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(The FASEB Journal. 2001;15:2230-2240.)
© 2001 FASEB

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

CARLOS FERNANDEZ-PATRON*,{dagger},1, CHRISTINE ZOUKI*,1, RANDY WHITTAL{ddagger}, JOHN S. D. CHAN*, SANDRA T. DAVIDGE{dagger} and JÁNOS G. FILEP*2

* Research Center, Maisonneuve-Rosemont Hospital and Department of Medicine, University of Montréal, Montréal, Québec H1T 2M4 Canada;
{dagger} Perinatal Research Centre, and
{ddagger} Laboratory of Mass Spectrometry, Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2S2 Canada

2Correspondence: Research Center, Maisonneuve-Rosemont Hospital, University of Montréal, 5415 boulevard de l’Assomption, Montréal, Québec H1T 2M4 Canada. E-mail: janos.g.filep{at}umontreal.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We recently reported that matrix metalloproteinase 2 (MMP-2, gelatinase A) cleaves big endothelin 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-regulation 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 neutrophils 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].


Key Words: gelatinases • ETA receptor • L-selectin • CD11b/CD18 • leukocyte trafficking • endothelium • inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MATRIX METALLOPROTEINASE 2 (MMP-2, gelatinase A), expressed in various cells and tissues including the vascular smooth muscle and the endothelium (1) , is known mainly for its capacity to cleave specific bonds in extracellular matrix proteins such as collagen, elastin, fibronectin, and laminin 5 (1 2 3 4) , which results in a long-term process of vascular remodeling (2) . Recent evidence indicates novel roles for gelatinase A in vascular biology that are unrelated to long-term matrix breakdown. We have reported that gelatinase A modulates vascular reactivity by reducing the vasodilatory potency of calcitonin gene-related peptide through cleavage of the Gly14-Leu15 bond (6) . Moreover, gelatinase A cleaves the Gly32-Leu33 bond of big endothelin 1 (ET-1[1–38]), the inactive precursor of a potent vasoconstrictor, ET-1[1–21], and yields a novel vasoconstrictor peptide, ET-1[1–32] (7) . Simultaneous overexpression of gelatinase activity and big ET-1 were found to occur at sites of tissue injury and inflammation associated with a variety of pathological conditions, including myocardial ischemia (8 , 9) , neointima formation (10) , and atherosclerosis (11 , 12) , which are also characterized by increased adhesiveness of leukocytes to endothelial cells (13) .

Recent studies from several laboratories (including ours) have suggested that ET-1 may be an important autocrine/paracrine modulator of neutrophil functions. Human neutrophils possess ETA receptors (14) . ET-1 promotes neutrophil aggregation (15 , 16) , down-regulates surface expression of L-selectin (14) , up-regulates expression of CD11b/CD18 (14 , 17) , and augments their adhesion to cultured human coronary artery endothelial cells (HCAEC) through a CD18-dependent mechanism (14) . ET-1 causes a selective neutrophil leukocytopenia in guinea pigs (18) and induces neutrophil accumulation in the heart (17) , lung (19 , 20) , and kidney (21) predominantly via activation of ETA receptors (18 , 20) . ET-1-activated neutrophils migrate from the venous lumen into the tissue matrix of the human umbilical cord and induce a massive tissue destruction (22) probably through release of gelatinase B (MMP-9) (1) .

The regulation of neutrophil activation (specifically, release of gelatinase after activation of endothelin receptors) is not completely understood. Specific neutrophil functions appear to be regulated at least in part via distinct signaling mechanisms. Studies of the mitogen-activated protein kinases (MAPKs) in neutrophils indicate an association between chemoattractant, arachidonic acid, or peroxynitrite stimulation of the serine/threonine protein kinases p44 (Erk1) and p42 (Erk2) and the neutrophil adhesive function (23 24 25 26) . Erk activation is mediated partly through activation of Ras, Raf-1, and MAPK kinase (MEK) (24 , 27 , 28) , with kinetics concordant with rapid activation of neutrophils by these stimuli. These studies indicate a nonmitotic signaling function for Erk in neutrophils.

