Thornin Ear

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.

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)

Inflammatory Cytokine Expression Is Independent of the c-Jun N-Terminal Kinase/AP-1 Signaling Cascade in Human Neutrophils

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.

Constitutive Nuclear Expression of the IκB Kinase Complex and Its Activation in Human Neutrophils

A singular feature of human neutrophils is that they constitutively express substantial amounts of NF-κB/Rel proteins and IκB-α in the nucleus. In this study, we show that in these cells, IκB kinase α (IKKα), IKKβ, and IKKγ also partially localize to the nucleus, whereas IKK-related kinases (IKKε, TANK-binding kinase-1) are strictly cytoplasmic, and the NF-κB-inducing kinase is strictly nuclear. Following neutrophil activation, IKKβ and IKKγ become transiently phosphorylated in both the cytoplasm and nucleus, whereas IKKα transiently vanishes from both compartments in what appears to be an IKKβ-dependent process. These responses are paralleled by the degradation of IκB-α, and by the phosphorylation of RelA on serine 536, in both compartments. Although both proteins can be IKK substrates, inhibition of IKK prevented IκB-α phosphorylation, while that of RelA was mostly unaffected. Finally, we provide evidence that the nuclear IKK isoforms (α, β, γ) 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-κB/Rel proteins in primary neutrophils. More importantly, our findings unveil a hitherto unsuspected mode of activation for the IKK/IκB signaling cascade within the cell nucleus.

Molecular mechanisms underlying the synergistic induction of CXCL10 by LPS and IFN‐γ in human neutrophils

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‐γ, as well as by neutrophils, though the latter require a costimulation with IFN‐γ and LPS. In this study, we investigated the molecular mechanism(s) whereby IFN‐γ 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‐γ combination in neutrophils, consistent with previous studies showing that increased CXCL10 gene expression does not reflect enhanced mRNA stability. The IFN‐γ‐induced STAT1 activation and the lipopolysaccharide (LPS)‐induced NF‐κB activation were not enhanced if neutrophils were exposed to both stimuli, whereas both transcription factors were activated by IFN‐γ or LPS in monocytes. Finally, pharmacological inhibitors of NF‐κB demonstrated its role in the induction of CXCL10 expression by LPS plus IFN‐γ in neutrophils, and by LPS or IFN‐γ in monocytes. Together, these results suggest that in neutrophils, the synergy observed between LPS and IFN‐γ toward CXCL10 gene expression likely reflects the cooperative induction of the NF‐κB and STAT1 transcription factors by LPS and IFN‐γ, respectively.

Constitutive Association of TGF-β–Activated Kinase 1 with the IκB 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 IκB kinase (IKK)/IκB/NF-κB signaling cascade. A singular feature of neutrophils is that they express several components of this pathway (namely, NF-κB/Rel proteins and IκB-α) 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-β–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-α 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 IKKα/β, IκB-α, and RelA, and also impaired IκB-α degradation and NF-κB 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-α was reversed by TAK1 inhibition. By contrast, IKKγ 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.

Cytokine generation, promoter activation, and oxidant-independent NF-κB activation in a transfectable human neutrophilic cellular model

BackgroundHuman 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.ResultsHere, 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.ConclusionOur 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.

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‐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.

High efficiency transient transfection of genes in human umbilical vein endothelial cells by electroporation

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.