Leukocyte Engagement of Platelet Glycoprotein Ib via the Integrin Mac-1 Is Critical for the Biological Response to Vascular Injury
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《循环学杂志》
the Cardiovascular Division, Brigham and Women’s Hospital, Boston, Mass (Y.W., M.S., Z.C., C.S., K.C., A.C.Z., D.I.S.)
The Cleveland Clinic Foundation, Cleveland, Ohio (V.U., E.P.)
Thrombosis Research Section, Departments of Medicine and Molecular and Human Genetics, Baylor College of Medicine, Houston, Tex (J.L.)
Portola Pharmaceuticals, South San Francisco, Calif (P.A.).
Abstract
Background— Leukocyte-platelet interactions are critical in the initiation and progression of atherosclerosis as well as restenosis. Although the leukocyte integrin Mac-1 (M2, CD11b/CD18) has been implicated in the firm adhesion and transmigration of leukocytes at sites of platelet deposition, the precise M2 counterligand responsible for mediating adhesion-strengthening interactions between neutrophils and platelets in vivo has not previously been identified.
Methods and Results— Our previous studies have established the P201-K217 sequence in the MI domain as the binding site for platelet glycoprotein (GP) Ib. Here we report that antibody targeting of M(P201-K217) reduced M2-dependent adhesion to GP Ib but not other M2 ligands, including fibrinogen, intercellular adhesion molecule-1, and junctional adhesion molecule-3. Anti-M(P201-K217) inhibited the firm adhesion of both human and murine leukocytes to adherent platelets under laminar flow conditions. In a mouse femoral artery wire injury model, antibody targeting of M(P201-K217) reduced leukocyte accumulation after injury that was accompanied by inhibition of cellular proliferation and neointimal thickening.
Conclusions— This study demonstrates that GP Ib is a physiologically relevant ligand for M2 and that integrin engagement of GP Ib is critical to leukocyte function and the biological response to vascular injury. These observations establish a molecular target for selectively disrupting leukocyte-platelet complexes that promote inflammation in thrombosis and restenosis.
Key Words: cell adhesion molecules inflammation leukocytes platelets restenosis
Introduction
Leukocyte-platelet interactions are critical in the initiation and progression of atherosclerosis1 as well as restenosis.2 Platelet deposition precedes inflammatory cell accumulation in mouse models of atherogenesis, and inhibition of platelet adhesion dramatically reduces atherosclerotic lesion formation.1 However, the specific receptors responsible for mediating adhesive interactions between neutrophils and platelets in vivo are incompletely defined.
Recruitment of circulating leukocytes to vascular endothelium requires multistep adhesive and signaling events including selectin-mediated attachment and rolling, leukocyte activation, and integrin-mediated firm adhesion and diapedesis that result in the infiltration of inflammatory cells into the blood vessel wall.3 Firm attachment is mediated by members of the 2-integrin family, LFA-1 (L2, CD11a/CD18), Mac-1 (M2, CD11b/CD18), and p150,95 (X2, CD11c/CD18), which bind to endothelial counterligands (eg, intercellular adhesion molecule-1 [ICAM-1]), to endothelial-associated extracellular matrix proteins (eg, fibrinogen), or to glycosaminoglycans.4
Clinical Perspective p 3000
Importantly, leukocyte recruitment also occurs at sites of vascular injury where the lining endothelial cells have been denuded and platelets and fibrin have been deposited. A similar sequential adhesion model of leukocyte attachment to and transmigration across surface-adherent platelets has been proposed.5 The initial tethering and rolling of leukocytes on platelet P-selectin6 are followed by their firm adhesion and transplatelet migration, processes that are dependent on M2.5
Our laboratory has focused on identifying the platelet counterreceptor for M2. Evaluation of the structural features of integrins provides insight into candidate platelet counterreceptors for M2. Integrins are heterodimeric proteins composed of 1 and 1 subunit. A subset of integrin subunits, including M, contains an inserted domain (I domain) of &200 amino acids that is implicated in ligand binding7 and is strikingly similar to the A domains of von Willebrand factor (vWf),8 one of which, A1, mediates the interaction of vWf with its platelet receptor, the glycoprotein (GP) Ib-IX-V complex. Because of the similarity of the vWf A1 domain and the MI domain, we hypothesized that GP Ib might also be able to bind M2 and reported that GP Ib is indeed a constitutively expressed counterreceptor for M2.9 Furthermore, under the conditions used in these studies, the predominant interaction between neutrophils and platelets appeared to be between M2 and GP Ib.
The MI domain contributes broadly to the recognition of ligands by M27 and specifically to the binding of GP Ib.9 This region has also been implicated in the binding of ICAM-1,10 C3bi,11 and fibrinogen.10 We have recently localized the binding site for GP Ib within the MI domain segment M(P201-K217) using a strategy based on the differences in the binding of GP Ib to the MI and LI domains that involved several independent approaches, including screening of mutant cells, synthetic peptides, site-directed mutagenesis, and gain-in-function analyses.12 The grafting of only 2 amino acids within this segment into the LI domain converted it to a GP Ib binding protein. Thus, a small segment that has a defined structure within the MI domain is necessary and sufficient for GP Ib binding.
While M2 has a broad ligand binding repertoire, the precise ligand responsible for leukocyte accumulation at sites of platelet deposition remains to be defined. Central to unraveling the precise biological roles of M2 is defining the biologically relevant ligand(s) for this integrin. In this study we show that antibody targeting of M(P201-K217) reduced M2-dependent adhesion to GP Ib, but not other ligands, in vitro and leukocyte accumulation after vascular injury in vivo. In a mouse femoral artery injury model, treatment with anti-M(P201-K217) was accompanied by inhibition of cellular proliferation and neointimal thickening.
Methods
Materials
The soluble extracellular region of GP Ib (ie, glycocalicin or sGP Ib) was purified as we reported previously.9 Human fibrinogen depleted of plasminogen, vWf, and fibronectin was purchased from Enzyme Research Laboratories (South Bend, Ind). Human C3bi was obtained from Calbiochem (San Diego, Calif). Recombinant human ICAM-1 was purchased from R&D Systems, Inc (Minneapolis, Minn). Human junctional adhesion molecule-3 (JAM-3) was kindly provided by Dr Sentot Santoso (Justus-Liebig University, Giessen, Germany). The monoclonal antibody LPM19c, directed to the MI domain, was kindly provided by Dr Karen Pulford (Radcliffe, Oxford, UK). The monoclonal antibody 1B5, directed to murine IIb3 and capable of inhibiting murine platelet aggregation, was a generous gift of Dr Barry Coller (Rockefeller University, New York, NY). Peptides corresponding to the murine GP Ib binding site (SGSG-214LYFRHWLQENANNVYL229-C) for Mac-113 or scrambled control (SGSG-VEAFHLNYYRNNVWLQ-C) were obtained from the W.M. Keck Biotechnology Resource Center (Yale University, New Haven, Conn). The peptides were diluted in dimethyl sulfoxide and stored at –80°C.
Antibody Generation
An affinity purified peptide-specific polyclonal antibody (termed anti-M2) to the Mac-1 binding site for GP Ib was generated by immunizing rabbits with C-201PITQLLGRTHTATGIRK217 coupled to KLH (Zymed Laboratories, South San Francisco, Calif) corresponding to the human M(P201-K217) sequence. After test bleeds demonstrated high-titer anti-sera binding to solid-phase immobilized C-P201-K217, peptide-specific polyclonal antibody was purified by affinity matrix chromatography (Zymed Laboratories). Nonimmune rabbit IgG served as control antibody.
