Driss El Kebir

Resolvin E1 promotes phagocytosis-induced neutrophil apoptosis and accelerates resolution of pulmonary inflammation

Inappropriate neutrophil activation contributes to the pathogenesis of acute lung injury (ALI). Apoptosis is essential for removal of neutrophils from inflamed tissues and timely resolution of inflammation. Resolvin E1 (RvE1) is an endogenous lipid mediator derived from the ω-3 polyunsaturated fatty acid eicosapentaenoic acid that displays proresolving actions. Because the balance of prosurvival and proapoptosis signals determines the fate of neutrophils, we investigated the impact of RvE1 on neutrophil apoptosis and the outcome of neutrophil-mediated pulmonary inflammation in mice. Culture of human neutrophils with RvE1 accelerated apoptosis evoked by phagocytosis of opsonized Escherichia coli or yeast. RvE1 through the leukotriene B4 receptor BLT1 enhanced NADPH oxidase-derived reactive oxygen species generation and subsequent activation of caspase-8 and caspase-3. RvE1 also attenuated ERK and Akt-mediated apoptosis-suppressing signals from myeloperoxidase, serum amyloid A, and bacterial DNA, shifting the balance of pro- and anti-survival signals toward apoptosis via induction of mitochondrial dysfunction. In mice, RvE1 treatment enhanced the resolution of established neutrophil-mediated pulmonary injury evoked by intratracheal instillation or i.p. administration of live E. coli or intratracheal instillation of carrageenan plus myeloperoxidase via facilitating neutrophil apoptosis and their removal by macrophages. The actions of RvE1 were prevented by the pan-caspase inhibitor zVAD-fmk. These results identify a mechanism, promotion of phagocytosis-induced neutrophil apoptosis and mitigation of potent anti-apoptosis signals, by which RvE1 could enhance resolution of acute lung inflammation.

15-epi-lipoxin A4 inhibits myeloperoxidase signaling and enhances resolution of acute lung injury.

RATIONALE Apoptosis is essential for removal of neutrophils from inflamed tissues and efficient resolution of inflammation. Myeloperoxidase (MPO), abundantly expressed in neutrophils, not only generates cytotoxic oxidants but also signals through the beta(2) integrin Mac-1 to rescue neutrophils from constitutive apoptosis, thereby prolonging inflammation. OBJECTIVES Because aspirin-triggered 15-epi-lipoxin A(4) (15-epi-LXA(4)) modulates Mac-1 expression, we investigated the impact of 15-epi-LXA(4) on MPO suppression of neutrophil apoptosis and MPO-mediated neutrophil-dependent acute lung injury. METHODS Human neutrophils were cultured with MPO with or without 15-epi-LXA(4) to investigate development of apoptosis. Acute lung injury was produced by intratracheal injection of carrageenan plus MPO or intraperitoneal injection of live Escherichia coli in mice, and the animals were treated with 15-epi-LXA(4) at the peak of inflammation. MEASUREMENTS AND MAIN RESULTS 15-Epi-LXA(4) through down-regulation of Mac-1 expression promoted apoptosis of human neutrophils by attenuating MPO-induced activation of extracellular signal-regulated kinase and Akt-mediated phosphorylation of Bad and by reducing expression of the antiapoptotic protein Mcl-1, thereby aggravating mitochondrial dysfunction. The proapoptotic effect of 15-epi-LXA(4) was dominant over MPO-mediated effects even when it was added at 4 hours post MPO. In mice, treatment with 15-epi-LXA(4) accelerated the resolution of established carrageenan plus MPO-evoked as well as E. coli-induced neutrophil-dependent pulmonary inflammation through redirecting neutrophils to caspase-mediated cell death and facilitating their removal by macrophages. CONCLUSIONS These results demonstrate that aspirin-triggered 15-epi-LXA(4) enhances resolution of inflammation by overriding the powerful antiapoptosis signal from MPO, thereby demonstrating a hitherto unrecognized mechanism by which aspirin promotes resolution of inflammation.