In this study, we investigated whether ET-1[1–32], which is made by gelatinase A in the vasculature, could affect adhesion molecule expression on HCAEC and, consequently, adhesion of neutrophils to HCAEC. To gain better insight into the mechanism of action of ET-1[1–32], we also examined whether 1) ET-1[1–32] exerts its actions through activation of ETA or ETB receptors; 2) metabolism of ET-1[1–32] to ET-1[1–21] by neutrophils is required for its biological activity; 3) activation of the Ras/Raf-1/MEK/Erk signaling pathway mediates neutrophil activation by ET-1[1–32]. Finally, we characterized the adhesion molecules that mediate the effects of ET-1[1–32] on neutrophil-endothelial cell adhesion. Having found that ET-1[1–32] induced gelatinase B release from neutrophils, we investigated whether this matrix metalloproteinase could also increase formation of ET-1[1–32].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation and characterization of ET-1[1–32]
ET-1[1–32] was prepared as described previously (7) by cleaving synthetic human big ET-1 (Sigma-Aldrich, Oakville, ON, Canada) with highly pure 4-aminophenylmercuric acetate-activated human gelatinases A or B (140 nM, Chemicon International, Mississauga, ON, Canada). The cleavage reaction was conducted for 16 h (except where stated otherwise) at 37°C in HEPES-phosphate saline solution (in mM: NaCl 142, KCl 4.7, MgSO4 1.17, CaCl2 1.56, HEPES 10, KH2PO4 1.18; pH 7.4). The incubation mixture was separated on an HPLC chromatograph (Waters, Milford, MA) using a 12.5 cm x 4 mm C-18 column (LiChrospher, Merck, Rahway, NJ) with a (1%/min) gradient of 5% CH3CN in 0.1% aqueous TFA against 0.1% TFA in acetonitrile at 0.5 ml/min flow rate. HPLC resolved peaks were collected and the mass of peptides was determined with a Voyager Elite matrix-assisted laser desorption ionization (MALDI) mass spectrometer (Applied Biosystems, Framingham, MA) equipped with delayed extraction and a reflectron. The instrument was run in reflectron mode using 20 kV acceleration. External calibrations were completed using a mixture of known peptides. ET-1[1–32] stock solution (10-4 M) was diluted in phosphate-buffered saline (PBS), 0.1% BSA (pH 7.4) immediately before use. The ET-1[1–32] preparations did not contain detectable amounts of endotoxin; i.e., levels were below the detection limit (0.125 EU/ml) of the Limulus amebocyte lysate assay (E-Toxate, Sigma).

Isolation and treatment of neutrophil granulocytes
Venous blood (anticoagulated with sodium heparin, 50 U/ml) was obtained from nonsmoking healthy volunteers (male and female, 25–44 years) who had not taken any drugs for at least 10 days before the experiments; neutrophil granulocytes were isolated as described previously (14) . Informed consent was obtained from each volunteer, and the protocol was approved by the Clinical Research Committee. Neutrophils (107 cells/ml, purity >97%) were suspended in a modified Hanks’ balanced salt solution (in mM: NaCl 140, KCl 5, K2PO4 10, CaCl2 1.4, MgCl2 1.2, glucose 5, and 0.1% bovine serum albumin, pH 7.4), and preincubated with one of the following antagonists for 10 min at 37°C: the ETA receptor selective antagonist FR 139317 (1 µM, Fujisawa Pharmaceutical Co., Osaka, Japan) (29) , the ETB receptor selective antagonist BQ 788 (10 µM, Novabiochem Corp., San Diego, CA) (30) , or various enzyme inhibitors, as indicated, then challenged with ET-1[1–32] for 30 min at 37°C. As a negative control, neutrophils were challenged with biologically inactive big ET-1 in the presence of 100 µM phosphoramidon (which inhibits endothelin-converting enzymes, but not MMPs) and 100 µM o-phenanthroline (which potently inhibits MMPs) to prevent its cleavage into biologically active smaller peptides. As another negative control, neutrophils were challenged with an unrelated 32-amino acid peptide, bovine pTH (3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34) (0.1–1000 nM, SEIQFMHNLGKHLSSMERVEWLRKKLQNVHNF; Bachem Bioscience, King of Prussia, PA). The cells were then pelleted, and pellets and supernatants were collected for further analysis. In some experiments, whole blood aliquots were used instead of isolated neutrophils.

Flow cytometry analysis
Direct immunofluorescence labeling of control or treated neutrophil granulocytes was performed (31) . Leukocytes were stained with saturating concentration of FITC-labeled anti-human L-selectin monoclonal antibody (mAb) DREG-56 (IgG1; PharMingen, San Diego, CA), R-phycoerythrin-conjugated mouse anti-human CD11b mAb Leu-185 (IgG1; Becton Dickinson Immunocytometry Systems, Mountain View, CA), or R-phycoerythrin-labeled anti-human CD18 mAb (IgG1, Becton Dickinson). Appropriately labeled, class-matched irrelevant mouse IgG1 was used as a negative control for each staining. Single- or double-color immunofluorescence staining was analyzed by a FACScan Flow Cytometer (Becton Dickinson) with Lysis II software. Antibody binding was determined as mean fluorescence intensity after gating for neutrophils by their characteristic forward and side scatter properties.

Activation of the Ras/Raf-1/MEK/Erk signaling pathway
Neutrophils were lysed in an ice-cold lysis buffer (in mM: Tris 20, EGTA 1, Na3VO4, 2, NaF 25, PMSF 2, 0.5% (v/v) Triton X-100, 40 µg/ml aprotinin, and 10 µg/ml each of chymostatin, leupeptin, and pepstatin A, pH 7.4) for 15 min and centrifuged at 4°C for 10 min at 14,000 g. Western blot analysis of phosphorylated MEK and Erk1/2 (p44/42 MAPK) was performed as described (26) using the Phospho Plus MEK 1/2 and Erk 1/2 MAP kinase antibody kits (New England Biolabs, Beverly, MA). Raf-1 kinase activity was determined by a modification of the method of Gardner et al. (32) . Raf-1 was immunoprecipitated with an anti-Raf-1 antiserum (C-12, Santa Cruz Biotechnology, Santa Cruz, CA), antigen–antibody complexes were then isolated by protein A-Sepharose CL-4B and Raf-1 activity was measured using the Raf-1 Kinase Cascade Assay kit (Upstate Biotechnology, Lake Placid, NY) in accordance with the manufacturer’s protocol. Activated p21Ras (Ras-GTP) from neutrophil lysates was affinity precipitated with a GST-Ras binding domain of Raf-1 (residues 1–149) fusion protein conjugated to agarose (Upstate Biotechnology) as described elsewhere (33) . The beads were washed extensively and boiled in reducing sample buffer. The eluted proteins were resolved on a 10% SDS-polyacrylamide gel, transferred to a PVDF membrane, probed with a mouse anti-Ras mAb (clone RAS10, Upstate Biotechnology), and visualized using a goat anti-mouse secondary antibody conjugated to horseradish peroxidase (Bio-Rad, Mississauga, ON, Canada) and a chemiluminescence detection system.

