Search

Neutrophils play a critical role in innate immunity and also influence adaptive immune responses. This occurs in good part through their production of inflammatory and immunomodulatory cytokines, in conjunction with their prolonged survival at inflamed foci. While a picture of the signaling machinery underlying these neutrophil responses is now emerging, much remains to be uncovered. In this study, we report that neutrophils constitutively express various Src family isoforms (STKs), as well as Syk, and that inhibition of these protein tyrosine kinases selectively hinders inflammatory cytokine generation by acting posttranscriptionally. Accordingly, STK or Syk inhibition decreases the phosphorylation of signaling intermediates (e.g., eIF-4E, S6K, and MNK1) involved in translational control. By contrast, delayed apoptosis appears to be independent of either STKs or Syk. Our data therefore significantly extend our understanding of which neutrophil responses are governed by STKs and Syk and pinpoint some signaling intermediates that are likely involved. In view of the foremost role of neutrophils in several chronic inflammatory conditions, our findings identify potential molecular targets that could be exploited for future therapeutic intervention.

Background HIV-1 translation is modulated by the activation of the interferon (IFN)-inducible Protein Kinase RNA-activated (PKR). PKR phosphorylates its downstream targets, including the alpha subunit of the eukaryotic translation Initiation Factor 2 (eIF2α), which decreases viral replication. The PKR Activator (PACT) is known to activate PKR after a cellular stress. In lymphocytic cell lines, HIV-1 activates PKR only transiently and not when cells replicate the virus at high levels. The regulation of this activation is due to a combination of viral and cellular factors that have been only partially identified. Results PKR is transiently induced and activated in peripheral blood mononuclear cells after HIV-1 infection. The addition of IFN reduces viral replication, and induces both the production and phosphorylation of PKR. In lymphocytic Jurkat cells infected by HIV-1, a multiprotein complex around PKR contains the double-stranded RNA binding proteins (dsRBPs), adenosine deaminase acting on RNA (ADAR)1 and PACT. In HEK 293T cells transfected with an HIV-1 molecular clone, PACT unexpectedly inhibited PKR and eIF2α phosphorylation and increased HIV-1 protein expression and virion production in the presence of either endogenous PKR alone or overexpressed PKR. The comparison between different dsRBPs showed that ADAR1, TAR RNA Binding Protein (TRBP) and PACT inhibit PKR and eIF2α phosphorylation in HIV-infected cells, whereas Staufen1 did not. Individual or a combination of short hairpin RNAs against PACT or ADAR1 decreased HIV-1 protein expression. In the astrocytic cell line U251MG, which weakly expresses TRBP, PACT mediated an increased HIV-1 protein expression and a decreased PKR phosphorylation. In these cells, a truncated PACT, which constitutively activates PKR in non-infected cells showed no activity on either PKR or HIV-1 protein expression. Finally, PACT and ADAR1 interact with each other in the absence of RNAs. Conclusion In contrast to its previously described activity, PACT contributes to PKR dephosphorylation during HIV-1 replication. This activity is in addition to its heterodimer formation with TRBP and could be due to its binding to ADAR1. HIV-1 has evolved to replicate in cells with high levels of TRBP, to induce the expression of ADAR1 and to change the function of PACT for PKR inhibition and increased replication.

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.

Neutrophils influence innate and adaptative immunity, notably through the generation of numerous cytokines and chemokines and through the modulation of their constitutive apoptosis. Several signaling cascades are known to control neutrophil responses, including the MEK pathway, which is normally coupled to ERK. However, we show here that in human neutrophils stimulated with cytokines or TLR ligands, MEK and ERK are activated independently of each other. Pharmacological blockade of MEK had no effect on the induction of ERK kinase activity and vice versa. In autologous PBMC exposed to the same stimuli or in neutrophils exposed to chemoattractants, this uncoupling of MEK and ERK was not observed. Whereas we had shown before that MEK inhibition impairs cytokine generation translationally in LPS- or TNF-stimulated neutrophils, ERK inhibition affected this response transcriptionally and translationally. Transcriptional targets or ERK include the mitogen- and stress-activated protein kinase 1 (MSK-1) and its substrates, C/EBPβ and CREB, whereas translational targets include the S6 kinase and its substrate, the S6 ribosomal protein. In addition to affecting cytokine production, ERK inhibition interfered with how LPS or TNF promotes neutrophil survival and levels of the myeloid cell leukemia 1 (Mcl-1) antiapoptotic protein. Whereas the ERK-activating kinase was not identified, we found that the MAP3K, TGF-β-activated kinase 1 (TAK1), acts upstream of ERK and MEK in neutrophils. Our results document a functional uncoupling of the MEK/ERK module under certain stimulatory conditions and suggest that therapeutic strategies based on MEK inhibition might benefit from being complemented by ERK inhibition, particularly in chronic inflammatory conditions featuring a strong neutrophilic component.

