/activation of proproteins known to be involved in tumorigenesis.

Differential involvement of NF-κB and MAP kinase pathways in the generation of inflammatory cytokines by human neutrophils

The ability of human neutrophils to express a variety of genes encoding inflammatory mediators is well documented, and mounting evidence suggests that neutrophil‐derived cytokines and chemokines contribute to the recruitment of discrete leukocyte populations at inflammatory sites. Despite this, our understanding of the signaling intermediates governing the generation of inflammatory cytokines by neutrophils remains fragmentary. Here, we report that inhibitors of the p38 MAPK and MEK pathways substantially diminish the release of (and in the case of p38 inhibitors, the gene expression of) several inflammatory cytokines in neutrophils stimulated with LPS or TNF. In addition, various NF‐κB inhibitors were found to profoundly impede the inducible gene expression and release of inflammatory cytokines in these cells. The MAPK inhibitors did not affect NF‐κB activation; instead, the transcriptional effects of the p38 MAPK inhibitor appear to involve transcriptional factor IID. Conversely, the NF‐κB inhibitors failed to affect the activation of MAPKs. Finally, the MAPK inhibitors were found to prevent the activation a key component of the translational machinery, S6 ribosomal protein, in keeping with their post‐transcriptional impact on cytokine generation. To our knowledge, this constitutes the first demonstration that in neutrophils, the inducible expression of proinflammatory cytokines by physiological stimuli largely reflects the ability of the latter to activate NF‐κB and selected MAPK pathways. Our data also raise the possibility that NF‐κB or MAPK inhibitors could be useful in the treatment of inflammatory disorders in which neutrophils predominate.

The MYD88-Independent Pathway Is Not Mobilized in Human Neutrophils Stimulated via TLR4

LPS activates both MyD88-dependent and -independent signaling via TLR4, but the extent to which each cascade is operative in different cell types remains unclear. This prompted us to revisit the intriguing issue of CXCL10 production, which we previously showed to be inducible in neutrophils stimulated with LPS and IFN-γ but not with either stimulus alone, contrary to other myeloid cells. We now report that in neutrophils the MyD88-independent pathway is not activated by LPS. Indeed, microarray and real-time PCR experiments showed that neither IFNβ nor IFNβ-dependent genes (including CXCL10) are inducible in LPS-treated neutrophils, in contrast to monocytes. Further investigation into the inability of LPS to promote IFNβ expression in neutrophils revealed that the transcription factors regulating the IFNβ enhanceosome, such as IFN-regulatory factor-3 and AP-1, are not activated in LPS-treated neutrophils as revealed by lack of dimerization, nuclear translocation, confocal microscopy, and inducible binding to DNA. Moreover, we show that the upstream TANK-binding kinase-1 is not activated by LPS in neutrophils. A lack of IFNβ/CXCL10 mRNA expression and IFN-regulatory factor 3 activation was also observed in myeloid leukemia HL60 cells differentiated to granulocytes and then stimulated with LPS, indicating that the inability of neutrophils to activate the MyD88-independent pathway represents a feature of their terminal maturation. These results identify a disconnected activation of the two signaling pathways downstream of TLR4 in key cellular components of the inflammatory and immune responses and help us to better understand the primordial role of neutrophils in host defense against nonviral infections.

Hypoxia-inducible Factor Mediates Hypoxic and Tumor Necrosis Factor α-induced Increases in Tumor Necrosis Factor-α Converting Enzyme/ADAM17 Expression by Synovial Cells

Chronic hypoxia and inflammatory cytokines are hallmarks of inflammatory joint diseases like rheumatoid arthritis (RA), suggesting a link between this microenvironment and central pathological events. Because TACE/ADAM17 is the predominant protease catalyzing the release of tumor necrosis factor α (TNFα), a cytokine that triggers a cascade of events leading to RA, we examined the regulation of this metalloprotease in response to hypoxia and TNFα itself. We report that low oxygen concentrations and TNFα enhance TACE mRNA levels in synovial cells through direct binding of hypoxia-inducible factor-1 (HIF-1) to the 5′ promoter region. This is associated with elevated TACE activity as shown by the increase in TNFα shedding rate. By the use of HIF-1-deficient cells and by obliterating NF-κB activation, it was determined that the hypoxic TACE response is mediated by HIF-1 signaling, whereas the regulation by TNFα also requires NF-κB activation. As a support for the in vivo relevance of the HIF-1 axis for TACE regulation, immunohistological analysis of TACE and HIF-1 expression in RA synovium indicates that TACE is up-regulated in both fibroblast- and macrophage-like synovial cells where it localizes with elevated expression of both HIF-1 and TNFα. These findings suggest a mechanism by which TACE is increased in RA-affected joints. They also provide novel mechanistic clues on the influence of the hypoxic and inflammatory microenvironment on joint diseases.