Metabolism of ET-1[1–32]
Measurements were performed as described previously for ET-1[1–38] and ET-1[1–21] (7 , 34) . ET-1[1–32] (100 pmol) was incubated with preparations of neutrophil cytosol (0.5–5 µg protein) or membrane (0.2–2 µg protein) at 37°C for 2 h. The incubation mixture was separated on HPLC and the resolved peaks were collected and further characterized on a MALDI mass spectrometer, as described above.

Measurement of superoxide production and gelatinase release
Superoxide production was determined by measuring superoxide dismutase-inhibitable reduction of ferricytochrome c (35) . Gelatinase release was determined as the percentage of total enzyme units released from neutrophils treated with 0.1% Triton X-100 (36) .

Culture of endothelial cells
Normal HCAEC obtained from Clonetics Corp. (San Diego, CA) were cultured as described (36) . HCAEC (passages 3 to 6) seeded into 24-well or 96-well microplates and grown to confluence were used.

Neutrophil-endothelial cell adhesion assay
The adhesion assay was performed as in ref 36 . Monolayers of HCAEC in 96-well microplates were stimulated with lipopolysaccharide (LPS; 1 µg/ml, Escherichia coli, serotype O111:B4; Sigma Chemical Co., St. Louis, MO) to induce maximum expression of E-selectin and ICAM-1 or various concentrations of ET-1[1–32] for 4 h at 37°C in a 5% CO2 atmosphere. The cells were washed and 2 x 105 51Cr-labeled neutrophils in 100 µl were added. In some experiments, ET-1[1–32] was added together with neutrophils to HCAEC treated with LPS or ET-1[1–32] for 4 h. In another set of experiments, LPS-activated HCAEC were incubated for 15 min with the anti-E-selectin mAb ENA-2 (10 µg/ml, IgG1, purified F(ab)'2 fragments; Monosan, Uden, The Netherlands) or the irrelevant mAb MOPC-21 (20 µg/ml, IgG1, PharMingen) before addition of neutrophils. Radiolabeled neutrophils were incubated with the anti-L-selectin mAb DREG-56 (IgG1, PharMingen) at 20 µg/ml, the anti-CD18 mAb L130 (IgG1, Becton Dickinson) at 10 µg/ml, or MOPC-21 mAb for 15 min before addition to HCAEC. The mAb reactive with E-selectin was also added back to the neutrophil suspensions so that mAb ENA-2 was present throughout the assay. After incubation of HCAEC with neutrophils with or without ET-1[1–32] for 30 min at 37°C on an orbital shaker at 90 rpm, loosely adherent or unattached neutrophils were washed three times and the endothelial monolayer plus the adherent neutrophils were lysed in 150 µl of 0.1% Triton X-100. The number of adhered neutrophils in each experiment was estimated from the radioactivity of a control sample.

Expression of E-selectin and ICAM-1
After incubation for 4 h at 37°C in a 5% CO2 atmosphere with LPS (1 µg/ml) or ET-1[1–32], HCAEC were detached from the 24-well microplates by exposure to EDTA (0.01%) in PBS for 10 min at 37°C, followed by gentle trituration. The cells were then stained with saturating concentration of fluorescein dye-conjugated anti-E-selectin or anti-ICAM-1 mAb for 30 min at 4°C, washed, fixed in formaldehyde (3.9% in PBS), and immunofluorescence was analyzed with a flow cytometer as described (14) . Nonspecific binding was evaluated by using appropriately labeled mouse IgG1.

Statistical analysis
Results are expressed as means ± SE. Statistical comparisons were made by ANOVA using ranks (Kruskal-Wallis test), followed by Dunn’s multiple contrast hypothesis test to identify differences when various treatments were compared to the same control or by the Mann-Whitney U test for unpaired observations. P values <0.05 were considered significant for all tests.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
ET-1[1–32] regulates expression of L-selectin and CD11b/CD18 on neutrophils through activation of ETA receptor
Addition of ET-1[1–32] to isolated neutrophils resulted in down-regulation of neutrophil surface expression of L-selectin and up-regulation of CD11b expression in a concentration-dependent fashion (Fig. 1A ) with apparent EC50 concentrations of 1–3 nM. Similar increases were detected in CD18 expression (data not shown). The maximum changes achieved with ET-1[1–32] were similar to those evoked by 1 µM PAF (58±5% and 57±4% decreases in L-selectin expression by ET-1[1–32] and PAF, respectively, and 66±6% vs. 73±7% increases in CD11b expression by ET-1[1–32] and PAF, respectively, n=6, both P>0.1). Addition of ET-1[1–32] to whole blood evoked changes in neutrophil L-selectin and CD11b expression similar to those observed with isolated neutrophils (Fig. 1B ).