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.

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.

The CXCL10 chemokine is a critical chemoattractant for the recruitment of activated Th1 and NK cells into inflammatory sites. CXCL10 is typically produced by myeloid cells in response to IFN-gamma, as well as by neutrophils, though the latter require a costimulation with IFN-gamma and LPS. In this study, we investigated the molecular mechanism(s) whereby IFN-gamma and TLR4 ligation synergize to induce CXCL10 expression in neutrophils. By primary transcript real-time PCR analysis, we demonstrate that the CXCL10 gene is transcriptionally induced by the LPS plus IFN-gamma combination in neutrophils, consistent with previous studies showing that increased CXCL10 gene expression does not reflect enhanced mRNA stability. The IFN-gamma-induced STAT1 activation and the lipopolysaccharide (LPS)-induced NF-kappaB activation were not enhanced if neutrophils were exposed to both stimuli, whereas both transcription factors were activated by IFN-gamma or LPS in monocytes. Finally, pharmacological inhibitors of NF-kappaB demonstrated its role in the induction of CXCL10 expression by LPS plus IFN-gamma in neutrophils, and by LPS or IFN-gamma in monocytes. Together, these results suggest that in neutrophils, the synergy observed between LPS and IFN-gamma toward CXCL10 gene expression likely reflects the cooperative induction of the NF-kappaB and STAT1 transcription factors by LPS and IFN-gamma, respectively.

A singular feature of human neutrophils is that they constitutively express substantial amounts of NF-kappaB/Rel proteins and IkappaB-alpha in the nucleus. In this study, we show that in these cells, IkappaB kinase alpha (IKKalpha), IKKbeta, and IKKgamma also partially localize to the nucleus, whereas IKK-related kinases (IKKepsilon, TANK-binding kinase-1) are strictly cytoplasmic, and the NF-kappaB-inducing kinase is strictly nuclear. Following neutrophil activation, IKKbeta and IKKgamma become transiently phosphorylated in both the cytoplasm and nucleus, whereas IKKalpha transiently vanishes from both compartments in what appears to be an IKKbeta-dependent process. These responses are paralleled by the degradation of IkappaB-alpha, and by the phosphorylation of RelA on serine 536, in both compartments. Although both proteins can be IKK substrates, inhibition of IKK prevented IkappaB-alpha phosphorylation, while that of RelA was mostly unaffected. Finally, we provide evidence that the nuclear IKK isoforms (alpha, beta, gamma) associate with chromatin following neutrophil activation, which suggests a potential role in gene regulation. This is the first study to document IKK activation and the phosphorylation of NF-kappaB/Rel proteins in primary neutrophils. More importantly, our findings unveil a hitherto unsuspected mode of activation for the IKK/IkappaB signaling cascade within the cell nucleus.

In the last decade, the ability of neutrophils to generate proinflammatory cytokines has become firmly established. Because neutrophils typically infiltrate inflammatory sites in large numbers, they could significantly contribute to the cytokine environment and even represent a substantial source of cytokines in chronic inflammatory disorders in which they predominate over other cell types. To date, however, most studies have focused on identifying which mediators are produced by neutrophils, as opposed to elucidating the molecular bases underlying this process. We previously showed that most stimuli of cytokine production in neutrophils also activate NF-κB in these cells. In this report, we turned our attention to another transcription factor that plays a central role in inflammation, AP-1. Among Jun/Fos proteins, only JunD and c-Fos are abundantly expressed in neutrophils, and they are mainly cytoplasmic. Both the cellular levels and distribution of the Jun/Fos proteins remain unaffected by various neutrophil stimuli, including those that are known to increase the corresponding mRNA transcripts. Similarly, c-Jun N-terminal kinase (JNK) 1 is overwhelmingly cytoplasmic in neutrophils and does not translocate to the nucleus upon cell activation. Although JNK is not activatable under most circumstances, specific conditions do allow its phosphorylation in response to TNF. However, no experimental condition (even those leading to JNK activation) resulted in the induction of genuine AP-1 complexes in neutrophils. Accordingly, the potent JNK inhibitor, SP 600125, failed to inhibit inflammatory cytokine gene expression in neutrophils. Collectively, our findings strongly suggest that the JNK/AP-1 signaling pathway has little or no impact on the generation of inflammatory mediators in neutrophils.

