HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 15/1/25 00-0521fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by ZOUKI, C. Articles by FILEP, J. G. Search for Related Content PubMed PubMed Citation Articles by ZOUKI, C. Articles by FILEP, J. G.

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

Research Center, Maisonneuve-Rosemont Hospital and Department of Medicine, University of Montréal, Montréal, Québec, Canada H1T 2M4

²Correspondence: Research Center, Maisonneuve-Rosemont Hospital, 5415 boulevard de l’Assomption, Montreal, Quebec, Canada H1T 2M4. E-mail: filepj{at}ere.umontreal.ca

SPECIFIC AIMS

We investigated whether ONOO^- could affect adhesion molecule expression on human neutrophils (PMNs) and coronary artery endothelial cells (HCAEC) and consequently promote adhesion of PMNs to HCAEC, and addressed the underlying cellular and molecular mechanisms.

PRINCIPAL FINDINGS

1. ONOO^- induces L-selectin shedding and up-regulation of CD11b/CD18 expression on neutrophils
ONOO^- down-regulated neutrophil surface expression of L-selectin and up-regulated expression of CD11b in a concentration-dependent fashion (Fig. 1 ) with apparent EC₅₀ values of 10 to 15 µM. Similar increases were detected in CD18 expression. The maximum changes that could be achieved with ONOO^- were smaller than those evoked by 1 µM platelet-activating factor (PAF). Decomposed ONOO^- did not affect expression of L-selectin and CD11b expression. Furthermore, when tested in whole blood, ONOO^- evoked similar changes in neutrophil L-selectin and CD11b/CD18 expression as observed with isolated PMNs.

View larger version (17K): [in this window] [in a new window]   Figure 1. Concentration-dependent effect of ONOO- on surface expression of L-selectin and CD11b on human neutrophil granulocytes. Isolated PMNs were challenged with ONOO- or decomposed peroxynitrite (dP) for 30 min at 37°C. Fluorescence intensity (relative fluorescence unit, RFU) is presented as percentage of control, i.e., mean fluorescence intensity of neutrophils incubated in medium only. Results are the mean ± SE for 6 experiments using neutrophils from different donors. *P < 0.05; **P < 0.01; ***P < 0.001 vs. control.

2. Inhibition of MAPK kinase reverses ONOO^--induced changes in CD11b/CD18 expression on neutrophils
The MAPK kinase (MEK) inhibitor PD98059 prevented ONOO^--induced up-regulation of CD11b/CD18 expression in a concentration-dependent fashion, whereas it was a less effective inhibitor in reversing ONOO^--induced down-regulation of L-selectin expression. The tyrosine kinase inhibitor genistein and the selective p38 MAP kinase inhibitor SB 203580 had no significant effects on ONOO^--induced changes in CD11b/CD18 expression.

3. ONOO^- activates Erk via the Ras/Raf-1/MEK signaling cascade
Incubation of neutrophils with ONOO^- induced a time- and concentration-dependent increase in phosphorylation of Erk relative to unstimulated controls. Phosphorylation of Erk was rapid (near maximum increases within 5 min), concordant with the kinetics of ONOO^--stimulated up-regulation of CD11b/CD18 expression. Likewise, ONOO^- (100 µM) induced a 4.5-fold increase in Erk activity (measured by the ability of neutrophil lysates to phosphorylate Elk-1, a specific target for Erk).

ONOO stimulation of Erk depends on the sequential activation of Ras and the kinases Raf-1 and MEK. Phosphorylation of MEK was rapid in onset, maximal within 2 min after addition of ONOO^- to PMNs. ONOO^- also evoked increases in neutrophil Raf-1 kinase activity and stimulated association of the active (GTP-bound) form of Ras with the GST-Ras binding domain of Raf-1 (detected by immunoblotting of affinity precipitated GTP-bound Ras) within 2 min.

