Levente József

Conformational Rearrangement in C-Reactive Protein Is Required for Proinflammatory Actions on Human Endothelial Cells

Background—C-reactive protein (CRP) has been suggested to actively amplify the inflammatory response underlying coronary heart diseases by directly activating endothelial cells. In this study, we investigated whether loss of the cyclic pentameric structure of CRP, resulting in formation of modified or monomeric CRP (mCRP), is a prerequisite for endothelial cell activation. Methods and Results—We examined the impact of native CRP and mCRP on the production of monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8), key regulators of leukocyte recruitment, and on the expression of intercellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular adhesion molecule-1 (VCAM-1) in human cultured coronary artery endothelial cells (HCAECs). Incubation with mCRP for 4 hours increased MCP-1 and IL-8 secretion and mRNA levels and expression of ICAM-1, E-selectin, and VCAM-1 protein and mRNA. Significant induction occurred at 1 to 5 μg/mL, reached a maximum at 30 μg/mL, and did not require the presence of serum. Native CRP was without detectable effects at 4 hours, whereas it enhanced cytokine release after a 24-hour incubation. An anti-FcγRIII (CD16) but not an anti-FcγRII (CD32) antibody produced a 14% to 32% reduction of the mCRP effects (P <0.05). mCRP but not CRP evoked phosphorylation of p38 mitogen-activated protein kinase, and inhibition of this kinase with SB 203580 reversed the effects of mCRP. Furthermore, culture of HCAECs in the presence of SB203580 markedly decreased mCRP-stimulated E-selectin and ICAM-1–dependent adhesion of neutrophils to HCAECs (P <0.001). Conclusions—Loss of pentameric symmetry in CRP, resulting in formation of mCRP, promotes a proinflammatory HCAEC phenotype through a p38 MAPK–dependent mechanism.

Lipoxin A4 and aspirin-triggered 15-epi-lipoxin A4 inhibit peroxynitrite formation, NF-κB and AP-1 activation, and IL-8 gene expression in human leukocytes

Lipoxin A4 (LXA4) and aspirin-triggered 15-epi-LXA4 (ATL) are emerging as endogenous braking signals for neutrophil-mediated tissue injury. Recent studies indicate that peroxynitrite (ONOO−) may function as an intracellular signal for the production of IL-8, a potent proinflammatory cytokine in human leukocytes. In this study, we evaluated the impact of the metabolically stable analogues of LXA4/ATL on lipopolysaccharide (LPS)-induced ONOO− formation and ONOO−-mediated IL-8 gene expression in human leukocytes. At nanomolar concentrations, LXA4 analogues markedly reduced LPS-stimulated superoxide formation, evoked increases in intracellular diamino-fluorescein fluorescence (an indicator of NO formation), and consequently reduced ONOO− formation in isolated neutrophils, as well as in neutrophils, monocytes, and lymphocytes, in whole blood. LXA4/ATL analogues attenuated nuclear accumulation of activator protein-1 and nuclear factor-κB in both polymorphonuclear and mononuclear leukocytes and inhibited IL-8 mRNA expression and IL-8 release by 50–65% in response to LPS. The LXA4 inhibitory responses were concentration dependent and were not shared by 15-deoxy-LXA4. None of the LXA4 analogues studied affected neutrophil survival, nor reversed the apoptosis delaying action of LPS in neutrophils. In addition, LXA4 analogues had no significant effect on exogenous ONOO−-induced IL-8 gene and protein expression. These findings suggest that by attenuating ONOO− formation, LXA4 and ATL can oppose ONOO− signaling in leukocytes and provide a rationale for using stable synthetic analogues as antiinflammatory compounds in vivo.

Loss of Pentameric Symmetry of C-reactive Protein Is Associated with Delayed Apoptosis of Human Neutrophils

Human neutrophil granulocytes die rapidly, and their survival is contingent upon rescue from programmed cell death by signals from the environment. Here we report that a novel signal for delaying neutrophil apoptosis is the classic acute phase reactant, C-reactive protein (CRP). However, this anti-apoptotic activity is expressed only when the cyclic pentameric structure of CRP is lost, resulting in formation of modified or monomeric CRP (mCRP), which may be formed in inflamed tissues. By contrast, native pentameric CRP and CRP peptides 77–82, 174–185, and 201–206 failed to affect neutrophil apoptosis. The apoptosis delaying action of mCRP was markedly attenuated by an antibody against the low affinity IgG immune complex receptor (CD16) but not by an anti-CD32 antibody. mCRP evoked a transient concurrent activation of the extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase/Akt signaling pathways, leading to inhibition of caspase-3 and consequently to delaying apoptosis. Consistently, pharmacological inhibition of either ERK or Akt reversed the anti-apoptotic action of mCRP; however, they did not produce additive inhibition. Thus, mCRP, but not pentameric CRP or peptides derived from CRP, promotes neutrophil survival and may therefore contribute to amplification of the inflammatory response.

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.