IL-6, in Synergy with IL-7 or IL-15, Stimulates TCR-Independent Proliferation and Functional Differentiation of CD8+ T Lymphocytes

Recent reports have shown that IL-21, in synergy with IL-15, stimulates proliferation of CD8+ T lymphocytes in the absence of signaling via the TCR. In this study, we show that IL-6, which induces phosphorylation of STAT3 similarly to IL-21, also can stimulate proliferation of CD8+ T cells in synergy with IL-7 or IL-15. IL-6 displays a stronger synergy with IL-7 than with IL-15 to stimulate naive CD8+ T cells. Concomitant stimulation by IL-6 or IL-21 augments phosphorylation and DNA-binding activity of STAT5 induced by IL-7 or IL-15. Like IL-21, IL-6 reduces the TCR signaling threshold required to stimulate CD8+ T cells. Prior culture of P14 TCR transgenic CD8 T cells with IL-6 or IL-21 in the presence of IL-7 or IL-15 augments their proliferation and cytolytic activity upon subsequent stimulation by Ag. Furthermore, cytokine stimulation induces quantitatively and qualitatively distinct phenotypic changes on CD8+ T cells compared with those induced by TCR signaling. We propose that the ability of IL-6 to induce TCR-independent activation of CD8+ T cells in synergy with IL-7 or IL-15 may play an important role in the transition from innate to adaptive immunity.

Inflammatory Cytokine Production by Human Neutrophils Involves C/EBP Transcription Factors

A growing number of neutrophil-derived cytokines have proven to be crucial to various inflammatory and immune processes in vivo. Whereas C/EBP (CCAAT/enhancer-binding protein) transcription factors are important for neutrophil differentiation from myeloid precursors, we report herein that they also regulate cytokine production in mature neutrophils. All known C/EBP proteins but C/EBPγ are expressed in neutrophils; most isoforms localize to the nucleus, except for C/EBPα, which is cytoplasmic. Neutrophil stimulation does not alter the overall levels, cellular distribution, or turnover of C/EBP proteins; it also does not further induce the constitutive DNA-binding activity detected in nuclear extracts, consisting of C/EBPβ and C/EBPε. However, nuclear C/EBPβ is rapidly phosphorylated upon cell stimulation, suggesting that it can activate cytokine promoters. Indeed, the transactivation of an IL-8 promoter-luciferase construct in a human neutrophil-like cell line was impaired when its C/EBP or NF-κB sites were mutated. Overexpression of a C/EBP repressor also impeded IL-8 promoter transactivation, as well as the generation of IL-8, Mip-1α, and Mip-1β in this cellular model, whereas TNF-α generation was mostly unaffected. Finally, overexpression of a C/EBPβ mutant (T235A) as well as chromatin immunoprecipitation assays unveiled an important role for this residue in cytokine induction. This is the first demonstration that C/EBP factors are important regulators of cytokine expression in human neutrophils.

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 Alzheimers 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.

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.

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 cyto‐kines. Interleukin‐18 (IL‐18) is known to stimulate several neutrophil responses, and recent evidence sug‐gests 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‐κB 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 inflamma‐tory cytokines in neutrophils, including its own expression. We finally report that IL‐18 activates the p38 MAPK, MEK/ERK, and PI3K/Akt pathways in neutro‐phils. The IKK cascade is also activated by IL‐18, resulting in IκB‐α degradation, NF‐κB activation, and RelA phosphorylation. Accordingly, these pathways contribute to the generation of inflammatory cytokines in IL‐18‐stimulated neutrophils. By contrast, the phos‐phorylation and DNA‐binding activity of various STAT proteins were not induced by IL‐18. Collectively, our results unveil new interactions between IL‐18 and neutro‐phils and further support a role for these cells in influencing both innate and adaptive immunity.—Fortin, C. F., Ear, T., McDonald, P. P. Autocrine role of endogenous interleukin‐18 on inflammatory cytokine generation by human neutrophils. FASEB J. 23, 194‐203 (2009)