Tarek Khreiss

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.

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.

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.

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.

Opposing Effects of C-Reactive Protein Isoforms on Shear-Induced Neutrophil-Platelet Adhesion and Neutrophil Aggregation in Whole Blood

Background—Plasma C-reactive protein (CRP) level is a powerful predictor of cardiovascular events. However, it is not known whether CRP could affect neutrophil-platelet adhesion and neutrophil aggregation, key events in acute coronary syndromes. Emerging in vitro evidence suggests that some bioactivities of CRP are expressed on loss of the pentameric symmetry, resulting in formation of modified or monomeric CRP (mCRP). Methods and Results—We studied the impact of human native CRP and bioengineered mCRP that cannot rearrange into the pentameric structure on the kinetics of neutrophil-platelet adhesion and neutrophil aggregation in whole blood subjected to shear (≈100 s−1) using real-time flow cytometry. Shear resulted in upregulation of platelet P-selectin expression, leading to platelet capture of neutrophils and subsequent neutrophil aggregation, which was dependent on P-selectin, L-selectin, and CD18. Native CRP at clinically relevant concentrations markedly attenuated these changes. The residual amount of neutrophil adhesion was blocked with anti-CD18 or anti-CD11b antibody. By contrast, mCRP concentration-dependently enhanced shear-induced platelet P-selectin expression and increased the rate and extent of formation of both neutrophil-platelet and neutrophil-neutrophil aggregates. Complete abrogation of platelet-neutrophil adhesion and neutrophil aggregation required both anti–P-selectin and anti-CD18 antibodies but not anti–L-selectin antibody. The CRP action was markedly inhibited by an anti-CD32 antibody, whereas the mCRP effects were significantly attenuated by an anti-CD16 antibody. Conclusions—These results indicate that native CRP inhibits platelet activation and prevents platelet capture of neutrophils, whereas mCRP displays potent prothrombotic activities under low levels of shear. Thus, mCRP rather than native CRP may precipitate acute coronary syndromes.

Extracellular signal-regulated kinase plays an essential role in endothelin-1-induced homotypic adhesion of human neutrophil granulocytes.

Endothelin‐1 (ET‐1) stimulates integrin‐dependent adhesion of neutrophil granulocytes to endothelial cells, one of the early key events in acute inflammation. However, the signalling pathway(s) of ET‐1‐stimulated neutrophil adhesive responses has not been elucidated. Previous studies indicated that extracellular signal‐regulated kinase (ERK) activation could mediate rapid responses of neutrophil granulocytes to various stimuli. In this study, we investigated the role of ERK signalling in human neutrophil granulocytes challenged with ET‐1. ET‐1 rapidly down‐regulated the expression of L‐selectin and up‐regulated the expression of CD11b/CD18 on the neutrophil surface. Concomitantly, ET‐1 induced homotypic adhesion (aggregation) of neutrophils, that was blocked by a monoclonal antibody to CD18. ET‐1, through ETA receptors, evoked activation of Ras and subsequent phosphorylation of Raf‐1, mitogen‐activated protein kinase kinase (MAPK/ERK kinase) and ERK 1/2. ERK activation by ET‐1 was rapid, concordant with the kinetics of ET‐1‐stimulated neutrophil aggregation. Neutrophil responses to ET‐1 were markedly attenuated by the MAPK/ERK kinase inhibitor PD98059, whereas inhibitors of p38 MAPK, tyrosine kinases and phosphatidylinositol 3‐kinase had no detectable effects. We have observed a tight correlation between neutrophil ERK activation and homotypic adhesion. These data indicate an essential role for ERK in mediating ET‐1‐stimulated adhesive responses of human neutrophil granulocytes.