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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:528-535

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:528-535.)
© 1997 American Heart Association, Inc.

Articles

7E3 Monoclonal Antibody Directed Against the Platelet Glycoprotein IIb/IIIa Cross-reacts With the Leukocyte Integrin Mac-1 and Blocks Adhesion to Fibrinogen and ICAM-1

Daniel I. Simon; Hui Xu; Susan Ortlepp; Campbell Rogers; ; Navaneetha K. Rao

From the Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Mass (D.I.S., H.X., C.R., N.K.R.); Celltech Therapeutics Ltd, Slough, UK (S.O.); and Harvard-MIT Division of Health Sciences and Technology, Cambridge, Mass (C.R.).

Correspondence to Daniel I. Simon, MD, Cardiovascular Division PBB-A3, Brigham and Women's Hospital, 75 Francis St, Boston, MA 02115. E-mail disimon{at}bics.bwh.harvard.edu.


*    Abstract
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 *Abstract
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Abstract Recent clinical trials suggest that blockade of integrins is a promising strategy for the treatment of acute coronary syndromes. Administration of 7E3 monoclonal antibody (mAb) Fab fragment (c7E3 Fab) directed against platelet integrin IIb/IIIa ({alpha}IIbß3, CD41/CD61) reduces acute ischemic complications of coronary angioplasty and clinical restenosis at 6 months. However, 7E3 mAb is not selective for platelet IIb/IIIa but also cross-reacts with the leukocyte integrin Mac-1 ({alpha}Mß2, CD11b/CD18) and the vitronectin receptor ({alpha}vß3, CD51/CD61). Information regarding how this mAb may affect other cells important in vascular repair is scant. Potential interactions of c7E3 Fab with inflammatory (ie, monocytes and neutrophils), vascular smooth muscle, and endothelial cells may contribute to the in vivo actions of c7E3 Fab. In this study we explored the binding of 7E3 to monocytic cells and the functional effect of 7E3 and c7E3 Fab on Mac-1–mediated adhesion to fibrinogen (FGN) and intercellular adhesion molecule-1 (ICAM-1), ligands abundant in the injured vessel wall. Flow cytometry demonstrated that 7E3 bound to THP-1 monocytic cells and identified a subpopulation ({approx}10%) of Mac-1 that was qualitatively similar to that recognized by CBRM1/5, a mAb directed to an activation-specific neoepitope present on a subset of Mac-1 molecules. mAb 7E3 bound to K562 cells transfected with just the {alpha} subunit (CD11b) of Mac-1 but not to nontransfected cells, confirming a direct interaction between 7E3 and Mac-1. mAb 7E3 and c7E3 Fab blocked the adhesion of Mac-1–bearing cells to FGN (80±11% and 78±9% inhibition, respectively) and ICAM-1 (62±14% and 62±17%). Both 7E3 and c7E3 Fab significantly inhibited (70±6% and 62±26%) soluble FGN binding to human peripheral blood monocytes. Thus, c7E3 Fab cross-reacts with the CD11b subunit of Mac-1 and interrupts cell-extracellular matrix and cell-cell adhesive interactions and may thereby influence the recruitment of circulating monocytes to sites of vessel injury. Given the recent evidence that adherent and infiltrating monocyte number directly correlates with the extent of neointimal hyperplasia, inhibition of Mac-1–dependent adhesion and IIb/IIIa-dependent function by c7E3 Fab may jointly contribute to the regulation of vascular repair and to the sustained clinical benefits observed with c7E3 Fab after angioplasty.


Key Words: integrins • monocytes • cellular adhesion • restenosis • monoclonal antibody


*    Introduction
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 *Introduction
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Restenosis is the major limitation of all percutaneous coronary revascularization procedures, leading to recurrent anginal symptoms and repeat interventions in up to 40% of patients within 6 months.1 2 3 Restenosis is the arterial healing response to vascular injury, characterized pathologically by vessel elastic recoil, thrombus formation/incorporation, and neointimal hyperplasia secondary to smooth muscle cell proliferation and migration and excessive extracellular matrix production.4 5 6 7 8 Although various drugs (eg, heparin and angiotensin-converting enzyme inhibitors) have been successful in reducing neointimal thickening in animal models of restenosis,9 no agent has proved to be effective in large-scale clinical trials.10 Recently, however, the Evaluation of IIb/IIIa Platelet Receptor Antagonist 7E3 in Preventing Ischemic Complications (EPIC) trial has demonstrated that bolus and 12-hour infusion of a murine/human chimeric 7E3 mAb fragment (c7E3 Fab) directed against the platelet integrin IIb/IIIa not only reduced acute ischemic complications of coronary angioplasty by 35%,11 an effect attributed primarily to the ability of c7E3 Fab to potently inhibit platelet aggregation by blocking platelet fibrinogen binding, but also reduced clinical restenosis—major ischemic events and the need for repeat revascularization procedures—at 6 months by 23%.12 The sustained benefit of c7E3 Fab has been attributed to its inhibitory effect on IIb/IIIa and to vessel wall "passivation," a process by which the vascular response to injury is attenuated through poorly characterized effects on cells and extracellular matrix of the vessel wall.