Cell Lines and Culture Conditions
293 cells expressing human M2 receptors were established and maintained as previously described.12
Preparation of Murine Neutrophils
Neutrophils from wild-type and Mac-1–deficient C57Bl/J6 mice14 were harvested and purified (>90% neutrophils by cytospin) from the peritoneal cavity 6 hours after the intraperitoneal injection of 1 mL sterile 3% thioglycolate broth, as previously described.9 Animal care and procedures were reviewed and approved by the Harvard Medical School Standing Committee on Animals and performed in accordance with the guidelines of the American Association for Accreditation of Laboratory Animal Care and the National Institutes of Health.
Flow Cytometry
Wild-type and Mac-1–deficient neutrophils (106) were incubated with anti-M2 or nonimmune IgG (25 μg/mL). After they were washed, anti-M2 binding was assessed with the use of FITC-conjugated Fab2 anti-rabbit (BD Biosciences) by flow cytometry (FACScan, BD Biosciences-Immunocytometry Systems).
Blood Collection
Venous blood was obtained from volunteers who had not consumed aspirin or other nonsteroidal anti-inflammatory drugs for at least 10 days and was anticoagulated with 10 mmol/L trisodium citrate. All subjects gave written informed consent to the protocol, which was approved by the institutional review board. Venous blood was also collected from the vena cava of anesthetized mice and anticoagulated with 10 mmol/L trisodium citrate.
Adhesion Assays
293 cells expressing M2 were harvested with cell-dissociating buffer (Life Technologies) for 1 minute at 22°C, washed twice, resuspended in serum-free media, and loaded with BCECF AM [2',7'-bis-(2-carboyethyl)-5-(and-6)-carboxyfluorescein, acetoxy-methyl ester] (1 μmol/L) according to the manufacturer’s protocol (Molecular Probes). Cells (105 per well) were placed in 48-well tissue culture plates (Costar) coated with 200 μL of 5 nmol/L fibrinogen or 50 nmol/L C3bi, ICAM-1, JAM-3, or sGP Ib overnight at 4°C and then blocked with 0.5% polyvinylpyrrolidone for 1 hour at room temperature. Adhesion was stimulated with phorbol 12-myristate 13-acetate (PMA) (20 ng/mL) in the presence of 2 mmol/L Mg2+. Plates were washed with 0.9% NaCl (3 to 5 times), and adhesion was quantified by measuring the fluorescence of BCECF AM–loaded cells with a Cytofluor II fluorescence multiwell microplate reader (PerSeptive Biosystems). The effect of anti-M2 on adhesion was assessed by preincubating cells with anti-M2 or control antibody (10 μg/mL) for 30 minutes at 37°C before the addition of cells.
The effect of anti-M2 on murine neutrophil adhesion to a peptide corresponding to the murine GP Ib binding site for Mac-1 was also investigated. BCECF AM–loaded wild-type neutrophils (2x105 per well) were placed in 96-well tissue culture plates coated with 100 μL of GP Ib peptide (SGSG-214LYFRHWLQENANNVYL229-C) or scrambled control (SGSG-VEAFHLNYYRNNVWLQ-C) (50 μg/mL) overnight at 4°C and then blocked with 0.5% gelatin for 1 hour at room temperature. Plates were washed with PBS (3 to 5 times) and adhesion quantified. The effect of anti-M2 on adhesion was assessed by preincubating cells with anti-M2 or control antibody.
In Vitro Analysis of Cellular Adhesion Under Laminar Flow Conditions
The laminar flow chamber used in this assay has been described previously.15,16 Anticoagulated blood was incubated with rhodamine-6G (0.2 μg/mL, Sigma), the GP IIb/IIIa inhibitor eptifibatide (2.5 μg/mL, Millennium Pharmaceuticals), 2 mmol/L MgCl2, and anti-M2 or control antibody (1 to 25 μg/mL) and then perfused for 5 minutes through human type III collagen–coated rectangular capillaries at 625 s–1, resulting in the deposition of adherent platelets without large platelet aggregates. Leukocyte rolling and arrest were then monitored by perfusion for 10 minutes through the capillary at 125 s–1. The number of arrested cells, after perfusion with PBS at 625 s–1, was quantified in each of 5 random x10 fields (area &0.3 mm2).
For murine blood, anticoagulated blood was incubated with rhodamine-6G, 2 mmol/L MgCl2, and the anti-murine IIb3 monoclonal antibody 1B5 (5 μg/mL) and then perfused for 5 minutes through type III collagen–coated capillaries at 250 s–1, resulting in the deposition of adherent platelets. The firm arrest of murine neutrophils (106/mL) incubated with anti-M2 or control antibody (25 μg/mL) was then monitored by perfusion for 5 minutes through the capillary at 125 s–1. The number of arrested cells was then quantified as above. A minimum of 6 mice were used for each group studied.
Femoral Artery Injury
Male C57Bl/6J mice aged 8 to 10 weeks were anesthetized on day 0 with the use of ketamine (80 mg/kg IP) and xylazine (5 mg/kg IP), and wire injury (0.010 in) of the femoral artery was performed as described previously.17 All animals survived until the time of planned euthanasia without bleeding or infection.
Tissue Harvesting and Analysis
One day (control antibody: n=6; anti-M2: n=6), 5 days (control antibody: n=9; anti-M2: n=8), or 28 days (control antibody: n=9; anti-M2: n=9) after vascular injury, anesthesia was administered, the chest cavity was opened, and the animals were euthanized by right atrial exsanguination. A 22-gauge butterfly catheter was inserted into the left ventricle for in situ pressure perfusion at 100 mm Hg with 0.9% saline for 1 minute followed by fixation with 4% paraformaldehyde in 0.1 mol/L phosphate buffer, pH 7.3, for 10 minutes. The right and left femoral arteries were excised and immersed in buffered paraformaldehyde. Spleen and small intestine from 3 animals were harvested as control tissues for immunohistochemistry. All animals received BrdU, 50 mg/kg IP, 18 hours and 1 hour before euthanasia.
Femoral arteries were embedded, and 2 cross sections cut 1 mm apart were stained with hematoxylin and eosin and Verhoeff tissue elastin stain. A histologist blinded to treatment group measured the luminal, intimal, and medial areas of each cross-sectional plane using a microscope equipped with a CCD camera interfaced to a computer running NIH Image version 1.60 software. Results for the 2 planes of each artery were averaged. For baseline medial and external elastic lamina areas before injury or antibody treatment, a group of mice (n=5) was also euthanized for quantitative morphometry. For immunohistochemistry, standard avidin-biotin procedures for mouse CD45 (leukocyte common antigen, BD Biosciences, San Diego, Calif), mouse neutrophil-specific marker (monoclonal antibody 7/4, Serotec, Indianapolis, Ind), mouse macrophage-specific marker Mac-3 (mAb M3/84, BD Biosciences), and BrdU (DAKO, Carpinteria, Calif) were used. Immunostained sections were quantified as the number of immunostained positive cells per total number of nuclei.
Antibody Treatment
Antibody treatments were via intraperitoneal injection of 250 μg antibody 4 hours before injury (day 0) and then 100 μg antibody on days 2, 4, 6, 8, 10, and 12. Mice were divided into 2 treatment groups: (1) rabbit anti-M2 and (2) nonimmune rabbit IgG as control antibody.
