Christine Zouki

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.

Prevention of In vitro neutrophil adhesion to endothelial cells through shedding of L-selectin by C-reactive protein and peptides derived from C-reactive protein.

C-Reactive protein (CRP), the classic acute-phase reactant in humans, diminishes accumulation of neutrophils at inflammatory sites. To evaluate the underlying mechanisms, we have studied whether CRP and peptides derived from CRP could affect the first step of neutrophil extravasation, the L-selectin-mediated interaction of neutrophils with endothelial cells. CRP markedly attenuated attachment of human neutrophils to cultured LPS-activated human coronary artery and pulmonary microvascular endothelial cells with apparent IC50 values of 20 and 22 microg/ml, respectively. At similar concentrations, CRP rapidly downregulated the expression of L-selectin on the neutrophil surface, whereas it failed to affect expression of CD11b and CD45 or to induce granule enzyme release. The loss of L-selectin was due to cleavage and shedding of the molecule from the cell surface, as quantitated by the soluble form of L-selectin in cell-free supernatants. The effects of CRP could be prevented by an anti-CRP antiserum and by KD-IX-73-4, which inhibits shedding of L-selectin. Inhibition of adhesion with CRP was additive with function-blocking anti-E-selectin and anti-CD18 antibodies, but was not additive with anti-L-selectin antibody. Neutrophil attachment and L-selectin expression were also diminished by CRP peptides 174-185 and 201-206, but not peptide 77-82, albeit these peptides showed a weaker inhibitory effect than the parent protein. These studies indicate a specific activation-independent action of CRP and CRP peptides 174-185 and 201-206 on expression of L-selectin, and suggest that by attenuating neutrophil adhesion to the endothelium and consequently neutrophil traffic into tissues, native CRP and peptides 174-185 and 201-206 may be major mechanisms to attenuate or limit the inflammatory response.

Endothelin-1 enhances neutrophil adhesion to human coronary artery endothelial cells: role of ETA receptors and platelet-activating factor

The potent coronary vasoconstrictor, endothelin‐1 (ET‐1) may also regulate neutrophil traffic into tissues. The aim of the present study was to characterize the endothelin receptors responsible and to investigate the underlying mechanisms. ET‐1 (1u2003nM–1u2003μM) markedly enhanced attachment of human neutrophils to lipopolysaccharide‐, and to a lesser extent, to ET‐1‐activated human coronary artery endothelial cells (HCAEC). This can partially be blocked by monoclonal antibodies against E‐selectin, L‐selectin or CD18, whereas combination of the three antibodies inhibited adhesion by ∼83%. Increases in neutrophil adhesion evoked by ET‐1 were also blocked by the platelet‐activating factor (PAF) antagonists, BNu200352021 (50u2003μM) and WEBu2003286 (10u2003μM). ET‐1 downregulated the expression of L‐selectin and upregulated expression of CD11b/CD18 and CD45 on the neutrophil surface and induced gelatinase release with EC50 values of ∼2u2003nM. These actions of ET‐1 were almost completely prevented by the ETA receptor antagonist FRu2003139317 (1u2003μM) and the ETA/ETB receptor antagonist bosentan (10u2003μM), whereas the ETB receptor antagonist BQu2003788 (1u2003μM) had no effect. ET‐1 slightly increased the expression of E‐selectin and ICAM‐1 on HCAEC, that was prevented by BQu2003788, but not by FRu2003139317. Receptor binding studies indicated the presence of ETB receptors (KD: 40u2003pM) on phosphoramidon‐treated HCAEC and the predominant expression of ETA receptors (KD: 38u2003pM) on neutrophils. These results indicate that promotion by ET‐1 of neutrophil adhesion to HCAEC is predominantly mediated through activation of ETA receptors on neutrophils and subsequent generation of PAF.