Myeloperoxidase Delays Neutrophil Apoptosis Through CD11b/CD18 Integrins and Prolongs Inflammation

Polymorphonuclear neutrophil granulocytes have a central role in innate immunity and their programmed cell death and removal are critical for efficient resolution of acute inflammation. Myeloperoxidase (MPO), a heme protein abundantly expressed in neutrophils, is generally associated with killing of bacteria and oxidative tissue injury. Because MPO also binds to neutrophils, we investigated whether MPO could affect the lifespan of neutrophils. Here, we report that MPO independent of its catalytic activity through signaling via the adhesion molecule CD11b/CD18 rescued human neutrophils from constitutive apoptosis and prolonged their life span. MPO evoked a transient concurrent activation of extracellular signal-regulated kinase and Akt, leading to phosphorylation of Bad at both Ser112 and Ser136, prevention of mitochondrial dysfunction, and subsequent activation of caspase-3. Consistently, pharmacological inhibition of extracellular signal-regulated kinase, Akt, or caspase-3 reversed the antiapoptosis action of MPO. Acute increases in plasma MPO delayed murine neutrophil apoptosis assayed ex vivo. In a mouse model of self-resolving inflammation, MPO also prolonged the duration of carrageenan-induced acute lung injury, as evidenced by enhanced alveolar permeability and accumulation of neutrophils parallel with suppression of neutrophil apoptosis. Our results indicate that MPO functions as a survival signal for neutrophils and thereby contribute to prolongation of inflammation.

Aspirin-Triggered Lipoxins Override the Apoptosis-Delaying Action of Serum Amyloid A in Human Neutrophils: A Novel Mechanism for Resolution of Inflammation

Elevated plasma levels of the acute-phase reactant serum amyloid A (SAA) have been used as a marker and predictor of inflammatory diseases. SAA regulates leukocyte activation; however, it is not known whether it also modulates neutrophil apoptosis, which is critical to the optimal expression and resolution of inflammation. Culture of human neutrophils with SAA (0.1–20 μg/ml) markedly prolonged neutrophil longevity by delaying constitutive apoptosis. SAA evoked concurrent activation of the ERK and PI3K/Akt signaling pathways, leading to phosphorylation of BAD at Ser112 and Ser136, respectively, and to prevention of collapse of mitochondrial transmembrane potential, cytochrome c release, and caspase-3 activation. These actions were abrogated by pharmacological inhibition of the formyl peptide receptor, ERK or PI3K. Furthermore, aspirin-triggered 15-epi-lipoxin A4 (15-epi-LXA4) and its stable analog 15-epi-16-p-fluorophenoxy-LXA4, which binds to the same receptor as SAA, effectively overrode the antiapoptosis signal from SAA even when neutrophils were treated with 15-epi-LXA4 at either 1 or 4 h postculture with SAA. 15-Epi-LXA4 itself did not affect neutrophil survival and apoptosis. Our results indicate that SAA at clinically relevant concentrations promotes neutrophil survival by suppressing the apoptotic machinery, an effect that can be opposed by 15-epi-LXA4. The opposing actions of SAA and aspirin-triggered 15-epi-LXA4 may contribute to the local regulation of exacerbation and resolution of inflammation, respectively.

Activation of TLR-9 induces IL-8 secretion through peroxynitrite signaling in human neutrophils.

Bacterial DNA containing unmethylated CpG motifs is emerging as an important regulator of functions of human neutrophil granulocytes (polymorphonuclear leukocytes (PMN)). These motifs are recognized by TLR-9. Recent studies indicate that peroxynitrite (ONOO−) may function as an intracellular signal for the production of IL-8, one of the key regulators of leukocyte trafficking in inflammation. In this study we investigated whether bacterial DNA (CpG-DNA) could induce ONOO− signaling in human PMN. Human whole blood, isolated PMN (purity, >95%), and high purity (>99%) PMN respond to CpG-DNA, but not to calf thymus DNA, with secretion of IL-8 and, to a lesser extent, IL-6 and TNF. Methylation of cytosines in CpG-DNA resulted in a complete loss of activity. The endosomal acidification inhibitors, bafilomycin A and chloroquine, inhibited CpG-DNA-induced cytokine release from PMN. CpG-DNA-induced IL-8 mRNA expression and release was also blocked by the NO synthase inhibitor Nω-nitro-l-arginine methyl ester. CpG-DNA evoked concomitant increases in intracellular superoxide and NO levels, leading to enhanced ONOO− formation and, consequently, nuclear accumulation of c-Fos and NF-κB. Pharmacological inhibition of NF-κB activation attenuated ∼75% of CpG-DNA-evoked IL-8 release. These results identify ONOO−-dependent activation of NF-κB and c-Fos as an important mechanism that mediates PMN responses, including IL-8 gene expression and release, to bacterial DNA and unmethylated CpG motifs in particular. Enhanced ONOO− formation represents a mechanism by which bacterial DNA may contribute to prolongation and amplification of the inflammatory response.