Statistical Analysis
Data are presented as mean±SD. Comparisons between groups were performed by unpaired t test. Probability values <0.05 were considered significant.
Results
Antibody Targeting M2–GP Ib
Although M2 is capable of binding a broad repertoire of ligands, the precise ligand responsible for leukocyte accumulation at sites of vascular injury remains to be defined. We hypothesized that the M2–GP Ib interaction would likely dominate on the basis of our in vitro observations9 that leukocyte adhesion to platelets was largely abrogated by sGP Ib, antibodies to GP Ib, and pretreatment of platelets with the snake venom metalloprotease mocarhagin, whose major platelet substrate is GP Ib. Furthermore, agonist-stimulated leukocyte-platelet complexes were significantly decreased in whole blood from a patient with Bernard-Soulier syndrome compared with a normal control.9 Our prior report12 identified the M(P201-K217) segment as the M2 binding site for GP Ib. A synthetic peptide (termed M2) duplicating the P201-K217 sequence, but not scrambled versions, directly bound sGP Ib and inhibited M2-dependent adhesion to sGP Ib and adherent platelets. Therefore, as a first step in targeting M2–GP Ib for in vivo experiments, an affinity-purified peptide-specific polyclonal antibody to this site (anti-M2) was generated by immunizing rabbits with C-201PITQLLGRTHTATGIRK217 coupled to KLH corresponding to the human M(P201-K217) sequence. The human sequence (M 201PITQLLGRTHTATGIRK217) is highly homologous to the corresponding murine M201PIKQLNGRTKTASGIRK217 sequence with 13 of 17 identical residues.
We first examined the effect of anti-M2 antibody on M2-dependent static adhesion to sGP Ib, ICAM-1, fibrinogen, and JAM-3 (Figure 1). Anti-M2 antibody was preincubated with M2-expressing 293 cells, and adhesion was stimulated with PMA in the presence of Mg2+. M2-expressing 293 cells adhered to sGP Ib, ICAM-1, fibrinogen, and JAM-3, and this adhesion was blocked by the anti-M monoclonal antibody LPM19c (data not shown), indicating that adhesion is predominantly M2 dependent. Anti-M2 strongly inhibited PMA-stimulated adhesion to sGP Ib (percent inhibition versus control=80±4; P<0.001). In contrast, anti-M2 had little or no effect on M2-expressing 293 cell adhesion to fibrinogen (percent inhibition versus control=21±8), JAM-3 (13±18%), or ICAM-1 (0±9%). This is consistent with our prior observation that M2 peptide itself only inhibited adhesion to sGP Ib but not other ligands.12 Taken together, these observations suggest that anti-M2, directed to the M(P201-K217) sequence within the MI domain, is capable of selectively blocking M2 binding to GP Ib.
Anti-M2 Abrogates the Firm Adhesion of Leukocytes Under Flow
To evaluate the potential for anti-M2 to modulate the adhesion of human blood cells under laminar flow conditions, we perfused whole blood, in the presence of the platelet aggregation inhibitor eptifibatide, over collagen-coated capillaries at a shear rate of 625 s–1, resulting in the deposition of adherent platelets without large platelet aggregates.16 Leukocyte rolling and firm arrest on adherent platelets were observed by lowering the shear rate to 125 s–1. The number of arrested cells was unaffected by nonimmune IgG (control versus nonimmune IgG, P>0.05) (Figure 2). In contrast, anti-M2 dose-dependently inhibited leukocyte arrest under flow (maximal percent inhibition=92±8; P<0.001).
Anti-M2 Binds to Murine Neutrophils and Blocks Adhesion to Murine Platelets
We next verified that anti-M2 would bind to murine Mac-1 and block murine neutrophil adhesion to murine GP Ib or murine platelets. As determined by FACS, anti-M2 bound to wild-type neutrophils (mean fluorescence intensity [MFI]= 252±7) but not Mac-1–deficient neutrophils (MFI=36±15); moreover, binding of anti-M2 to Mac-1–deficient neutrophils (MFI=53±12) was equivalent to that observed with nonimmune IgG (MFI=68±9).
We next turned to examining the effect of anti-M2 on murine neutrophil adhesion. Thioglycolate-elicited neutrophils were added to wells coated with a peptide corresponding to murine GP Ib binding site for Mac-1 (SGSG-214LYFRHWLQENANNVYL229-C).13 Wild-type neutrophils bound to GP Ib peptide but not to scrambled peptide control (Figure 3A). Anti-M2, but not nonimmune IgG, significantly inhibited neutrophil adhesion to murine GP Ib peptide (maximal percent inhibition=70±3).
We next investigated whether anti-M2 would inhibit murine leukocyte adhesion to murine platelets under laminar flow conditions. Anticoagulated murine blood, in the presence of 1B5 monoclonal antibody that blocks IIb3 integrin–dependent murine platelet aggregation, was perfused over type III collagen–coated capillaries at a shear rate of 250 s–1, resulting in the deposition of adherent platelets without large platelet aggregates. The firm arrest of murine neutrophils (106/mL) incubated with anti-M2 or nonimmune IgG was then monitored by perfusion for 10 minutes through the capillary at 125 s–1. Anti-M2 inhibited (percent inhibition=81±4) leukocyte arrest under laminar flow conditions (Figure 3B). Taken together, these observations indicate that anti-M2 is capable of inhibiting murine Mac-1 binding to murine GP Ib peptide and murine leukocyte adhesion to murine platelets.
Anti-M2 Reduces Leukocyte Recruitment and Neointimal Thickening After Vascular Injury
To determine whether the M2–GP Ib interaction is required for neointimal formation, we performed femoral artery wire injury in mice treated with anti-M2 or nonimmune IgG control before injury and then every other day through 12 days after injury. Wire injury is accompanied by endothelial denudation, platelet and fibrin deposition, and prominent vascular inflammation.17 This model has been useful in demonstrating that inflammatory cell recruitment and function modulate neointimal formation.18–20
We first examined leukocyte recruitment after injury over time. A role for M2–GP Ib in this process was implicated because altered leukocyte accumulation within vessels was observed in injured vessels from anti-M2 compared with control mice. Inflammatory cells (CD45-positive) invading the media at 1 day were reduced by 75% from 7.9±5.0% to 2.0±1.6% of total cells (P=0.021) in anti-M2–treated compared with control antibody–treated mice (Figures 4 and 5). Accumulation of leukocytes in the developing neointima was also reduced significantly by anti-M2, with a 42% reduction at 5 days (P=0.047) and 58% reduction at 28 days (P=0.012).
We expanded the CD45 analysis by immunostaining using cell-specific markers. Neutrophil (ie, monoclonal antibody 7/4-positive cells) accumulation within the media at 1 day was reduced by 75% in anti-M2–treated compared with control antibody–treated mice (control: 8.9±6.7% versus anti-M2: 2.2±1.6%; P=0.043). Macrophages (ie, Mac-3–positive cells) were essentially undetectable (<0.5% of total medial cells) 1 day after injury. Accumulation of neutrophils in the developing neointima at 5 days was also reduced by 47% with anti-M2 (control: 26.2±8.0% versus anti-M2: 13.9±4.9%; P=0.036). Concordant with the CD45 staining, intimal macrophages (Mac-3–positive cells) were reduced at both 5 days (13.8±6.2% versus 6.5±4.7%; P=0.040) and 28 days (7.4±3.7% versus 3.1±1.2%; P=0.022) in anti-M2–treated compared with control antibody–treated mice. Intimal and medial neutrophils were undetectable at 28 days in both treatment groups.