Matrix metalloproteinases regulate neutrophil-endothelial cell adhesion through generation of endothelin-1[1–32]

We recently reported that matrix metal‐loproteinase 2 (MMP‐2, gelatinase A) cleaves big endo‐thelin 1 (ET‐1), yielding the vasoactive peptide ET‐1[1–32]. We tested whether ET‐1[1–32] could affect the adhesion of human neutrophils to coronary artery endothelial cells (HCAEC). ET‐1[1–32] rapidly down‐regulated the expression of L‐selectin and up‐regulated expression of CD11b/CD18 on the neutrophil surface, with EC50 values of 1–3 nM. These actions of ET‐1[1–32] were mediated via ETA receptors and did not require conversion of ET‐1[1–32] into ET‐1 by neutrophil proteases, as revealed by liquid chromatography and mass spectroscopy. Moreover, ET‐1[1–32] evoked release of neutrophil gelatinase B, which cleaved big ET‐1 to yield ET‐1[1–32], thus revealing a positive feedback loop for ET‐1[1–32] generation. Up‐regula‐tion of CD11b/CD18 expression and gelatinase release was tightly associated with activation of extracellular signal‐regulated kinase (Erk). Stimulation of Erk activity was due to activation of Ras, Raf‐1, and MEK (MAPK kinase). ET‐1[1–32] also produced slight increases in the expression of ICAM‐1 and E‐selectin on HCAEC, and markedly enhanced ß2 integrin‐dependent adhesion of neutrophils to activated HCAEC. These results are the first indication that gelatinolytic MMPs via cleavage of big ET‐1 to yield ET‐1[1–32] activate neu‐trophils and promote leukocyte‐endothelial cell adhesion and, consequently, neutrophil trafficking into inflamed tissues.—Fernandez‐Patron, C., Zouki, C., Whittal, R., Chan, J. S. D., Davidge, S. T., Filep, J. G. Matrix metalloproteinases regulate neutrophil‐endothelial cell adhesion through generation of endothelin‐ 1[1–32]. FASEB J. 15, 2230–2240 (2001)

Peroxynitrite induces integrin-dependent adhesion of human neutrophils to endothelial cells via activation of the Raf-1/MEK/Erk pathway

Accumulating evidence suggests that enhanced peroxynitrite (ONOO‐) formation occurs during inflammation. We have studied the impact and the mechanisms of ONOO‐ action on expression of adhesion molecules on human neutrophils and coronary artery endothelial cells (HCAEC) and binding of neutrophils to HCAEC. Addition of ONOO‐ (0.1 to 200 µΜ) to isolated neutrophils resulted in a concentration‐dependent down‐regulation of L‐selectin expression, and up‐regulation of CD11b/CD18 expression. ONOO‐ stimulation of Erk activity was accompanied by activation of Ras, Raf‐1 and MEK (mitogen‐activated protein kinase kinase), and was sensitive to the MEK inhibitor PD 98059. We have observed a tight association between Erk activation and changes in CD11b/CD18 expression. ONOO‐ also evoked activation of neutrophil p38 MAPK. Neither ONOO‐‐induced up‐regulation of CD11b/CD18 expression nor Erk activation was affected by SB 203580, a selective inhibitor of p38 MAPK. ONOO‐ by itself had little effect on expression of ICAM‐1 and E‐selectin on HCAEC, whereas it markedly enhanced attachment of neutrophils to lipopolysaccharide‐activated HCAEC only when it was added together with neutrophils. Increases in neutrophil adhesion evoked by ONOO‐ were blocked by an anti‐CD18 monoclonal antibody. These data suggest that ONOO‐ activates Erk in neutrophils via the Ras/Raf‐1/MEK signal transduction pathway, leading to up‐regulation of surface expression of CD11b/CD18 and consequently to increased neutrophil adhesion to endothelial cells.