Receptors of the integrin family are an important class of cell adhesion molecules that mediate cell-cell and cell–extracellular matrix interactions that are central to inflammation, wound healing, and hemostasis.13 Since integrins share common {alpha} and ß subunits and because there is a high degree of homology between subunit families,14 it is not surprising that some mAbs bind to more than one integrin. c7E3 Fab is not selective for platelet IIb/IIIa ({alpha}IIbß3, CD41/CD61) but also cross-reacts with the vitronectin receptor ({alpha}vß3, CD51/CD61),15 found on endothelial and smooth muscle cells, and the integrin Mac-1 ({alpha}Mß2, CD11b/CD18),16 found on monocytes and neutrophils.

Mac-1, a leukocyte integrin that is mobilized from intracellular storage pools in response to a variety of agonists, including ADP, C5a, fMLP, and phorbol esters,17 18 is capable of binding heterogeneous ligands including ICAM-1,19 fibrin(ogen),20 21 22 and factor X.23 The binding of Mac-1 to ICAM-1 and fibrin(ogen) results in the adhesion of neutrophils/monocytes to the endothelium and to sites of fibrin deposition, respectively. After binding to factor X, Mac-1 coordinates the activation of factor X independent of tissue factor and factor VII, culminating in rapid fibrin formation.23 mAbs to Mac-1 interrupt the adhesive and migratory capability of leukocytes and reduce tissue injury in models of inflammation.24 25 26 27

Circulating monocytes are among the earliest cells recruited to sites of vessel injury28 29 and have the potential to interact with other vascular cells by secreting growth factors and cytokines.30 31 32 In fact, adherent and infiltrating monocyte number directly correlates with the extent of neointimal hyperplasia after deep vessel injury.33 Our demonstration in this study that 7E3 and c7E3 Fab inhibit Mac-1–mediated adhesion to FGN and ICAM-1 in vitro may provide an additional mechanism for the beneficial effects of this mAb therapy.


*    Methods
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Materials
ADP and the chemotactic peptide fMLP were purchased from Sigma Chemical Company. Human plasminogen-free FGN was purchased from Enzyme Research Laboratories. FGN was iodinated with Iodobeads as previously described.34 TGF-ß1 was from Collaborative Research Inc, and 1,25-(OH)2 vitamin D3 was a gift of Dr M. Uskokvic (Hoffman-LaRoche Laboratories, Nutley, NJ). LPM19c, a mAb to the {alpha}M subunit of Mac-1 (CD11b) that blocks FGN binding to Mac-1,35 was purchased from DAKO Corp. The stimulating CD18 mAb KIM127 was a generous gift of Dr Martyn Robinson (Celltech Ltd, Slough, England).36 Additional Mac-1 mAbs included TS1/18, a mAb to the common ß2 subunit (CD18) of Mac-1, and CBRM1/5, a mAb to an activation-specific neoepitope in a subpopulation of Mac-1 molecules (kindly provided by Dr Timothy A. Springer, Harvard Medical School, Boston, Mass).37 mAbs 7E3 and 10E5,38 murine mAbs to glycoprotein IIb and/or IIIa that block platelet FGN binding, and c7E3 Fab,39 a Fab fragment of a human-mouse genetic reconstruction of murine 7E3, in which mouse heavy and light chain variable regions were linked to the constant domains of a human IgG1 heavy chain and {kappa} light chain, respectively, were kindly provided by Dr Barry S. Coller (Mt Sinai Medical Center, New York, NY). Soluble human ICAM-1/Ig, a recombinant fusion protein comprising the extracellular domain of human ICAM-1 linked to the constant region of human IgG1, was obtained from Chiron Corp. FITC-conjugated goat anti-mouse IgG, F(ab')2 antibody was obtained from Boehringer Mannheim Corp.