Role of Neutrophil Apoptosis in the Resolution of Inflammation

Neutrophil granulocytes play a central role in host defense to infection and tissue injury. Their timely removal is essential for resolution of inflammation. Increasing evidence identified neutrophil apoptosis as an important control point in the development and resolution of inflammation. Delayed apoptosis and/or impaired clearance of neutrophils aggravate and prolong tissue injury. This review will focus on outside-in signals that provide survival cues for neutrophils, the hierarchy of pro- and antiapoptotic signals, and molecular targets in the antiapoptotic signaling network that can be exploited by endogenously produced bioactive lipids, such as lipoxins or pharmacological inhibitors, including cyclin-dependent kinase inhibitors, to redirect neutrophils to apoptosis in vivo, thus promoting resolution of inflammation.

Neutrophil apoptosis: a target for enhancing the resolution of inflammation.

Neutrophils are essential for host defense and their programmed cell death and removal are critical for the optimal expression as well as for efficient resolution of inflammation. Delayed neutrophil apoptosis or impaired clearance of apoptotic neutrophils by macrophages contributes to the progression of chronic inflammation. Under most conditions, neutrophils are exposed to multiple factors and their fate would ultimately depend on the balance between pro‐survival and pro‐apoptotic signals. Life or death decisions are tightly controlled by a complex network of intracellular signaling pathways. Accumulating data indicate that receptors, such as the formyl peptide receptor 2/lipoxin receptor or β2‐integrins can generate contrasting cues in neutrophils in a ligand‐specific manner and suggest a hierarchy among these signals. In this article, we review recent advances on how pro‐apoptosis and pro‐survival signals interact to determine the fate of neutrophils and the inflammatory response, and highlight novel pharmacological strategies that could be used to enhance the resolution of inflammation by redirecting neutrophils to apoptosis. J. Cell. Biochem. 108: 1039–1046, 2009.

A redox switch in C-reactive protein modulates activation of endothelial cells

C‐reactive protein (CRP) has been implicated in the regulation of inflammation underlying coronary artery disease; however, little is known about the molecular mechanisms responsible for the expression of its pro‐ or anti‐inflammatory activities. Here, we have identified the intrasubunit disulfide bond as a conserved switch that controls the structure and functions of CRP. Conformational rearrangement in human pentameric CRP to monomeric CRP (mCRP) is the prerequisite for this switch to be activated by reducing agents, including thioredoxin. Immunohistochemical analysis revealed 36‐79% colocalization of thioredoxin and mCRP in human advanced coronary atherosclerotic lesions. Nonreduced mCRP was largely inert in activating human coronary artery endothelial cells (HCAECs), whereas reduced or cysteine‐mutated mCRP evoked marked release of IL‐8 and monocyte chemoattractant protein‐1 from HCAECs, with ~50% increase at a concentration of 1 μg/ml. Reduced mCRP was ~4 to 40‐fold more potent than mCRP in up‐regulating adhesion molecule expression, promoting U937 monocyte adhesion to HCAECs, and inducing cytokine release from rabbit arteries ex vivo and in mice. These actions were primarily due to unlocking the lipid raft interaction motif. Therefore, expression of proinflammatory properties of CRP on endothelial cells requires sequential conformational changes, i.e., loss of pentameric symmetry followed by reduction of the intrasubunit disulfide bond.—Wang M‐Y., Ji, S‐R., Bai, C‐J., El Kebir, D., Li, H‐Y., Shi, J‐M., Zhu, W., Costantino, S., Zhou, H‐H., Potempa, L. A., Zhao, J., Filep, J. G., Wu, Y. A redox switch in C‐reactive protein modulates activation of endothelial cells. FASEB J. 25, 3186‐3196 (2011). www.fasebj.org