Peroxynitrite mediates cytokine-induced IL-8 gene expression and production by human leukocytes

Recent studies indicate that nitric oxide (NO) or related compounds mayregulate the production of interleukin (IL)‐8, a potent proinflammatorychemokine. Here we report that peroxynitrite (ONOO−)formed by a reaction of NO with superoxide mediates IL‐8 geneexpression and IL‐8 production in IL‐1β‐ and TNF‐α‐stimulated humanleukocytes in whole blood. The NO synthase inhibitors aminoguanidineand NG‐nitro‐l‐arginine methylester blocked nuclear accumulation of activator protein‐1 (AP‐1) andnuclear factor (NF)‐κB in both polymorphonuclear (PMN) andmononuclear leukocytes and inhibited IL‐8 mRNA expression and IL‐8release by ∼90% in response to IL‐1β and TNF‐α. EnhancedONOO− formation was detected in granulocytes, monocytes, and lymphocytes after challenge with IL‐1β or TNF‐α. The additionof ONOO− (0.2–80 μM) to whole blood increased nuclearaccumulation of AP‐1 and NF‐κB in PMN and mononuclear leukocytes andaugmented IL‐8 mRNA expression and IL‐8 production in aconcentration‐dependent fashion. Pyrrolidine dithiocarbamate, aninhibitor of NF‐κB activation, attenuated ∼70% of IL‐8 releaseevoked by IL‐1β, TNF‐α, or ONOO−. These resultsindicate that ONOO− formation may underlie the action ofcytokines towards IL‐8 gene expression in human leukocytes.

Loss of Pentameric Symmetry of C-Reactive Protein Is Associated with Promotion of Neutrophil-Endothelial Cell Adhesion

The classic acute-phase reactant C-reactive protein (CRP) is a cyclic pentameric protein that diminishes neutrophil accumulation in inflamed tissues. When the pentamer is dissociated, CRP subunits undergo conformational rearrangement that results in expression of a distinctive isomer with unique antigenic and physicochemical characteristics (termed modified CRP (mCRP)). Recently, mCRP was detected in the wall of normal human blood vessels. We studied the impact and mechanisms of action of mCRP on expression of adhesion molecules on human neutrophils and their adhesion to human coronary artery endothelial cells. Both CRP and mCRP (0.1–200 μg/ml) down-regulated neutrophil l-selectin expression in a concentration-dependent fashion. Furthermore, mCRP, but not CRP, up-regulated CD11b/CD18 expression and stimulated neutrophil extracellular signal-regulated kinase activity, which was accompanied by activation of p21ras oncoprotein, Raf-1, and mitogen-activated protein kinase kinase. These actions of mCRP were sensitive to the mitogen-activated protein kinase kinase inhibitor PD98059. mCRP markedly enhanced attachment of neutrophils to LPS-activated human coronary artery endothelial when added together with neutrophils. This effect of mCRP was attenuated by an anti-CD18 mAb. Thus, loss of pentameric symmetry in CRP is associated with appearance of novel bioactivities in mCRP that enhance neutrophil localization and activation at inflamed or injured vascular sites.

The anti-inflammatory peptides, antiflammins, regulate the expression of adhesion molecules on human leukocytes and prevent neutrophil adhesion to endothelial cells

Antiflammin‐1 and antiflammin‐2 are nonapeptides corresponding to the region of highest similarity between glucocorticoid‐inducible proteins lipocortin‐1 and uteroglobin. We have studied whether antiflammins could affect expression of adhesion molecules on human leukocytes and coronary artery endothelial cells (HCAEC) and binding of neutrophils (PMNs) to HCAEC. Although neither antiflammin‐1 nor antiflammin‐2 affected expression of adhesion molecules on resting PMNs, monocytes, and lymphocytes in whole blood, they attenuated changes in L‐selectin and CD11/CD18 expression evoked by platelet‐activating factor or interleukin‐8 with IC50 values of 4–20 μmol/l. The maximum inhibition was similar to those seen with human recombinant lipocortin‐1 (100 μg/ml). Unlike dexa‐methasone (100 nmol/l), the antiflammins had little effect on LPS‐stimulated expression of E‐selectin and ICAM‐1 on HCAEC. Consistently, culture of HCAEC with dexamethasone, but not with antiflam‐mins, decreased PMN binding to endothelial cells. Preincubation of PMNs with antiflammins markedly decreased their adhesion to LPS‐activated HCAEC. Inhibition of adhesion was additive with function blocking anti‐E‐selectin and anti‐L‐selectin antibodies, but was not additive with anti‐CD18 antibody. These results show that antiflammins inhibit PMN adhesion to HCAEC by attenuating activation‐induced up‐regulation of CD11/CD18 expression on leukocytes, and suggest that antiflammins may represent a novel therapeutic approach in blocking leukocyte trafficking in host defense and inflammation.—Zouki, C., Ouellet, S., Filep, J. G. The antiinflammatory peptides, antiflammins, regulate the expression of adhesion molecules on human leukocytes and prevent neutrophil adhesion to endothelial cells. FASEB J. 14, 572–580 (2000)