To provide further evidence that ONOO^- stimulation of neutrophil Erk is mediated via Raf-1 and MEK, we tested whether PD 98059 has an effect on Erk activation. Preincubation of PMNs with PD 98059 (100 µM) resulted in 91 ± 3% and 75 ± 5% inhibition of ONOO^- (100 µM) -stimulated Erk activity and up-regulation of CD11b/CD18 expression, respectively.

4. ONOO^- induces slight increases in E-selectin and ICAM-1 expression on HCAEC
Flow cytometry analysis revealed that approximately 50% and 4% of untreated HCAEC expressed ICAM-1 and E-selectin, respectively. Culture of HCAEC for 4 h with ONOO^- resulted in slight increases in the overall expression of these adhesion molecules and in the percentage of cells expressing E-selectin without affecting cell viability.

5. ONOO^- enhances CD18-dependent adhesion of neutrophils to HCAEC
Though only a few PMNs were able to bind to unstimulated HCAEC, neutrophil adherence was enhanced fivefold by activation of HCAEC with lipopolysaccharides (LPS) (Fig. 2A ). Addition of ONOO^- (0.1 to 100 µM initial concentrations) to HCAEC resulted in only 15% to 80% increases in the number of adherent neutrophils (Fig. 2A ). The number of adherent neutrophils was further enhanced when PMNs were added together with ONOO^- to LPS-activated HCAEC (Fig. 2B ), whereas only slight increases in neutrophil adherence were detected when ONOO^- was added together with PMNs to HCAEC incubated with ONOO^- for 4 h (Fig. 2C ).

View larger version (29K): [in this window] [in a new window]   Figure 2. ONOO- enhances CD18-dependent adhesion of human neutrophils (PMNs) to coronary artery endothelial cells (HCAEC). Confluent HCAEC monolayers were cultured in medium only (control) or activated with ONOO- (A, B) or LPS (1 µg/ml) for 4 h (A, C), as indicated. Radiolabeled PMNs (A) or PMNs together with ONOO- (B, C) were then added and incubated with HCAEC for 30 min at 37°C on an orbital shaker. Values are expressed as the mean ± SE of 6 experiments in triplicate using neutrophils from different donors. *P < 0.05; ***P < 0.001 vs. control; #P < 0.05 vs. LPS-activated HCAEC. D) PMNs were added together with 100 µM ONOO- to LPS-activated HCAEC and incubated for 30 min in the absence (medium) or presence of function blocking monoclonal antibodies (mAb) directed against CD18, CD62L, and CD62E, as indicated on the Y axis. The irrelevant mAb MOPC-21 (IgG1) was used as a negative control. Results are expressed as the mean ± SE of 3 experiments in triplicate using neutrophils from different donors.

A significant proportion of neutrophil adhesion to LPS-activated HCAEC was blocked by mAbs binding to E-selectin (29±1%, n=3), L-selectin (22±1%), and CD18 (28±1%) (Fig. 2D ). The combination of these mAbs inhibited neutrophil adhesion by 80% (Fig. 2D ). ONOO^--stimulated PMN adhesion to LPS-activated HCAEC was blocked by mAbs binding to E-selectin (22±2%, n=3), L-selectin (10±2%) and CD18 (55±5%) (Fig. 2D ), indicating that ONOO^- promoted CD18-dependent attachment. The combination of these three mAbs inhibited PMN adhesion by 80–88% (Fig. 2D ).

CONCLUSIONS

Inflammation, ischemia-reperfusion injury and sepsis are associated with enhanced peroxynitrite formation. In this report, we describe a novel mechanism by which ONOO^- can affect the inflammatory process, namely, via the modulation of surface expression of adhesion molecules on human neutrophils leading to increased CD18-dependent adhesion of PMNs to endothelial cells.

We demonstrate that at micromolar concentrations, ONOO^- is capable of down-regulating L-selectin and up-regulating CD11/CD18 expression in human isolated neutrophils. Of particular interest, ONOO^- clearly remains bioactive in the microenvironment of whole blood to regulate expression of adhesion molecules on neutrophils. We suggest that this activation is most likely mediated via activation of Erk. Peroxynitrite-induced phosphorylation of Erk and increases in Erk activity were concentration and time dependent. ONOO^- stimulated Ras and Raf-1 kinase activity, evoked phosphorylation of MEK, and the specific MEK inhibitor PD 98059 inhibited Erk activation by ONOO^-, although this inhibition was incomplete (80%).