Inhaled nitric oxide in 2003: a review of its mechanisms of action

PurposeTo review the pulmonary and systemic effects of endogenous nitric oxide and inhaled nitric oxide administered to patients.SourceA systematic search for experimental data, human case reports, and randomized clinical trials since 1980, the year of discovery of endothelium-derived relaxing factor.Principal findingsNitric oxide has pulmonary and systemic effects. Inhaled nitric oxide not only causes selective pulmonary vasodilation but also results in pulmonary vasoconstriction of the vessels perfusing non-ventilated alveolae. The systemic effects of inhaled nitric oxide, which include modulation of the distribution of systemic blood flow, increase in renal output, interaction with coagulation, fibrinolysis and platelet functions, alteration of the inflammatory response, are described and the mechanisms of nitric oxide transport are explained. The possible toxicity of inhaled nitric oxide is also discussed.ConclusionThe multiple effects of inhaled nitric oxide support its role as a pulmonary and extra-pulmonary medication.RésuméObjectifRevoir les effets pulmonaires et généraux de l’oxyde nitrique (NO) endogène et du NO inhalé, administré aux patients.SourceUne recherche systématique de données expérimentales, d’études de cas humains et d’essais cliniques randomisés réalisés depuis 1980, année de la découverte de ce facteur relaxant d’origine endothéliale, dénomination qui lui fût attribuée à l;époque.Constatations principalesLe monoxyde d’azote a des effets pulmonaires et généraux. Le monoxyde d’azote inhalé ne cause pas seulement une vasodilatation pulmonaire sélective, mais il provoque aussi une vasoconstriction pulmonaire des vaisseaux perfusant les alvéoles non ventilées. Les effets généraux de monoxyde d’azote, y compris la modulation de la distribution du débit sanguin, l’augmentation du débit rénal, l’interaction avec la coagulation, la capacité fonctionnelle de la fibrinolyse et des plaquettes, la modification de la réponse inflammatoire, sont décrits et les mécanismes du transport de monoxyde d’azote sont expliqués. On discute également de la toxicité de monoxyde d’azote inhalé.ConclusionLes multiples effets de l’oxyde nitrique inhalé expliquent l’intérêt qu’il présente comme médication pulmonaire et extrapulmonaire.

Bacterial DNA Activates Endothelial Cells and Promotes Neutrophil Adherence through TLR9 Signaling

TLR9 detects bacterial DNA (CpG DNA) and elicits both innate and adoptive immunity. Recent evidence indicates that TLR9 is expressed in more diverse cell types than initially thought. In this study, we report that HUVECs constitutively express TLR9 and selectively recognize unmethylated CpG motifs in bacterial DNA and synthetic immune stimulatory CpG oligodeoxynucleotides. HUVECs respond to CpG DNA with rapid phosphorylation of IκB-α and NF-κB-mediated gene transcription and surface expression of the adhesion molecules ICAM-1 and E-selectin independent of MAPK signaling. The telomere-derived TLR9 inhibitory oligonucleotide 5′-TTT AGG GTT AGG GTT AGG G-3′, agents that block endosomal acidification such as chloroquine and bafilomycin A, and NF-κB inhibitors abrogated CpG DNA-induced signaling. HUVEC activation by CpG DNA led to markedly enhanced neutrophil adhesion under nonstatic conditions that was further enhanced when neutrophils were stimulated with CpG DNA. The adhesive interactions were blocked by Abs against CD18 and, to a lesser degree, by anti-E-selectin and anti-L-selectin Abs. Our findings demonstrate that bacterial DNA promotes β2 integrin and E-selectin-mediated HUVEC-neutrophil adherence, and indicate the ability of CpG DNA to initiate and/or maintain the inflammatory response.