In mice receiving control antibody, intimal thickening began by 5 days after injury and progressed significantly between 5 days (921±534 μm2) and 28 days (10 395±3549 μm2) (Table). Anti-M2 antibody reduced intimal thickening at 28 days by 56% (P=0.012) (Figure 5, Table). Medial area was unaffected by anti-M2 treatment. Intimal-to-medial area ratio at 28 days in control antibody–treated mice was reduced 56% by anti-M2 (P=0.036). Intimal and medial thickening were accompanied by progressive vessel enlargement (ie, "positive remodeling"), as determined by external elastic lamina area measurements over time, that was comparable in control and anti-M2 antibody–treated mice.
Quantitative Morphometry and Immunohistochemistry
Because increasing evidence suggests that leukocytes play an important role in regulating cellular proliferation,20 we assessed cellular proliferation by quantifying incorporation of BrdU. Substantial proliferation was observed 5 days after injury in control vessels (5.0% of medial cells), and proliferation was still evident at 28 days (3.8% of intimal cells). Anti-M2 reduced medial proliferation at 5 days by 64% (P=0.043) and intimal proliferation at 28 days by 47% (P=0.047) (Table).
Discussion
In this study we have shown that antibody targeting of the P201-K217 segment of the MI domain reduces leukocyte adhesion to platelets under flow in vitro and leukocyte accumulation and neointimal thickening after vascular injury in vivo. These observations demonstrate that GP Ib is a physiologically relevant ligand for M2 and that integrin engagement of GP Ib is critical to leukocyte function in vivo.
By virtue of binding diverse ligands including, among others, fibrinogen,21,22 ICAM-1,23 factor X,24 C3bi,21 platelet GP Ib,9,12 and JAM-3,25 M2 regulates important leukocyte functions including adhesion, migration, coagulation, proteolysis, phagocytosis, oxidative burst, and signaling.4,14,26 However, identification of the precise ligand responsible for leukocyte accumulation at sites of injury with adherent platelets was heretofore undefined. Although previous studies have shown that M2 directly facilitates the recruitment of leukocytes at sites of platelet and fibrin deposition,5 the true biological importance of platelet counterreceptors for M2, such as GP Ib,9 JAM-3,25 and fibrinogen bound to GP IIb/IIIa, was unknown. Although we have targeted M2 with the monoclonal antibody M1/70 in prior studies,27,28 these studies were uninformative with regard to identifying the physiological counterreceptor for M2 because M1/70 is a pan ligand–blocking antibody.
Our prior report has identified M2 as a molecular determinant of neointimal thickening after endothelial-denuding injury. We found that selective absence of M2 impaired transplatelet leukocyte migration into the vessel wall, diminishing medial leukocyte accumulation and neointimal thickening after experimental angioplasty.2 Deficiency of M2 was associated with a 67% reduction in early leukocyte accumulation. Interestingly, the magnitude of this reduction is similar to that of targeting M2–GP Ib alone with anti-M2 (75% inhibition of CD45-positive cells), suggesting that leukocyte recruitment is largely dependent on the interaction between M2 and GP Ib. This is consistent with our prior in vitro observation that the predominant interaction between neutrophils and surface-adherent platelets after vigorous washing appeared to be mediated by M2 binding to GP Ib, based on the ability of M2 peptide to inhibit >80% of neutrophil adhesion.12
The relative specificity of anti-M2 inhibitory action toward GP Ib (ie, noninhibitory toward ICAM-1, fibrinogen, and JAM-3) suggests a minor contribution of other ligands for M2 in the context of endothelial-denuding injury and platelet deposition. Other potential M2 ligands present on the platelet membrane include fibrinogen (bound to GP IIb/IIIa),21,22 ICAM-2,29 and JAM-3.25 However, a leukocyte-platelet interaction mediated by fibrinogen bridging between M2 and GP IIb/IIIa has been largely discounted by Ostrovsky and colleagues,30 who found that neither RGDS peptides nor the replacement of normal platelets with thrombasthenic platelets (ie, lacking GP IIb/IIIa) affected the accumulation of the leukocytes on platelets. Importantly, deficiency of 3 (ie, GP IIIa subunit) did not affect leukocyte recruitment or neointimal formation in mice subjected to the identical femoral artery injury model.18 Although M2 binds ICAM-1, this receptor is not found on platelets. Platelets express a related receptor, ICAM-2,29 but Diacovo et al5 have shown that ICAM-2 blockade has no effect on the firm adhesion of neutrophils on monolayers of activated platelets under flow. Santoso and coworkers25 have reported recently that M2 may also bind to platelet JAM-3, cooperating with GP Ib to mediate neutrophil-platelet adhesive contacts in vitro. However, anti-M2 had minimal inhibitory effect (13% inhibition) on M2-dependent adhesion to JAM-3 (Figure 1).
Limitations of the Study
We have not focused directly on the adventitia as a source of infiltrating inflammatory cells. Leukocytes may infiltrate the vessel wall both from the lumen as well as from the adventitia, but adventitial recruitment is difficult to assess reliably in this model that requires dissection and isolation of the artery from surrounding tissues in order to perform wire injury. Furthermore, although intimal and medial thickening were accompanied by progressive vessel enlargement or positive remodeling that was comparable in control and anti-M2 antibody–treated mice, adventitial cells influence vascular remodeling,31 suggesting that alternative injury models will be necessary to investigate adventitial responses to injury. Finally, we cannot discount the contribution of other M2 ligands and functions or other cellular adhesion molecules in the biological response to vascular injury. Treatment with anti-M2 reduced neointimal thickening by 56%, a less robust inhibitory effect compared with complete deficiency of M2 (80% inhibition compared with wild-type mice).2 Deficiency of P-selectin also markedly reduces leukocyte recruitment and neointimal thickening (73% inhibition compared with wild-type mice) after femoral artery wire injury.18 Inflammatory cells contribute to neointimal thickening because of their direct bulk within the intima, generation of injurious reactive oxygen intermediates, elaboration of growth and chemotactic factors, or production of enzymes (eg, matrix metalloproteinases, cathepsin S) capable of degrading extracellular constituents and thereby facilitating cell migration,31 processes that are both M2 dependent and independent.
Conclusion
Thus, these studies demonstrate that GP Ib is a physiologically relevant ligand for M2 and that integrin engagement of GP Ib is critical to leukocyte function in vivo. The present observations also suggest a possible target for therapeutic intervention. In particular, the specificity of anti-M2 inhibitory action toward GP Ib suggests that it might be possible to prevent leukocyte attachment to platelets by targeting GP Ib without inhibiting other M2 functions. The M(P201-K217) sequence is an obvious molecular target for disrupting leukocyte-platelet complexes that promote vascular inflammation in thrombosis,32 atherosclerosis,1 and restenosis.2 Since platelets also orchestrate inflammatory cell recruitment and play a pathophysiological role in lung33 and glomerular34 injury, targeting M(P201-K217) is likely to be of broader disease interest.
Acknowledgments
This work was supported in part by grants from the National Institutes of Health (HL65967 and HL64796 to Dr Lopez, HL66197 to Dr Plow, and HL57506 and HL60942 to Dr Simon). Dr Ustinov received a postdoctoral research fellowship from the Ohio Valley Affiliate, American Heart Association (0120394B). The authors acknowledge the technical expertise of Craig Muir, Hans Luedemann, and Golnaz Shapurian for supporting the laminar flow experiments.