ONOO^- crosses cell membranes at a rate significantly faster than the rates of its known decomposition pathways. The present observations suggest that after diffusion into PMNs (and perhaps after intracellular generation), ONOO^- initiates Erk activation via Ras, Raf-1, and MEK (Fig. 3 ), similarly to that previously observed with other stimuli. Although this model represents a best-fit for our data, it fails to provide a mechanism whereby ONOO^- leads to activation of Ras. Whether ONOO^- activation of Ras is G-protein dependent and the mechanisms whereby G-protein activation leads to Ras and/or Raf-1 activation remain to be investigated.

View larger version (36K): [in this window] [in a new window]   Figure 3. A model for peroxynitrite-stimulated neutrophil-endothelial cell adhesion. Peroxynitrite diffuses into neutrophils where it activates Erk via the Ras/Raf-1/MEK signaling pathway, leading to down-regulation of L-selectin and up-regulation of CD11b/CD18 (Mac-1) expression, and consequently to increased neutrophil adhesion to human coronary artery endothelial cells (HCAEC). Broken arrow indicates an undefined pathway.

The present data further suggest that Erk activation is required for ONOO^- stimulation of CD11b/CD18 expression. The degree of Erk inhibition by PD 98059 was similar to the degree of inhibition of CD11b/CD18 expression. However, PD 98059 appeared to be a less potent inhibitor of the ONOO^- down-regulation of L-selectin expression, indicating the involvement of other, Erk-independent pathway(s) in adhesion signaling.

Despite inducing L-selectin shedding from neutrophils, which was reported to attenuate PMN-HCAEC attachment, ONOO^- actually promoted neutrophil adhesion to HCAEC. This augmentation was largely due to its action on neutrophils rather than on HCAEC, because ONOO^- evoked only slight increases in the intensity of staining for E-selectin and ICAM-1 and in the number of adherent PMNs to ONOO^--activated HCAEC. Our findings on ICAM-1 expression are consistent with observations that ONOO^- decomposition catalysts reduced mesenteric ICAM-1 staining evoked by ischemia-reperfusion in the rat.

Taken together, the present and previous studies suggest a dual role for ONOO^- in the regulation of neutrophil trafficking. At nanomolar concentrations, ONOO^- may exert an antiadhesive action, whereas at higher concentrations (1–100 µM), which conceivably could be achieved in vivo, it promotes neutrophil adhesion. Such diametrically opposed actions of ONOO^- can be understood by appreciating that the target(s) (i.e., the reaction substrate) of ONOO^- when it is proadhesive is different from that when it is protective. Indeed, low amounts of ONOO^- may react with serum albumin or glutathione to form nitrosothiols, which release NO. Higher amounts of ONOO^- may freely diffuse into cells to activate intracellular signaling pathways, including Ras and/or Raf-1, when the protection conferred by albumin, glutathione, and other antioxidants is overwhelmed. This would lead to changes in neutrophil adhesion molecule expression and ultimately to increased neutrophil adhesiveness. Inflammation is often associated with depletion of antioxidant capacity, thereby unmasking and aggravating the deleterious actions of ONOO^-.

Since the half-life of ONOO^- at neutral pH is on the order of seconds, we cannot now conclude whether the observed effects were a direct action of ONOO^- or one of its more stable decomposition products. These decomposition products are still a subject of debate. Hydroxyl radical, peroxynitrous acid (or its activated isomer), and nitroxyl anion have been proposed as active intermediates to mediate the reactivity of ONOO^-. Thus, it is unlikely for ONOO^- to function as an NO-donating compound as previously suggested.