LIPOXIN A4 AND ASPIRIN-TRIGGERED 15-EPI­ LIPOXIN A4 MODULATE ADHESION MOLECULE EXPRESSION ON HUMAN LEUKOCYTES IN WHOLE BLOOD AND INHIBIT NEUTROPHIL-ENDOTHELIAL CELL ADHESION

Leukocyte recruitment and activation play a central role in host defense in response to infection or tissue injury. Leukocyte migration into inflamed areas involves a multistep interaction of leukocytes and endothelial cells via regulated expression of surface adhesion molecules and their counter-receptors on leukocytes and endothelial cells.1,2To prevent over-exuberant inflammatory response and damage to the host, leukocyte recruitment is tightly regulated. Conversely, failure of the host to resolve ongoing inflammation culminates in tissue destruction and remodeling, key events in the pathogenesis of a variety of diseases. The host mediators serving as endogenous anti-inflammatory signals are only recently being appreciated.3Among lipid mediators, lipoxins (trihydroxytetraene-containing eicosanoids) represent a unique class with potent anti-inflammatory actions.3Lipoxins are produced from arachidonate by the combined actions of 5-lipoxygease and 12- or 15-lipoxygenase (for review see reference 4). Aspirin also triggers the production of 15-epimer of LXA4.5One lipoxin in particular, LXA4, controls leukocyte responses via its own G-protein-coupled receptor4which also engages 15-epi-LXA4. LXA4 and its stable analogs were found to inhibit neutrophil (PMN) chemotaxis,b7P-selectin-dependent adhesion and rolling on the mesenteric endothelium8transmigration across epithelial and endothelial monolayers9,10and entry into inflamed renal tissues.11In this study, we investigated whether stable analogs of native LXA4 and aspirin-triggered 15-epi-LXA4 are active within the microenvironment of human whole blood, and studied the cellular mechanisms that account for their novel inhibitory actions in leukocyte trafficking.

Methods for analysis of matrix metalloproteinase regulation of neutrophil-endothelial cell adhesion

Recent evidence indicates novel role for matrix metalloproteinases (MMPs), in particular gelatinase A (MMP-2), in the regulation of vascular biology that are unrelated to their well-known proteolytic breakdown of matrix proteins. We have previously reported that MMP-2 can modulate vascular reactivity by cleavage of the Gly32-Leu33 bound in big endothelin-1 (ET-1) yielding a novel vasoactive peptide ET-1[1–32]. These studies were conducted to investigate whether gelatinolytic MMPs could affect neutrophil-endothelial cell attachment. ET-1[1–32] produced by MMP-2 up-regulated CD11b/CD18 expression on human neutrophils, thereby promoted their adhesion to cultured endothelial cells. ET-1[1–32] evoked release of gelatinase B (MMP-9), which in turn cleaved big ET-1 to yield ET-1[1–32], thus revealing a self-amplifying loop for ET-1[1–32] generation. ET-1[1–32] was rather resistant to cleavage by neutrophil proteases and further metabolism of ET-1[1–32] was not a prerequisite for its biological actions on neutrophils. The neutrophil responses to ET-1[1–32] were mediated via activation of ETA receptors through activation of the Ras/Raf-1/MEK/ERK signaling pathway. These results suggest a novel role for gelatinase A and B in the regulation of neutrophil functions and their interactions with endothelial cells. Here we describe the methods in detail as they relate to our previously published work.