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  		Figure 1. ET-1[1–32]-induced changes in adhesion molecule expression on human neutrophils. A) Concentration-dependent effect of ET-1[1–32] on surface expression of L-selectin and CD11b. Isolated neutrophils were challenged with ET-1 or ET-1[1–32] for 30 min at 37°C, then stained with fluorescein-labeled anti-L-selectin and anti-CD11b mAbs. Fluorescence intensity 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. B) Isolated neutrophils or whole blood aliquots were incubated with 30 nM ET-1[1–32] for 30 min at 37°C, then stained as just described. Antibody binding to leukocytes in whole blood was determined after gating for neutrophils by their characteristic forward and side scatter properties. Each histogram also displays the negative control of immunostaining with class-matched irrelevant antibodies (c). A representative of 6 experiments using blood from different donors is shown.

The ETA receptor selective antagonist FR 139317 (1 µM) markedly attenuated ET-1[1–32] (100 nM) -induced changes in L-selectin and CD11b expression (Fig. 2 ). In contrast, the ETB receptor antagonist BQ 788 (10 µM) had no detectable effects (Fig. 2) . Neither FR139317 nor BQ788 on its own affected expression of adhesion molecules on resting neutrophils.



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  		Figure 2. Effects of ET receptor antagonists on neutrophil adhesion molecules. Isolated neutrophils were preincubated with the ETA receptor-selective antagonist FR139317 (1 µM) or the ETB receptor-selective antagonist BQ788 (10 µM) for 20 min at 37°C, then challenged with 30 nM ET-1[1–32] for 30 min. Adhesion molecule expression is presented as the percentage of control. Mean fluorescence intensity for control samples: L-selectin, 75 ± 4; CD11b, 1059 ± 47. Values are the mean ± SE of 6 independent experiments. **P < 0.01 vs. unstimulated (open columns); #P < 0.05 vs. ET-1[1–32]-stimulated.

ET-1[1–32] induces the release of neutrophil gelatinase, which cleaves big ET-1[1–38] to ET-1[1–32]
To assess further neutrophil activation by ET-1[1–32], we studied superoxide production and gelatinase. ET-1[1–32] did not induce superoxide production (unstimulated neutrophils produced 0.6±0.6 nmol superoxide/5x106 cells/min vs. 0.3±0.2 nmol superoxide/5x106 cells/min in response to 100 nM ET-1[1–32], n=4, P>0.1), but evoked a concentration-dependent release of gelatinase that was prevented by the MEK inhibitor PD98059 (Fig. 3A ). Gelatin zymography revealed the presence of gelatinase B, but not gelatinase A, in cytosol and membrane preparations of human neutrophils (Fig. 3B ). Since gelatinase A can cleave big ET-1 to ET-1[1–32] (7) , we tested whether gelatinase B also possesses similar activity. Indeed, 4-aminophenylmercuric acetate-activated gelatinase B cleaved big ET-1 to yield ET-1[1–32], as demonstrated using HPLC and MALDI-TOF mass spectroscopy (Fig. 3C ).



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  		Figure 3. ET-1[1–32] induces gelatinase release from neutrophils. A) Neutrophils were challenged with ET-1[1–32] at 37°C for 30 min. Gelatinase release is expressed as percentage of total cellular enzyme activity released by neutrophils to the culture medium after incubation with ET-1[1–32] in the absence and presence of PD98059 (100 µM) for 30 min at 37°C. Values are means ± SE of four independent experiments. *P < 0.05 vs. control (unstimulated neutrophils). B) Elastin zymographic analysis showed that gelatinase B (MMP-9, 92 kDa), but not gelatinase A (MMP-2, 72 kDa), is a major gelatinase in cytosol (c) as well as membrane (m) preparations of neutrophils. C) Gelatinase B cleaved big ET-1[1–32] to yield ET-1[1–32] as demonstrated by HPLC (left panel) combined with mass spectrometric analysis (right panel).

Metabolism of ET-1[1–32] by neutrophils
We next investigated whether further cleavage of ET-1[1–32] was required for its bioactivity. ET-1[1–32] (100 pmol) was incubated with preparations of neutrophil cytosol (0.5–5 µg protein) or membrane (0.2–2 µg protein) and assessed for conversion into smaller peptides. A combination of HPLC with mass spectroscopy showed that ET-1[1–32] was minimally converted into other smaller peptides such as ET-1[1–22], ET-1[1–21], ET-1[1–20], and ET-1[1–19] (Fig. 4 ). In line with this, the effects of ET-1[1–32] on adhesion molecule expression were insensitive to phosphoramidon, an inhibitor of the metal-dependent ECEs (L-selectin expression was 35±6% and 31±7% of control, CD11b expression 178±17% and 184±16% of control in response to 100 nM ET-1[1–32] in the absence or presence of phosphoramidon, respectively, n=5, P>0.1).