Endothelial cells derived from the human umbilical vein (HUVEC) are used to study the mechanisms involved in EC response to various stimuli as well as to investigate the basis of pathological conditions of the vascular system such as altered endothelium permeability, tumor-induced angiogenesis, atherosclerosis and leukocyte extravasation in chronic inflammatory responses. However, investigations of gene involvement related to these conditions have progressed slowly because of the difficulty of transfecting HUVEC with high efficiency. Whereas several technical approaches have been described, they usually result in low levels of transfected cells or they require several steps or sophisticated instrumentation. We describe here a straightforward protocol of transfection of freshly isolated HUVEC that is based on the simple technique of electroporation. Efficiencies of gene transfection greater than 40% were routinely obtained by using a combination of optimized conditions of HUVEC isolation, composition of the electroporation medium and homogeneity of the plasmids. The protocol has been applied to the functional transient transfection of functional genes in HUVEC as illustrated in the case of the cDNA encoding GFP, protein kinase C (alpha and epsilon isotypes) and beta-galactosidase.

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-1β. Further investigation showed that Mip-1α and Mip-1β 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.

Background Human neutrophils are key players of innate immunity, and influence inflammatory and immune reactions through the production of numerous cytokines and chemokines. Despite major advances in our understanding of this important functional response of neutrophils, the short lifespan of these cells and their resistance to transfection have always been an obstacle to the detailed dissection of signaling pathways and effector responses that is often possible in other cell types. Results Here, we report that granulocytic differentiation of human PLB-985 cells with DMSO yields cells that are neutrophil-like with respect to surface markers, acquisition of responsiveness to physiological neutrophil stimuli (fMLP, LPS), cytokine expression and production profile, and transcription factor activation profile (NF-κB, C/EBP, AP-1, STAT). We also show that granulocytic PLB-985 cells can be reliably tranfected by nucleofection in a rapid and efficient manner. Indeed, we overexpressed several proteins and luciferase constructs into these cells. In particular, overexpression of a dominant negative IκB-α confirmed the central role of NF-κB in the production of cytokines by granulocytes. Moreover, the use of PLB-985 granulocytes in which the NADPH oxidase is inactive due to the targeted disruption of a key component (gp91phox) revealed that NF-κB activation and κB-dependent responses are independent of endogenous reactive oxygen intermediates in these cells. Antioxidant studies performed in primary human neutrophils support this conclusion. Conclusion Our results unveil a new facet of the NF-κB system of human granulocytes, and pave the way for deciphering signal transduction pathways and promoter activation in these cells.

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.

The ability of human neutrophils to ex- press a variety of genes encoding inflammatory mediators is well documented, and mounting evi- dence suggests that neutrophil-derived cytokines and chemokines contribute to the recruitment of discrete leukocyte populations at inflammatory sites. Despite this, our understanding of the signal- ing intermediates governing the generation of in- flammatory cytokines by neutrophils remains frag- mentary. 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 ad- dition, various NF-B inhibitors were found to pro- foundly impede the inducible gene expression and release of inflammatory cytokines in these cells. The MAPK inhibitors did not affect NF-B activa- tion; instead, the transcriptional effects of the p38 MAPK inhibitor appear to involve transcriptional factor IID. Conversely, the NF-B inhibitors failed to affect the activation of MAPKs. Finally, the MAPK inhibitors were found to prevent the activa- tion a key component of the translational machin- ery, S6 ribosomal protein, in keeping with their post-transcriptional impact on cytokine genera- tion. To our knowledge, this constitutes the first demonstration that in neutrophils, the inducible expression of proinflammatory cytokines by phys- iological stimuli largely reflects the ability of the latter to activate NF-B and selected MAPK path- ways. Our data also raise the possibility that NF-B or MAPK inhibitors could be useful in the treat- ment of inflammatory disorders in which neutro- phils predominate. J. Leukoc. Biol. 81: 000-000; 2007.

Responsiveness of human neutrophils to IL-10 was recently shown to be strictly dependent on the levels of IL-10R1 expression. Activation of signal transducer and activator of transcription 3 (STAT3) phosphorylation and induction of suppressor of cytokine signaling (SOCS)-3 protein by IL-10 are in fact negligible in circulating or freshly isolated ("time 0") neutrophils, but become readily measurable in neutrophils cultured for 4 h in the presence or absence of LPS. In this study, we show that modulation by IL-10 of LPS-induced TNF-alpha, CXCL8/IL-8 and IL-1 receptor antagonist (IL-1ra) mRNA accumulation in neutrophils already expressing a functional IL-10R and antigenic SOCS-3 (i.e. in "4-h-cultured" neutrophils) occurs with kinetics that are similar to those observed in "time 0" neutrophils, depends on de novo protein synthesis, but does not require SOCS-1, SOCS-3, heme oxygenase and Bcl-3 induction. By contrast, we show that IL-10 alone rapidly modulates the expression of TNF-alpha, CXCL8/IL-8 and IL-1ra mRNA, without any new protein synthesis requirement, if neutrophils have been previously exposed to LPS for at least 4 h. These findings suggest that LPS prepares neutrophils to optimally respond to IL-10 in terms of rapid gene modulation via mechanisms that, presumably, depend on specific LPS-induced protein(s).