Absence of Akt1 Reduces Vascular Smooth Muscle Cell Migration and Survival and Induces Features of Plaque Vulnerability and Cardiac Dysfunction During Atherosclerosis

Objective—Deletion of Akt1 leads to severe atherosclerosis and occlusive coronary artery disease. Vascular smooth muscle cells (VSMCs) are an important component of atherosclerotic plaques, responsible for promoting plaque stability in advanced lesions. Fibrous caps of unstable plaques contain less collagen and ECM components and fewer VSMCs than caps from stable lesions. Here, we investigated the role of Akt1 in VSMC proliferation, migration, and oxidative stress–induced apoptosis. In addition, we also characterized the atherosclerotic plaque morphology and cardiac function in an atherosclerosis-prone mouse model deficient in Akt1. Methods and Results—Absence of Akt1 reduces VSMC proliferation and migration. Mechanistically, the proliferation and migratory phenotype found in Akt1-null VSMCs was linked to reduced Rac-1 activity and MMP-2 secretion. Serum starvation and stress-induced apoptosis was enhanced in Akt1 null VSMCs as determined by flow cytometry using Annexin V/PI staining. Immunohistochemical analysis of atherosclerotic plaques from Akt1−/−ApoE−/− mice showed a dramatic increase in plaque vulnerability characteristics such as enlarged necrotic core and reduced fibrous cap and collagen content. Finally, we show evidence of myocardial infarcts and cardiac dysfunction in Akt1−/−ApoE−/− mice analyzed by immunohistochemistry and echocardiography, respectively. Conclusion—Akt1 is essential for VSMC proliferation, migration, and protection against oxidative stress–induced apoptosis. Absence of Akt1 induces features of plaque vulnerability and cardiac dysfunction in a mouse model of atherosclerosis.

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.

Loss of Pentameric Symmetry in C-Reactive Protein Induces Interleukin-8 Secretion Through Peroxynitrite Signaling in Human Neutrophils

Plasma levels of C-reactive protein (CRP), nitrotyrosine, and interleukin-8 (IL-8) are known predictors of acute cardiovascular events. Peroxynitrite (ONOO−) may function as an intracellular signal for the production of IL-8; however, it is not known whether CRP regulates these events. Emerging evidence suggests that some bioactivities of CRP are expressed only when the pentameric structure of CRP is lost, resulting in formation of monomeric or modified CRP (mCRP). We studied the impact of human native CRP and bioengineered mCRP that cannot rearrange into the pentameric structure on ONOO− formation and ONOO−-mediated IL-8 gene expression in human leukocytes. Incubation of human whole blood or isolated neutrophils with mCRP (0.1 to 100 &mgr;g/mL) for 4 hours increased IL-8 gene expression and secretion that was blocked ≈70% by the NO synthase inhibitor N&ohgr;-nitro-l-arginine methyl ester (L-NAME). In neutrophils, mCRP simultaneously increased superoxide production and endothelial nitric oxide synthase-mediated NO formation, leading to enhanced ONOO− formation, and consequently activation of nuclear factor-&kgr;B and activator protein-1. Native CRP had no detectable effect at 4 hours, whereas it enhanced IL-8 release after a 24-hour incubation that was blocked by L-NAME. An anti-CD16 antibody, but not an anti-CD32 antibody, produced 60% to 70% reductions in mCRP-stimulated NO formation and IL-8 release (both P<0.05). These results suggest that loss of the pentameric symmetry in CRP, resulting in formation of mCRP, leads to IL-8 release from human neutrophils via peroxynitrite-mediated activation of nuclear factor-&kgr;B and activator protein-1.

CpG motifs in bacterial DNA delay apoptosis of neutrophil granulocytes

Human neutrophil granulocytes die rapidly, and their survival is contingent upon rescue from programmed cell death by signals from the environment. We now show that a novel signal for delaying neutrophil apoptosis is unmethylated CpG motifs prevalent in bacterial DNA (CpG‐DNA). Human neutrophils express toll‐like receptor 9 that recognizes these motifs. CpG‐DNA, but not mammalian DNA or methylated bacterial DNA, markedly enhanced neutrophil viability by delaying spontaneous apoptosis. Endosomal maturation of CpG‐DNA is prerequisite for these actions and was coupled to concurrent activation of the extracellular signal‐regulated kinase (ERK) and phosphatidylinositol 3‐kinase/Akt signaling pathways, leading to phosphorylation of BAD at Ser112 and Ser136, respectively, and to prevention of decreases in mitochondrial transmembrane potential, cytochrome c release and caspase‐3 activation. Consistently, pharmacological inhibition of either ERK or phosphatidylinositol 3‐kinase partially reversed these actions of CpG‐DNA; however, they did not produce additive inhibition. Furthermore, intravenous injection of CpG‐DNA (200 μg/kg) into rats evoked slight decreases in blood pressure and induced a modest leukocytosis, whereas it effectively suppressed neutrophil apoptosis as assayed ex vivo. Our results indicate that unmethylated CpG motifs in bacterial DNA promote neutrophil survival by suppressing the apoptotic machinery and may therefore contribute to prolongation and amplification of inflammation.