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  		Figure 4. Metabolism of ET-1[1–32] by neutrophil proteases. ET-1[1–32] was incubated for 2 h with neutrophil membrane and cytosol preparations. The resulting peptides were analyzed by HPLC, followed by MALDI-TOF mass spectrometry. In brackets: sequences of ET-1[1–32]-derived peptides deduced from the mass spectrometry analysis. Bars: peaks in the region of the HPLC chromatogram corresponding to neutrophil proteins, not ET-1 peptides.

To further confirm that the biological effects of ET-1[1–32] were due to the inherent bioactivity of this peptide, neutrophils were challenged with big ET-1 (100 nM), the inactive precursor of ET-1 peptides, in the presence of phosphoramidon (an endothelin-converting enzyme inhibitor) and o-phenanthroline (an MMP inhibitor). There were no detectable changes in adhesion molecule expression on neutrophils under these conditions (L-selectin and CD11b expression were 110±6% and 102±3% of control, respectively, n=4, P>0.1). Bovine pTH[3–34] (0.1 to 1000 nM), which has 32 residues like ET-1[1–32], also failed to evoke significant changes in L-selectin and CD11b expression (data not shown).

ET-1[1–32] activates the Ras/Raf-1/MEK/Erk signaling pathway
Initial studies indicated that PD98059 prevented ET-1[1–32]-induced up-regulation of CD11b/CD18 expression but was less effective in reversing changes in L-selectin expression (Fig. 5 ). The phosphatidylinositol-3-kinase inhibitor wortmannin, the tyrosine kinase inhibitor genistein, or the selective p38 MAP kinase inhibitor SB 203580 did not significantly attenuate ET-1[1–32]-induced changes in adhesion molecule expression even though, by themselves, wortmannin and genistein each down-regulated L-selectin expression (Fig. 5) .



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  		Figure 5. Effects of protein kinase inhibitors on expression of neutrophil adhesion molecules. Isolated neutrophils were preincubated with PD98059, SB203580, wortmannin, or genistein for 20 min at 37°C, then challenged with 30 nM ET-1[1–32] for 30 min. Adhesion molecule expression is presented as the percentage of control. Mean fluorescence intensity for control samples: L-selectin, 62 ± 6; CD11b, 966 ± 116. Values are the mean ± SE of 6 independent experiments.

ET-1[1–32] induced phosphorylation of MEK and Erk relative to unstimulated controls (Fig. 6 A). MEK and Erk phosphorylation was rapid in onset (peak at between 2 and 5 min) and occurred in a concentration-dependent fashion (Fig. 6A ). The relative degree of Erk phosphorylation evoked by 1 µM PAF is shown for comparison. Erk phosphorylation was sensitive to PD98059 (Fig. 6B ). Within 2 min, ET-1[1–32] induced association of GTP-bound active Ras with the glutathione-S-transferase-Ras binding domain of Raf-1 (Fig. 6C ), which indicated Ras activation, and increased neutrophil Raf-1 kinase activity in a concentration-dependent fashion (Fig. 6D ).



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  		Figure 6. Activation of the Ras/Raf-1/MEK/Erk signaling pathway in neutrophils by ET-1[1–32]. A) Phosphorylation of MEK and Erk1/2. Neutrophils were challenged with ET-1[1–32] or PAF (1 µM) for the times indicated at 37°C and lysed; phosphorylation of kinases was assessed in immunoblots using polyclonal phospho-specific antibodies. Results are representative of 4 independent experiments. B) Inhibition of Erk phosphorylation by PD98059. Neutrophils were preincubated with PD98059 (100 µM) for 10 min and challenged with ET-1[1–32]. Shown is a representative immunoblot; the experiment was repeated three times. C) GTP-bound active Ras was isolated from neutrophil lysates by affinity precipitation with a GST-Ras binding domain fusion protein, followed by immunoblot analysis with an anti-Ras antibody. Shown is a representative result; the experiment was repeated three times. D) Activation of Raf-1 kinase. Neutrophils were challenged with ET-1[1–32] for 2 min at 37°C, lysed, Raf-1 was immunoprecipitated, and Raf-1 kinase activity was measured as described in Materials and Methods. Control experiments performed in the absence of substrate or using a nonspecific control antiserum resulted in levels of Raf-1 activity below those of unstimulated lysate. Results shown are the mean ± SE of 4 experiments. *P < 0.05; **P < 0.01 vs. unstimulated.

To further assess the role of Erk in mediating neutrophil responses to ET-1[1–32], we compared the effects of PD98059, a specific inhibitor that binds to MEK and prevents its phosphorylation by Raf-1 or other kinases (37) on neutrophil responses to ET-1[1–32]. Preincubation of neutrophils with PD98059 (100 µM) inhibited ET-1[1–32]-stimulated Erk phosphorylation, up-regulation of CD11b expression, and gelatinase release by 75 ± 6%, 72 ± 5%, and 80 ± 5%, respectively.