Disclosure
Dr Andre is an employee of Portola Pharmaceuticals, comanufacturer of the dynamic adhesion system used in the laminar flow experiments.
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Cameron JS. Platelets in glomerular disease. Annu Rev Med. 1984; 35: 175–180.(Yunmei Wang, PhD; Masashi)
The Cleveland Clinic Foundation, Cleveland, Ohio (V.U., E.P.)
Thrombosis Research Section, Departments of Medicine and Molecular and Human Genetics, Baylor College of Medicine, Houston, Tex (J.L.)
Portola Pharmaceuticals, South San Francisco, Calif (P.A.).
Abstract
Background— Leukocyte-platelet interactions are critical in the initiation and progression of atherosclerosis as well as restenosis. Although the leukocyte integrin Mac-1 (M2, CD11b/CD18) has been implicated in the firm adhesion and transmigration of leukocytes at sites of platelet deposition, the precise M2 counterligand responsible for mediating adhesion-strengthening interactions between neutrophils and platelets in vivo has not previously been identified.
Methods and Results— Our previous studies have established the P201-K217 sequence in the MI domain as the binding site for platelet glycoprotein (GP) Ib. Here we report that antibody targeting of M(P201-K217) reduced M2-dependent adhesion to GP Ib but not other M2 ligands, including fibrinogen, intercellular adhesion molecule-1, and junctional adhesion molecule-3. Anti-M(P201-K217) inhibited the firm adhesion of both human and murine leukocytes to adherent platelets under laminar flow conditions. In a mouse femoral artery wire injury model, antibody targeting of M(P201-K217) reduced leukocyte accumulation after injury that was accompanied by inhibition of cellular proliferation and neointimal thickening.
Conclusions— This study demonstrates that GP Ib is a physiologically relevant ligand for M2 and that integrin engagement of GP Ib is critical to leukocyte function and the biological response to vascular injury. These observations establish a molecular target for selectively disrupting leukocyte-platelet complexes that promote inflammation in thrombosis and restenosis.
Key Words: cell adhesion molecules inflammation leukocytes platelets restenosis
Introduction
Leukocyte-platelet interactions are critical in the initiation and progression of atherosclerosis1 as well as restenosis.2 Platelet deposition precedes inflammatory cell accumulation in mouse models of atherogenesis, and inhibition of platelet adhesion dramatically reduces atherosclerotic lesion formation.1 However, the specific receptors responsible for mediating adhesive interactions between neutrophils and platelets in vivo are incompletely defined.
Recruitment of circulating leukocytes to vascular endothelium requires multistep adhesive and signaling events including selectin-mediated attachment and rolling, leukocyte activation, and integrin-mediated firm adhesion and diapedesis that result in the infiltration of inflammatory cells into the blood vessel wall.3 Firm attachment is mediated by members of the 2-integrin family, LFA-1 (L2, CD11a/CD18), Mac-1 (M2, CD11b/CD18), and p150,95 (X2, CD11c/CD18), which bind to endothelial counterligands (eg, intercellular adhesion molecule-1 [ICAM-1]), to endothelial-associated extracellular matrix proteins (eg, fibrinogen), or to glycosaminoglycans.4
Clinical Perspective p 3000
Importantly, leukocyte recruitment also occurs at sites of vascular injury where the lining endothelial cells have been denuded and platelets and fibrin have been deposited. A similar sequential adhesion model of leukocyte attachment to and transmigration across surface-adherent platelets has been proposed.5 The initial tethering and rolling of leukocytes on platelet P-selectin6 are followed by their firm adhesion and transplatelet migration, processes that are dependent on M2.5
Our laboratory has focused on identifying the platelet counterreceptor for M2. Evaluation of the structural features of integrins provides insight into candidate platelet counterreceptors for M2. Integrins are heterodimeric proteins composed of 1 and 1 subunit. A subset of integrin subunits, including M, contains an inserted domain (I domain) of &200 amino acids that is implicated in ligand binding7 and is strikingly similar to the A domains of von Willebrand factor (vWf),8 one of which, A1, mediates the interaction of vWf with its platelet receptor, the glycoprotein (GP) Ib-IX-V complex. Because of the similarity of the vWf A1 domain and the MI domain, we hypothesized that GP Ib might also be able to bind M2 and reported that GP Ib is indeed a constitutively expressed counterreceptor for M2.9 Furthermore, under the conditions used in these studies, the predominant interaction between neutrophils and platelets appeared to be between M2 and GP Ib.
The MI domain contributes broadly to the recognition of ligands by M27 and specifically to the binding of GP Ib.9 This region has also been implicated in the binding of ICAM-1,10 C3bi,11 and fibrinogen.10 We have recently localized the binding site for GP Ib within the MI domain segment M(P201-K217) using a strategy based on the differences in the binding of GP Ib to the MI and LI domains that involved several independent approaches, including screening of mutant cells, synthetic peptides, site-directed mutagenesis, and gain-in-function analyses.12 The grafting of only 2 amino acids within this segment into the LI domain converted it to a GP Ib binding protein. Thus, a small segment that has a defined structure within the MI domain is necessary and sufficient for GP Ib binding.
While M2 has a broad ligand binding repertoire, the precise ligand responsible for leukocyte accumulation at sites of platelet deposition remains to be defined. Central to unraveling the precise biological roles of M2 is defining the biologically relevant ligand(s) for this integrin. In this study we show that antibody targeting of M(P201-K217) reduced M2-dependent adhesion to GP Ib, but not other ligands, in vitro and leukocyte accumulation after vascular injury in vivo. In a mouse femoral artery injury model, treatment with anti-M(P201-K217) was accompanied by inhibition of cellular proliferation and neointimal thickening.
Methods
Materials
The soluble extracellular region of GP Ib (ie, glycocalicin or sGP Ib) was purified as we reported previously.9 Human fibrinogen depleted of plasminogen, vWf, and fibronectin was purchased from Enzyme Research Laboratories (South Bend, Ind). Human C3bi was obtained from Calbiochem (San Diego, Calif). Recombinant human ICAM-1 was purchased from R&D Systems, Inc (Minneapolis, Minn). Human junctional adhesion molecule-3 (JAM-3) was kindly provided by Dr Sentot Santoso (Justus-Liebig University, Giessen, Germany). The monoclonal antibody LPM19c, directed to the MI domain, was kindly provided by Dr Karen Pulford (Radcliffe, Oxford, UK). The monoclonal antibody 1B5, directed to murine IIb3 and capable of inhibiting murine platelet aggregation, was a generous gift of Dr Barry Coller (Rockefeller University, New York, NY). Peptides corresponding to the murine GP Ib binding site (SGSG-214LYFRHWLQENANNVYL229-C) for Mac-113 or scrambled control (SGSG-VEAFHLNYYRNNVWLQ-C) were obtained from the W.M. Keck Biotechnology Resource Center (Yale University, New Haven, Conn). The peptides were diluted in dimethyl sulfoxide and stored at –80°C.
Antibody Generation
An affinity purified peptide-specific polyclonal antibody (termed anti-M2) to the Mac-1 binding site for GP Ib was generated by immunizing rabbits with C-201PITQLLGRTHTATGIRK217 coupled to KLH (Zymed Laboratories, South San Francisco, Calif) corresponding to the human M(P201-K217) sequence. After test bleeds demonstrated high-titer anti-sera binding to solid-phase immobilized C-P201-K217, peptide-specific polyclonal antibody was purified by affinity matrix chromatography (Zymed Laboratories). Nonimmune rabbit IgG served as control antibody.