In summary, these data indicate that ONOO^- activates neutrophil Erk via the Ras/Raf-1/MEK signaling cascade, leading to up-regulation of CD11b/CD18 expression and increased neutrophil adhesion to HCAEC via a CD18-dependent mechanism (Fig. 3) . Our results also support the hypothesis that Erk activation in human neutrophils plays a critical role in the heterotypic adhesive responses of these cells.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0521fje To cite this article, use (November 9, 2000) FASEB J. 10.1096/fj.00-0521fje

This article has been cited by other articles:

				P. Pacher, J. S. Beckman, and L. Liaudet Nitric Oxide and Peroxynitrite in Health and Disease Physiol Rev, January 1, 2007; 87(1): 315 - 424. [Abstract] [Full Text] [PDF]

				Y.-J. Zhang, Y.-F. Xu, Y.-H. Liu, J. Yin, H.-L. Li, Q. Wang, and J.-Z. Wang Peroxynitrite induces Alzheimer-like tau modifications and accumulation in rat brain and its underlying mechanisms FASEB J, July 1, 2006; 20(9): 1431 - 1442. [Abstract] [Full Text] [PDF]

				K. S. Farley, L. F. Wang, H. M. Razavi, C. Law, M. Rohan, D. G. McCormack, and S. Mehta Effects of macrophage inducible nitric oxide synthase in murine septic lung injury Am J Physiol Lung Cell Mol Physiol, June 1, 2006; 290(6): L1164 - L1172. [Abstract] [Full Text] [PDF]

				F. Zare, M. Magnusson, T. Bergstrom, M. Brisslert, E. Josefsson, A. Karlsson, and A. Tarkowski Uric acid, a nucleic acid degradation product, down-regulates dsRNA-triggered arthritis J. Leukoc. Biol., March 1, 2006; 79(3): 482 - 488. [Abstract] [Full Text] [PDF]

				L. Jozsef, T. Khreiss, D. El Kebir, and J. G. Filep Activation of TLR-9 Induces IL-8 Secretion through Peroxynitrite Signaling in Human Neutrophils J. Immunol., January 15, 2006; 176(2): 1195 - 1202. [Abstract] [Full Text] [PDF]

				M. Haley, C. Parent, X. Cui, A. Kalil, Y. Fitz, R. Correa-Araujo, C. Natanson, R. L. Danner, S. M. Banks, and P. Q. Eichacker Neutrophil inhibition with L-selectin-directed MAb improves or worsens survival dependent on the route but not severity of infection in a rat sepsis model J Appl Physiol, June 1, 2005; 98(6): 2155 - 2162. [Abstract] [Full Text] [PDF]

				S Muzaffar, N Shukla, G D Angelini, and J Y Jeremy Acute hypoxia simultaneously induces the expression of gp91phox and endothelial nitric oxide synthase in the porcine pulmonary artery Thorax, April 1, 2005; 60(4): 305 - 313. [Abstract] [Full Text] [PDF]

				S. Muzaffar, N. Shukla, G. Angelini, and J. Y. Jeremy Nitroaspirins and Morpholinosydnonimine but Not Aspirin Inhibit the Formation of Superoxide and the Expression of gp91phox Induced by Endotoxin and Cytokines in Pig Pulmonary Artery Vascular Smooth Muscle Cells and Endothelial Cells Circulation, August 31, 2004; 110(9): 1140 - 1147. [Abstract] [Full Text] [PDF]

				S. Lancel, S. Tissier, S. Mordon, X. Marechal, F. Depontieu, A. Scherpereel, C. Chopin, and R. Neviere Peroxynitrite decomposition catalysts prevent myocardial dysfunction and inflammation in endotoxemic rats J. Am. Coll. Cardiol., June 16, 2004; 43(12): 2348 - 2358. [Abstract] [Full Text] [PDF]

				T. Wang, D. El Kebir, and G. Blaise Inhaled nitric oxide in 2003: a review of its mechanisms of action: [L'inhalation de monoxyde d'azote en 2003 : une revue de ses mecanismes et de son action] Can J Anesth, October 1, 2003; 50(8): 839 - 846. [Abstract] [Full Text] [PDF]