ET-1[1–32] enhances neutrophil adhesion to human coronary artery endothelial cells
Only a few neutrophils were able to bind to unstimulated HCAEC. Neutrophil adherence was enhanced 12-fold by activation of HCAEC with LPS for 4 h (Fig. 7A ). The number of adherent neutrophils was further enhanced when neutrophils were added together with ET-1[1–32] to LPS-activated HCAEC (Fig. 7A ). ET-1[1–32] did not enhance neutrophil adhesion to unstimulated HCAEC. Culture of HCAEC with ET-1[1–32] for 4 h produced increases, albeit to a markedly less extent than LPS, in the number of adherent neutrophils (Fig. 7B ). For instance, on average 2.7-fold more neutrophils adhered to HCAEC cultured with 100 nM ET-1[1–32] for 4 h than to unstimulated HCAEC (Fig. 7B ). The number of adherent neutrophils to ET-1[1–32]-activated HCAEC was further enhanced when the adhesion assay was performed in the presence of ET-1[1–32] (Fig. 7B ), suggesting an activation of both neutrophils and endothelial cells by ET-1[1–32]. Overall, these results indicate that ET-1[1–32] by itself is capable of enhancing neutrophil adhesion to HCAEC.



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  		Figure 7. ET-1[1–32] promotes adhesion of neutrophils to coronary artery endothelial cells (HCAEC). Confluent HCAEC monolayers were cultured in medium only (control) or activated for 4 h with A) 1 µg/ml LPS or B) ET-1[1–32]. Radiolabeled neutrophils (PMNs) without or together with ET-1[1–32] were then added and incubated with HCAEC on an orbital shaker for 30 min at 37°C. Values are expressed as mean ± SE of six experiments using neutrophils from different donors. *P < 0.05; **P < 0.01 vs. LPS-stimulated (filled column); #P < 0.05; ##P < 0.01 vs. unstimulated (open column). C) Inhibition of ET-1[1–32]-stimulated neutrophil attachment to LPS-activated HCAEC by function-blocking, anti-E-selectin, anti-L-selectin, and anti-CD18 monoclonal antibodies (mAb). HCAEC were cultured with LPS for 4 h, then neutrophils together with 30 nM ET-1[1–32] were added for 30 min at 37°C in the absence (medium) or presence of function-blocking mAbs, as indicated. The irrelevant mAb MOPC-21 (IgG) was used as a negative control. Results are expressed as mean ± SE of 6 experiments using neutrophils from different donors. +P < 0.05; ++P < 0.01; +++P < 0.001 vs. ET-1[1–32] without mAbs.

Preincubation of neutrophils with the selective ETA receptor antagonist FR 139317 (1 µM) reduced ET-1[1–32] (30 nM) -stimulated neutrophil adherence to LPS-activated HCAEC (from 6.6±0.5x104 adherent cells/well, n=7, to 4.2±0.3x104 adherent cells/well, n=4, P<0.01). The number of adherent neutrophils did not differ significantly from those observed when neutrophils were added to LPS-activated HCAEC in the absence of ET-1[1–32] (4.1±0.2x104 adherent cells/well, n=7, P>0.1). On the other hand, the selective ETB receptor antagonist BQ 788 (10 µM) had no detectable effect on ET-1[1–32]-stimulated adhesion (6.3±0.4x104 adherent cells/well, n=4, P>0.1), further indicating ETA receptors as the relevant ET receptor subtype responsible for the adhesion enhancing effect of ET-1[1–32].

Since multiple receptors are involved in neutrophil adhesion to activated endothelial cells even under nonstatic conditions (36 , 38) and ET-1[1–32] affected both L-selectin and CD11b/CD18 expression, we assayed the contribution of L-selectin, CD18 integrins, and E-selectin to the binding interaction by using function-blocking mAbs to these adhesion molecules. ET-1[1–32]-stimulated neutrophil attachment to LPS-activated HCAEC was blocked by mAbs binding to E-selectin (43±7%, n=6) and CD18 (59±3%), whereas the anti-L-selectin mAb produced only a slight inhibition (12±6%) (Fig. 7C ). The combination of these mAbs inhibited the neutrophil adhesion by 90% (Fig. 7C ).

Effects of ET-1[1–32] on expression of ICAM-1 and E-selectin
Under our experimental conditions, 43% and 1% of untreated HCAEC expressed ICAM-1 and E-selectin on average, respectively (Table 1 ). Treatment of HCAEC for 4 h with ET-1[1–32] slightly increased the overall expression of E-selectin and ICAM-1 as well as the percentage of cells expressing these adhesion molecules (Table 1) . As a positive control, LPS treatment produced on average 14- and 9-fold increases in E-selectin and ICAM-1 expression, respectively (Table 1) .


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  		Table 1. E-selectin and ICAM-1 expression on human coronary artery endothelial cells challenged with ET-1[1-32] and LPSa


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study identifies a novel role for gelatinase-generated ET-1[1–32] in the regulation of leukocyte–endothelial cell adhesion. ET-1[1–32] down-regulated neutrophil L-selectin expression, up-regulated CD11b/CD18 expression, and promoted neutrophil adhesion to activated HCAEC predominantly via a CD18-dependent mechanism. Moreover, ET-1[1–32] evoked gelatinase B release from neutrophils, which cleaved big ET-1, yielding ET-1[1–32], and suggests a new feed-forward loop for subsequent neutrophil activation and gelatinase release. We also investigated the mechanisms of ET-1[1–32] signaling in human neutrophils, suggesting a possible role for Erk in ET-1[1–32]-stimulated neutrophil responses.