Cell Lines and Culture Conditions
293 cells expressing human M2 receptors were established and maintained as previously described.12
Preparation of Murine Neutrophils
Neutrophils from wild-type and Mac-1–deficient C57Bl/J6 mice14 were harvested and purified (>90% neutrophils by cytospin) from the peritoneal cavity 6 hours after the intraperitoneal injection of 1 mL sterile 3% thioglycolate broth, as previously described.9 Animal care and procedures were reviewed and approved by the Harvard Medical School Standing Committee on Animals and performed in accordance with the guidelines of the American Association for Accreditation of Laboratory Animal Care and the National Institutes of Health.
Flow Cytometry
Wild-type and Mac-1–deficient neutrophils (106) were incubated with anti-M2 or nonimmune IgG (25 μg/mL). After they were washed, anti-M2 binding was assessed with the use of FITC-conjugated Fab2 anti-rabbit (BD Biosciences) by flow cytometry (FACScan, BD Biosciences-Immunocytometry Systems).
Blood Collection
Venous blood was obtained from volunteers who had not consumed aspirin or other nonsteroidal anti-inflammatory drugs for at least 10 days and was anticoagulated with 10 mmol/L trisodium citrate. All subjects gave written informed consent to the protocol, which was approved by the institutional review board. Venous blood was also collected from the vena cava of anesthetized mice and anticoagulated with 10 mmol/L trisodium citrate.
Adhesion Assays
293 cells expressing M2 were harvested with cell-dissociating buffer (Life Technologies) for 1 minute at 22°C, washed twice, resuspended in serum-free media, and loaded with BCECF AM [2',7'-bis-(2-carboyethyl)-5-(and-6)-carboxyfluorescein, acetoxy-methyl ester] (1 μmol/L) according to the manufacturer’s protocol (Molecular Probes). Cells (105 per well) were placed in 48-well tissue culture plates (Costar) coated with 200 μL of 5 nmol/L fibrinogen or 50 nmol/L C3bi, ICAM-1, JAM-3, or sGP Ib overnight at 4°C and then blocked with 0.5% polyvinylpyrrolidone for 1 hour at room temperature. Adhesion was stimulated with phorbol 12-myristate 13-acetate (PMA) (20 ng/mL) in the presence of 2 mmol/L Mg2+. Plates were washed with 0.9% NaCl (3 to 5 times), and adhesion was quantified by measuring the fluorescence of BCECF AM–loaded cells with a Cytofluor II fluorescence multiwell microplate reader (PerSeptive Biosystems). The effect of anti-M2 on adhesion was assessed by preincubating cells with anti-M2 or control antibody (10 μg/mL) for 30 minutes at 37°C before the addition of cells.
The effect of anti-M2 on murine neutrophil adhesion to a peptide corresponding to the murine GP Ib binding site for Mac-1 was also investigated. BCECF AM–loaded wild-type neutrophils (2x105 per well) were placed in 96-well tissue culture plates coated with 100 μL of GP Ib peptide (SGSG-214LYFRHWLQENANNVYL229-C) or scrambled control (SGSG-VEAFHLNYYRNNVWLQ-C) (50 μg/mL) overnight at 4°C and then blocked with 0.5% gelatin for 1 hour at room temperature. Plates were washed with PBS (3 to 5 times) and adhesion quantified. The effect of anti-M2 on adhesion was assessed by preincubating cells with anti-M2 or control antibody.
In Vitro Analysis of Cellular Adhesion Under Laminar Flow Conditions
The laminar flow chamber used in this assay has been described previously.15,16 Anticoagulated blood was incubated with rhodamine-6G (0.2 μg/mL, Sigma), the GP IIb/IIIa inhibitor eptifibatide (2.5 μg/mL, Millennium Pharmaceuticals), 2 mmol/L MgCl2, and anti-M2 or control antibody (1 to 25 μg/mL) and then perfused for 5 minutes through human type III collagen–coated rectangular capillaries at 625 s–1, resulting in the deposition of adherent platelets without large platelet aggregates. Leukocyte rolling and arrest were then monitored by perfusion for 10 minutes through the capillary at 125 s–1. The number of arrested cells, after perfusion with PBS at 625 s–1, was quantified in each of 5 random x10 fields (area &0.3 mm2).
For murine blood, anticoagulated blood was incubated with rhodamine-6G, 2 mmol/L MgCl2, and the anti-murine IIb3 monoclonal antibody 1B5 (5 μg/mL) and then perfused for 5 minutes through type III collagen–coated capillaries at 250 s–1, resulting in the deposition of adherent platelets. The firm arrest of murine neutrophils (106/mL) incubated with anti-M2 or control antibody (25 μg/mL) was then monitored by perfusion for 5 minutes through the capillary at 125 s–1. The number of arrested cells was then quantified as above. A minimum of 6 mice were used for each group studied.
Femoral Artery Injury
Male C57Bl/6J mice aged 8 to 10 weeks were anesthetized on day 0 with the use of ketamine (80 mg/kg IP) and xylazine (5 mg/kg IP), and wire injury (0.010 in) of the femoral artery was performed as described previously.17 All animals survived until the time of planned euthanasia without bleeding or infection.
Tissue Harvesting and Analysis
One day (control antibody: n=6; anti-M2: n=6), 5 days (control antibody: n=9; anti-M2: n=8), or 28 days (control antibody: n=9; anti-M2: n=9) after vascular injury, anesthesia was administered, the chest cavity was opened, and the animals were euthanized by right atrial exsanguination. A 22-gauge butterfly catheter was inserted into the left ventricle for in situ pressure perfusion at 100 mm Hg with 0.9% saline for 1 minute followed by fixation with 4% paraformaldehyde in 0.1 mol/L phosphate buffer, pH 7.3, for 10 minutes. The right and left femoral arteries were excised and immersed in buffered paraformaldehyde. Spleen and small intestine from 3 animals were harvested as control tissues for immunohistochemistry. All animals received BrdU, 50 mg/kg IP, 18 hours and 1 hour before euthanasia.
Femoral arteries were embedded, and 2 cross sections cut 1 mm apart were stained with hematoxylin and eosin and Verhoeff tissue elastin stain. A histologist blinded to treatment group measured the luminal, intimal, and medial areas of each cross-sectional plane using a microscope equipped with a CCD camera interfaced to a computer running NIH Image version 1.60 software. Results for the 2 planes of each artery were averaged. For baseline medial and external elastic lamina areas before injury or antibody treatment, a group of mice (n=5) was also euthanized for quantitative morphometry. For immunohistochemistry, standard avidin-biotin procedures for mouse CD45 (leukocyte common antigen, BD Biosciences, San Diego, Calif), mouse neutrophil-specific marker (monoclonal antibody 7/4, Serotec, Indianapolis, Ind), mouse macrophage-specific marker Mac-3 (mAb M3/84, BD Biosciences), and BrdU (DAKO, Carpinteria, Calif) were used. Immunostained sections were quantified as the number of immunostained positive cells per total number of nuclei.
Antibody Treatment
Antibody treatments were via intraperitoneal injection of 250 μg antibody 4 hours before injury (day 0) and then 100 μg antibody on days 2, 4, 6, 8, 10, and 12. Mice were divided into 2 treatment groups: (1) rabbit anti-M2 and (2) nonimmune rabbit IgG as control antibody.
Statistical Analysis
Data are presented as mean±SD. Comparisons between groups were performed by unpaired t test. Probability values <0.05 were considered significant.