				S. S. Akimov and A. M. Belkin Opposing Roles of Ras/Raf Oncogenes and the MEK1/ERK Signaling Module in Regulation of Expression and Adhesive Function of Surface Transglutaminase J. Biol. Chem., September 12, 2003; 278(37): 35609 - 35619. [Abstract] [Full Text] [PDF]

				S Muzaffar, J Y Jeremy, G D Angelini, K Stuart-Smith, and N Shukla Role of the endothelium and nitric oxide synthases in modulating superoxide formation induced by endotoxin and cytokines in porcine pulmonary arteries Thorax, July 1, 2003; 58(7): 598 - 604. [Abstract] [Full Text] [PDF]

				E. Nabeyrat, G. E. Jones, P. S. Fenwick, P. J. Barnes, and L. E. Donnelly Mitogen-activated protein kinases mediate peroxynitrite-induced cell death in human bronchial epithelial cells Am J Physiol Lung Cell Mol Physiol, June 1, 2003; 284(6): L1112 - L1120. [Abstract] [Full Text] [PDF]

				H. W. M. NIESSEN, P. A. J. KRIJNEN, C. A. VISSER, C. J. L. M. MEIJER, and C. E. HACK Intercellular Adhesion Molecule-1 in the Heart Ann. N.Y. Acad. Sci., November 1, 2002; 973(1): 573 - 585. [Abstract] [Full Text] [PDF]

				T. Khreiss, L. Jozsef, S. Hossain, J. S. D. Chan, L. A. Potempa, and J. G. Filep Loss of Pentameric Symmetry of C-reactive Protein Is Associated with Delayed Apoptosis of Human Neutrophils J. Biol. Chem., October 18, 2002; 277(43): 40775 - 40781. [Abstract] [Full Text] [PDF]

				L. Jozsef, C. Zouki, N. A. Petasis, C. N. Serhan, and J. G. Filep Lipoxin A4 and aspirin-triggered 15-epi-lipoxin A4 inhibit peroxynitrite formation, NF-kappa B and AP-1 activation, and IL-8 gene expression in human leukocytes PNAS, October 1, 2002; 99(20): 13266 - 13271. [Abstract] [Full Text] [PDF]

				L. You, F. E. Kruse, S. Bacher, and M. L. Schmitz Lipoteichoic Acid Selectively Induces the ERK Signaling Pathway in the Cornea Invest. Ophthalmol. Vis. Sci., July 1, 2002; 43(7): 2272 - 2277. [Abstract] [Full Text] [PDF]

				E. de Lamirande and C. Gagnon The extracellular signal-regulated kinase (ERK) pathway is involved in human sperm function and modulated by the superoxide anion Mol. Hum. Reprod., February 1, 2002; 8(2): 124 - 135. [Abstract] [Full Text] [PDF]

				C. Zouki, B. Haas, J. S. D. Chan, L. A. Potempa, and J. G. Filep Loss of Pentameric Symmetry of C-Reactive Protein Is Associated with Promotion of Neutrophil-Endothelial Cell Adhesion J. Immunol., November 1, 2001; 167(9): 5355 - 5361. [Abstract] [Full Text] [PDF]

				S. L. de Menezes and O. Augusto EPR Detection of Glutathionyl and Protein-tyrosyl Radicals during the Interaction of Peroxynitrite with Macrophages (J774) J. Biol. Chem., October 19, 2001; 276(43): 39879 - 39884. [Abstract] [Full Text] [PDF]

				B. M. Matata and M. Galinanes Peroxynitrite Is an Essential Component of Cytokines Production Mechanism in Human Monocytes through Modulation of Nuclear Factor-kappa B DNA Binding Activity J. Biol. Chem., January 11, 2002; 277(3): 2330 - 2335. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 15/1/25 00-0521fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by ZOUKI, C. Articles by FILEP, J. G. Search for Related Content PubMed PubMed Citation Articles by ZOUKI, C. Articles by FILEP, J. G.