The effects of ET-1[1–32] on neutrophils were unlikely to be due to a conversion of ET-1[1–32] into ET-1, since ET-1[1–32] lacks the COOH terminus of big ET-1 required for recognition and cleavage by endothelin-converting enzymes (39) . Indeed, the endothelin-converting enzyme inhibitor phosphoramidon did not affect neutrophil responses to ET-1[1–32], and HPLC and mass spectrometric analysis revealed that incubation of ET-1[1–32] with neutrophil cytosol or membrane fractions resulted in only a negligible amount of ET-1. It is also unlikely that the biological actions of ET-1[1–32] can be attributed to the small quantities of ET-1[1–22], ET-1[1–20] and ET-1[1–19], because they display negligible bioactivity (39 , 40) . Neutrophils did not metabolize ET-1[1–32] to ET-1[1–31], a vasoactive peptide cleaved by mast cell chymase from big ET-1 (41) . These results suggest that ET-1[1–32] is somewhat resistant to cleavage by neutrophil proteases and that further metabolism may not be a prerequisite for its biological actions on human neutrophils. The neutrophil stimulatory actions of ET-1[1–32] are specific for this peptide sequence because big ET-1 had no stimulatory effect when its cleavage to ET-1[1–21] or ET-1[1–32] was prevented. Furthermore, an unrelated 32-residues peptide pTH[3–34] failed to mimic the biological effects of ET-1[1–32].

On a molar basis, ET-1[1–32] appears to be a more potent neutrophil agonist than ET-1. At nanomolar concentrations, ET-1[1–32] down-regulated L-selectin expression and released gelatinase from tertiary granules, reflecting neutrophil activation. Because the most readily mobilizable store of CD11b/CD18 is in a granule distinct from the classic azurophil and secondary granules (42) but may be associated with tertiary granules, up-regulation of CD11b/CD18 expression can occur without degranulation of azurophil and specific granules. Furthermore, like ET-1 (43) , ET-1[1–32] does not induce superoxide production. Therefore, ET-1[1–32] may function as a selective neutrophil agonist. The present observations that ET-1[1–32]-induced changes in adhesion molecule expression and gelatinase release can be effectively attenuated by PD98059 coupled with those of Yu et al. (44) , who have reported a dissociation between neutrophil Erk activation and superoxide generation, are consistent with this notion.

Pharmacological and receptor binding studies showed that human neutrophils predominantly express ETA receptors (14) . The actions of ET-1[1–32] on neutrophil expression of L-selectin and CD11b as well as gelatinase release were significantly inhibited by the selective ETA receptor antagonist FR 139317, but not by the ETB receptor antagonist BQ 788. These findings clearly point to the ETA receptor as being the relevant receptor subtype responsible for these actions of ET-1[1–32].

Erk phosphorylation in response to some G-protein-coupled receptors proceeds via Ras, Raf-1, and MEK (24 , 27 , 28) . Our results suggest that ET-1[1–32] stimulation of Erk also involves this signaling pathway, since ET-1[1–32] 1) stimulated association of Ras with Raf-1; 2) increased Raf-1 kinase activity; and 3) resulted in phosphorylation of MEK. The specific MEK inhibitor PD98059 also inhibited Erk phosphorylation and neutrophil responses to ET-1[1–32], although the inhibition was incomplete. These latter findings are consistent with previous studies reporting a tight correlation between Erk activation and neutrophil aggregation (homotypic adhesion) or neutrophil adhesion to endothelial cells in response to chemoattractants (24) , arachidonic acid (25) , and peroxynitrite (26) . Taken together, these observations indicate an essential role for Erk in signaling neutrophil adhesive responses.

ET-1[1–32] markedly enhanced neutrophil adhesion to activated HCAEC. About 3.5-fold more neutrophils adhered to LPS-stimulated than to ET-1[1–32]-stimulated HCAEC, indicating that ET-1[1–32] is a considerably less potent activator of HCAEC than LPS. Since neutrophils were incubated in the adhesion assays for 30 min with activated HCAEC, stimulation of neutrophil adhesion by ET-1[1–32] can be attributed primarily to the effects of this peptide on neutrophils. No adhesion experiments were performed with neutrophils alone preincubated with ET-1[1–32] since, by up-regulating CD11b/CD18 expression, this peptide may induce neutrophil aggregation similar to that observed with ET-1 (15 , 16) , making interpretation of the results difficult.