Results
Antibody Targeting M2–GP Ib
Although M2 is capable of binding a broad repertoire of ligands, the precise ligand responsible for leukocyte accumulation at sites of vascular injury remains to be defined. We hypothesized that the M2–GP Ib interaction would likely dominate on the basis of our in vitro observations9 that leukocyte adhesion to platelets was largely abrogated by sGP Ib, antibodies to GP Ib, and pretreatment of platelets with the snake venom metalloprotease mocarhagin, whose major platelet substrate is GP Ib. Furthermore, agonist-stimulated leukocyte-platelet complexes were significantly decreased in whole blood from a patient with Bernard-Soulier syndrome compared with a normal control.9 Our prior report12 identified the M(P201-K217) segment as the M2 binding site for GP Ib. A synthetic peptide (termed M2) duplicating the P201-K217 sequence, but not scrambled versions, directly bound sGP Ib and inhibited M2-dependent adhesion to sGP Ib and adherent platelets. Therefore, as a first step in targeting M2–GP Ib for in vivo experiments, an affinity-purified peptide-specific polyclonal antibody to this site (anti-M2) was generated by immunizing rabbits with C-201PITQLLGRTHTATGIRK217 coupled to KLH corresponding to the human M(P201-K217) sequence. The human sequence (M 201PITQLLGRTHTATGIRK217) is highly homologous to the corresponding murine M201PIKQLNGRTKTASGIRK217 sequence with 13 of 17 identical residues.
We first examined the effect of anti-M2 antibody on M2-dependent static adhesion to sGP Ib, ICAM-1, fibrinogen, and JAM-3 (Figure 1). Anti-M2 antibody was preincubated with M2-expressing 293 cells, and adhesion was stimulated with PMA in the presence of Mg2+. M2-expressing 293 cells adhered to sGP Ib, ICAM-1, fibrinogen, and JAM-3, and this adhesion was blocked by the anti-M monoclonal antibody LPM19c (data not shown), indicating that adhesion is predominantly M2 dependent. Anti-M2 strongly inhibited PMA-stimulated adhesion to sGP Ib (percent inhibition versus control=80±4; P<0.001). In contrast, anti-M2 had little or no effect on M2-expressing 293 cell adhesion to fibrinogen (percent inhibition versus control=21±8), JAM-3 (13±18%), or ICAM-1 (0±9%). This is consistent with our prior observation that M2 peptide itself only inhibited adhesion to sGP Ib but not other ligands.12 Taken together, these observations suggest that anti-M2, directed to the M(P201-K217) sequence within the MI domain, is capable of selectively blocking M2 binding to GP Ib.
Anti-M2 Abrogates the Firm Adhesion of Leukocytes Under Flow
To evaluate the potential for anti-M2 to modulate the adhesion of human blood cells under laminar flow conditions, we perfused whole blood, in the presence of the platelet aggregation inhibitor eptifibatide, over collagen-coated capillaries at a shear rate of 625 s–1, resulting in the deposition of adherent platelets without large platelet aggregates.16 Leukocyte rolling and firm arrest on adherent platelets were observed by lowering the shear rate to 125 s–1. The number of arrested cells was unaffected by nonimmune IgG (control versus nonimmune IgG, P>0.05) (Figure 2). In contrast, anti-M2 dose-dependently inhibited leukocyte arrest under flow (maximal percent inhibition=92±8; P<0.001).
Anti-M2 Binds to Murine Neutrophils and Blocks Adhesion to Murine Platelets
We next verified that anti-M2 would bind to murine Mac-1 and block murine neutrophil adhesion to murine GP Ib or murine platelets. As determined by FACS, anti-M2 bound to wild-type neutrophils (mean fluorescence intensity [MFI]= 252±7) but not Mac-1–deficient neutrophils (MFI=36±15); moreover, binding of anti-M2 to Mac-1–deficient neutrophils (MFI=53±12) was equivalent to that observed with nonimmune IgG (MFI=68±9).
We next turned to examining the effect of anti-M2 on murine neutrophil adhesion. Thioglycolate-elicited neutrophils were added to wells coated with a peptide corresponding to murine GP Ib binding site for Mac-1 (SGSG-214LYFRHWLQENANNVYL229-C).13 Wild-type neutrophils bound to GP Ib peptide but not to scrambled peptide control (Figure 3A). Anti-M2, but not nonimmune IgG, significantly inhibited neutrophil adhesion to murine GP Ib peptide (maximal percent inhibition=70±3).
We next investigated whether anti-M2 would inhibit murine leukocyte adhesion to murine platelets under laminar flow conditions. Anticoagulated murine blood, in the presence of 1B5 monoclonal antibody that blocks IIb3 integrin–dependent murine platelet aggregation, was perfused over type III collagen–coated capillaries at a shear rate of 250 s–1, resulting in the deposition of adherent platelets without large platelet aggregates. The firm arrest of murine neutrophils (106/mL) incubated with anti-M2 or nonimmune IgG was then monitored by perfusion for 10 minutes through the capillary at 125 s–1. Anti-M2 inhibited (percent inhibition=81±4) leukocyte arrest under laminar flow conditions (Figure 3B). Taken together, these observations indicate that anti-M2 is capable of inhibiting murine Mac-1 binding to murine GP Ib peptide and murine leukocyte adhesion to murine platelets.
Anti-M2 Reduces Leukocyte Recruitment and Neointimal Thickening After Vascular Injury
To determine whether the M2–GP Ib interaction is required for neointimal formation, we performed femoral artery wire injury in mice treated with anti-M2 or nonimmune IgG control before injury and then every other day through 12 days after injury. Wire injury is accompanied by endothelial denudation, platelet and fibrin deposition, and prominent vascular inflammation.17 This model has been useful in demonstrating that inflammatory cell recruitment and function modulate neointimal formation.18–20
We first examined leukocyte recruitment after injury over time. A role for M2–GP Ib in this process was implicated because altered leukocyte accumulation within vessels was observed in injured vessels from anti-M2 compared with control mice. Inflammatory cells (CD45-positive) invading the media at 1 day were reduced by 75% from 7.9±5.0% to 2.0±1.6% of total cells (P=0.021) in anti-M2–treated compared with control antibody–treated mice (Figures 4 and 5). Accumulation of leukocytes in the developing neointima was also reduced significantly by anti-M2, with a 42% reduction at 5 days (P=0.047) and 58% reduction at 28 days (P=0.012).
We expanded the CD45 analysis by immunostaining using cell-specific markers. Neutrophil (ie, monoclonal antibody 7/4-positive cells) accumulation within the media at 1 day was reduced by 75% in anti-M2–treated compared with control antibody–treated mice (control: 8.9±6.7% versus anti-M2: 2.2±1.6%; P=0.043). Macrophages (ie, Mac-3–positive cells) were essentially undetectable (<0.5% of total medial cells) 1 day after injury. Accumulation of neutrophils in the developing neointima at 5 days was also reduced by 47% with anti-M2 (control: 26.2±8.0% versus anti-M2: 13.9±4.9%; P=0.036). Concordant with the CD45 staining, intimal macrophages (Mac-3–positive cells) were reduced at both 5 days (13.8±6.2% versus 6.5±4.7%; P=0.040) and 28 days (7.4±3.7% versus 3.1±1.2%; P=0.022) in anti-M2–treated compared with control antibody–treated mice. Intimal and medial neutrophils were undetectable at 28 days in both treatment groups.