Leukocyte–endothelial cell interaction involves a complex interplay among adhesion molecules (45) . Indeed, the experiments with function-blocking mAbs revealed the involvement of CD18 integrins, E-selectin, and L-selectin in mediating ET-1[1–32]-induced neutrophil–HCAEC adhesion. Thus, in the presence of ET-1[1–32], neutrophil adhesion to LPS-stimulated HCAEC was only slightly inhibited by the anti-L-selectin mAb whereas ~60% of the adhesion was blocked by the anti-CD18 mAb. By contrast, anti-L-selectin mAb and anti-CD18 mAb inhibited 22 and 28% of neutrophil attachment to LPS-activated HCAEC in the absence of ET-1[1–32] (26) . ET-1[1–32] also produced small increases in overall expression of E-selectin and ICAM-1 on HCAEC and slightly increased the number of cells that stained positive for these adhesion molecules. Previously we had found HCAEC to express ETB receptors (14) , suggesting that ET-1[1–32] may not function as an ETA receptor-selective agonist. ETA and ETB receptors both mediate the vasoconstrictor action of ET-1[1–32] in mesenteric circulation (7) . The role of ETB receptors in mediating neutrophil adhesive responses to ET-1 appears to be controversial, as both antiadhesive (46) and proadhesive functions (47 48 49 50) have been reported. Sanz et al. (49) have reported that superfusion of rat mesenteric postcapillary venules with 1 nM ET-1 resulted in P-selectin-dependent leukocyte rolling. When administered intravenously, however, much higher doses of ET-1 (0.2 to 3 nmol/kg) were required to evoke leukocyte rolling and firm adhesion in the same vascular bed (50) . This latter study also showed the effectiveness of ETA receptor antagonists in blocking the actions of ET-1, consistent with an action of leukocytes (which express ETA receptors) (14) . On the other hand, the receptor subtype responsible for P-selectin expression has not yet been characterized. In our experiments, ET-1[1–32] augmented E-selectin and ICAM-1 expression at concentrations 30- to 100-fold higher than those described for ET-1 to induce P-selectin expression (49) , raising the possibility of species and/or receptor subtype differences. It is also possible that short- and long-term exposure of endothelial cells to ET-1 (and perhaps to ET-1[1–32]) might affect endothelial adhesiveness differently; the underlying mechanisms remain to be investigated.

The simultaneous overexpression of gelatinolytic MMPs (MMP-2 and MMP-9) as well as of big ET-1 at the sites of tissue injury and inflammation (1 , 8 9 10 11 12) would suggest that high amounts of ET-1[1–32] may be formed locally. Endothelial damage, in particular, and a consequent loss of endothelin-converting enzyme activity may favor the conversion of big ET-1 into ET-1[1–32] by gelatinase A localized in the intima and media of blood vessels (7) . Rapid release of bioactive gelatinase A could occur in response to thrombin stimulation (51) , raising the possibility of ET-1[1–32] formation under conditions associated with thrombin generation.

Based on these observations, we propose a model for neutrophil activation by ET-1[1–32] (Fig. 8 ). We suggest that ET-1[1–32] binds to neutrophil ETA receptors and that G-protein activity then initiates Erk phosphorylation via Ras, Raf-1, and MEK. Our results further suggest that Erk activation is required for ET-1[1–32] up-regulation of CD11b/CD18 expression and, consequently, stimulation of neutrophil adhesion to activated endothelial cells. ET-1[1–32] also induces release of gelatinase B from neutrophils, which in turn cleaves big ET-1 to yield ET-1[1–32]. These findings suggest a novel positive feedback loop for the generation of ET-1[1–32] and a self-amplifying loop for stimulation of gelatinase B release and neutrophil activation/adhesion. It should be noted, however, that ET-1[1–32] could also induce a modest up-regulation of ICAM-1 and E-selectin expression on endothelial cells, thereby promoting neutrophil adhesion. Whether these actions are mediated via activation of the ETB receptors present on HCAEC (14) and the underlying signaling pathways remain to be investigated. However, our data clearly indicate that ET-1[1–32] promotes neutrophil adhesion predominantly through activation of ETA receptors expressed on neutrophils.



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  		Figure 8. Positive feedback loop for neutrophil activation by gelatinase-generated ET-1[1–32]. See text for details. Broken arrows indicate yet undefined pathways.

In summary, the present results indicate a novel role for two MMPs (gelatinase A and gelatinase B) in converting big ET-1[1–38] into ET-1[1–32] to regulate the expression of adhesion molecules on the neutrophil surface, leading to their increased attachment to the endothelium. To our knowledge, this constitutes the first indication that MMPs via cleavage of big ET-1 could affect leukocyte–endothelial cell interactions; as such, it adds a new facet to our understanding of the functions of MMPs in acute tissue injury and inflammation.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the Medical Research Council of Canada/Canadian Institutes of Health Research (MRC/CIHR, MT-12573 to J.G.F., MT-13420 to J.S.D.C. and J.G.F., and MT-13404 to S.T.D.) and the Heart and Stroke Foundation of Canada (to S.T.D. and J.G.F.). C.F.-P. is a postdoctoral fellow of the CIHR and Alberta Heritage Foundation for Medical Research; C.Z. is the recipient of a Ph.D. studentship award from the MRC/CIHR.


   FOOTNOTES
 
1 C.F.-P. and C.Z. contributed equally to this study.


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K. J. Greenlee, Z. Werb, and F. Kheradmand
Matrix Metalloproteinases in Lung: Multiple, Multifarious, and Multifaceted
Physiol Rev, January 1, 2007; 87(1): 69 - 98.
[Abstract] [Full Text] [PDF]

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