In mice receiving control antibody, intimal thickening began by 5 days after injury and progressed significantly between 5 days (921±534 μm2) and 28 days (10 395±3549 μm2) (Table). Anti-M2 antibody reduced intimal thickening at 28 days by 56% (P=0.012) (Figure 5, Table). Medial area was unaffected by anti-M2 treatment. Intimal-to-medial area ratio at 28 days in control antibody–treated mice was reduced 56% by anti-M2 (P=0.036). Intimal and medial thickening were accompanied by progressive vessel enlargement (ie, "positive remodeling"), as determined by external elastic lamina area measurements over time, that was comparable in control and anti-M2 antibody–treated mice.
Quantitative Morphometry and Immunohistochemistry
Because increasing evidence suggests that leukocytes play an important role in regulating cellular proliferation,20 we assessed cellular proliferation by quantifying incorporation of BrdU. Substantial proliferation was observed 5 days after injury in control vessels (5.0% of medial cells), and proliferation was still evident at 28 days (3.8% of intimal cells). Anti-M2 reduced medial proliferation at 5 days by 64% (P=0.043) and intimal proliferation at 28 days by 47% (P=0.047) (Table).
Discussion
In this study we have shown that antibody targeting of the P201-K217 segment of the MI domain reduces leukocyte adhesion to platelets under flow in vitro and leukocyte accumulation and neointimal thickening after vascular injury in vivo. These observations demonstrate that GP Ib is a physiologically relevant ligand for M2 and that integrin engagement of GP Ib is critical to leukocyte function in vivo.
By virtue of binding diverse ligands including, among others, fibrinogen,21,22 ICAM-1,23 factor X,24 C3bi,21 platelet GP Ib,9,12 and JAM-3,25 M2 regulates important leukocyte functions including adhesion, migration, coagulation, proteolysis, phagocytosis, oxidative burst, and signaling.4,14,26 However, identification of the precise ligand responsible for leukocyte accumulation at sites of injury with adherent platelets was heretofore undefined. Although previous studies have shown that M2 directly facilitates the recruitment of leukocytes at sites of platelet and fibrin deposition,5 the true biological importance of platelet counterreceptors for M2, such as GP Ib,9 JAM-3,25 and fibrinogen bound to GP IIb/IIIa, was unknown. Although we have targeted M2 with the monoclonal antibody M1/70 in prior studies,27,28 these studies were uninformative with regard to identifying the physiological counterreceptor for M2 because M1/70 is a pan ligand–blocking antibody.
Our prior report has identified M2 as a molecular determinant of neointimal thickening after endothelial-denuding injury. We found that selective absence of M2 impaired transplatelet leukocyte migration into the vessel wall, diminishing medial leukocyte accumulation and neointimal thickening after experimental angioplasty.2 Deficiency of M2 was associated with a 67% reduction in early leukocyte accumulation. Interestingly, the magnitude of this reduction is similar to that of targeting M2–GP Ib alone with anti-M2 (75% inhibition of CD45-positive cells), suggesting that leukocyte recruitment is largely dependent on the interaction between M2 and GP Ib. This is consistent with our prior in vitro observation that the predominant interaction between neutrophils and surface-adherent platelets after vigorous washing appeared to be mediated by M2 binding to GP Ib, based on the ability of M2 peptide to inhibit >80% of neutrophil adhesion.12
The relative specificity of anti-M2 inhibitory action toward GP Ib (ie, noninhibitory toward ICAM-1, fibrinogen, and JAM-3) suggests a minor contribution of other ligands for M2 in the context of endothelial-denuding injury and platelet deposition. Other potential M2 ligands present on the platelet membrane include fibrinogen (bound to GP IIb/IIIa),21,22 ICAM-2,29 and JAM-3.25 However, a leukocyte-platelet interaction mediated by fibrinogen bridging between M2 and GP IIb/IIIa has been largely discounted by Ostrovsky and colleagues,30 who found that neither RGDS peptides nor the replacement of normal platelets with thrombasthenic platelets (ie, lacking GP IIb/IIIa) affected the accumulation of the leukocytes on platelets. Importantly, deficiency of 3 (ie, GP IIIa subunit) did not affect leukocyte recruitment or neointimal formation in mice subjected to the identical femoral artery injury model.18 Although M2 binds ICAM-1, this receptor is not found on platelets. Platelets express a related receptor, ICAM-2,29 but Diacovo et al5 have shown that ICAM-2 blockade has no effect on the firm adhesion of neutrophils on monolayers of activated platelets under flow. Santoso and coworkers25 have reported recently that M2 may also bind to platelet JAM-3, cooperating with GP Ib to mediate neutrophil-platelet adhesive contacts in vitro. However, anti-M2 had minimal inhibitory effect (13% inhibition) on M2-dependent adhesion to JAM-3 (Figure 1).
Limitations of the Study
We have not focused directly on the adventitia as a source of infiltrating inflammatory cells. Leukocytes may infiltrate the vessel wall both from the lumen as well as from the adventitia, but adventitial recruitment is difficult to assess reliably in this model that requires dissection and isolation of the artery from surrounding tissues in order to perform wire injury. Furthermore, although intimal and medial thickening were accompanied by progressive vessel enlargement or positive remodeling that was comparable in control and anti-M2 antibody–treated mice, adventitial cells influence vascular remodeling,31 suggesting that alternative injury models will be necessary to investigate adventitial responses to injury. Finally, we cannot discount the contribution of other M2 ligands and functions or other cellular adhesion molecules in the biological response to vascular injury. Treatment with anti-M2 reduced neointimal thickening by 56%, a less robust inhibitory effect compared with complete deficiency of M2 (80% inhibition compared with wild-type mice).2 Deficiency of P-selectin also markedly reduces leukocyte recruitment and neointimal thickening (73% inhibition compared with wild-type mice) after femoral artery wire injury.18 Inflammatory cells contribute to neointimal thickening because of their direct bulk within the intima, generation of injurious reactive oxygen intermediates, elaboration of growth and chemotactic factors, or production of enzymes (eg, matrix metalloproteinases, cathepsin S) capable of degrading extracellular constituents and thereby facilitating cell migration,31 processes that are both M2 dependent and independent.
Conclusion
Thus, these studies demonstrate that GP Ib is a physiologically relevant ligand for M2 and that integrin engagement of GP Ib is critical to leukocyte function in vivo. The present observations also suggest a possible target for therapeutic intervention. In particular, the specificity of anti-M2 inhibitory action toward GP Ib suggests that it might be possible to prevent leukocyte attachment to platelets by targeting GP Ib without inhibiting other M2 functions. The M(P201-K217) sequence is an obvious molecular target for disrupting leukocyte-platelet complexes that promote vascular inflammation in thrombosis,32 atherosclerosis,1 and restenosis.2 Since platelets also orchestrate inflammatory cell recruitment and play a pathophysiological role in lung33 and glomerular34 injury, targeting M(P201-K217) is likely to be of broader disease interest.
Acknowledgments
This work was supported in part by grants from the National Institutes of Health (HL65967 and HL64796 to Dr Lopez, HL66197 to Dr Plow, and HL57506 and HL60942 to Dr Simon). Dr Ustinov received a postdoctoral research fellowship from the Ohio Valley Affiliate, American Heart Association (0120394B). The authors acknowledge the technical expertise of Craig Muir, Hans Luedemann, and Golnaz Shapurian for supporting the laminar flow experiments.
Disclosure
Dr Andre is an employee of Portola Pharmaceuticals, comanufacturer of the dynamic adhesion system used in the laminar flow experiments.
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