Cell Transfection
Erythroleukemic K562 cells (European Collection of Animal Cell Cultures) were transfected with the {alpha}M (CD11b) subunit of Mac-1 as previously described.40 The CD11b gene was cloned by PCR amplification from first-strand cDNA prepared from U937 cells stimulated with 10 ng/mL phorbol myristate acetate for 48 hours. The gene was cloned into the expression vector EE6 hCMV carrying a G418-resistance marker for expression in K562 cells. This plasmid was linearized by using Sal I and then 40 µg of linearized DNA was transfected into 107 K562 cells by electroporation, using a BioRad Gene Pulser unit. Cells and DNA were subjected to one pulse at 250 V with a capacitance of 900 µF.

CHO cells transfected with human Mac-1 (CD11b/CD18) or the closely related leukocyte integrin p150,95 (CD11c/CD18) were provided by Dr Timothy A. Springer.35 Briefly, {alpha}M (CD11b) or {alpha}X (CD11c) and ß2 (CD18) subunits of Mac-1 in pCDM8 were cotransfected by electroporation with the pDCHIP plasmid, containing a CHO dihydrofolate reductase minigene, into CHO DG44 cells. Cells were selected with methotrexate, and a homogeneous population of positively expressing cells was obtained by immunopanning. Expression was then augmented by increasing the concentration of methotrexate (0.05 to 0.2 µmol/L) in culture. Both types of transfected cells, but not the nontransfected DG44 cells, acquired the ability to bind soluble 125I-labeled FGN. Furthermore, Mac-1 and p150,95 transfectants bound equivalent amounts of 125I-FGN, confirming equivalent expression of functional protein.

Cell Lines and Culture Conditions
The monocytic THP-1 cell line (American Type Culture Collection) was maintained in RPMI 1640 and supplemented with 10% (vol/vol) FBS. Unless otherwise indicated, medium containing 20 mmol/L HEPES, 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin was used in all cases. Differentiation of monocytic cells (106 per milliliter), which is accompanied by increased expression of Mac-1, was induced by treatment with 1 ng/mL TGF-ß1 and 50 nmol/L 1,2-(OH)2 vitamin D3 for 24 hours.41 K562 cells were cultured in DMEM supplemented with 10% FBS; CD11b K562 cells were cultured in DMEM containing G418 (1 mg/mL) and 10% FBS. Mac-1 and p150,95 CHO cells were cultured in {alpha}-MEM supplemented with 10% heat-inactivated, dialyzed FBS, 0.1 µmol/L methotrexate, and 10 µmol/L thymidine. Incubations were performed at 37°C, 5% CO2. Peripheral blood mononuclear cells were isolated from 10% (vol/vol) CPD-anticoagulated human blood by Ficoll-Hypaque centrifugation42 and human monocytes were separated from peripheral blood mononuclear cells by adherence to human AB serum-coated plastic Petri dishes. Monocytes were maintained in RPMI 1640 supplemented to 10% (vol/vol) with human AB serum.

Adhesion Assays
Adherent cells were assayed by a colorimetric method, as previously described.43 44 Cytokine-primed monocytic THP-1 cells or Mac-1–transfected CHO cells were activated with ADP (10 µmol/L), fMLP (1 µmol/L), or the Mac-1–activating mAb KIM 127 (10 µg/mL). Activated cells were then added to wells precoated with the Mac-1 ligands FGN (100 µg/mL) or ICAM-1 (10 µg/mL) and blocked with gelatin. Cells (105 per well) were resuspended in serum-free RPMI containing 0.5% BSA at 106 per milliliter and were incubated in the presence or absence of the indicated mAbs (intact 7E3, c7E3 Fab, 10E5 [anti-IIb/IIIa, non–cross-reacting with Mac-1],45 and LPM19c [anti-CD11b]) at a concentration of 1 to 20 µg/mL and placed in 96-well microtiter plates for 1 to 2 hours at 37°C. Plates were washed with 0.9% NaCl three times, and adherent cells were fixed in methanol for 15 minutes, stained with Giemsa, and adhesion-quantified by measuring absorbance at 540 nm. Data are expressed as percent of maximum adherent responses of respective sets of treatment.

Flow Cytometry
K562 or THP-1 cells (2.5x106 per milliliter) were washed and resuspended in 200 µL of RPMI containing 2.5% BSA. Fc receptors were blocked by addition of human serum at a final concentration of 1%. The primary mAb was then added to the cell suspension followed by incubation on ice for 30 minutes. Cells were treated with ADP (10 µmol/L) or fMLP (1 µmol/L) for 15 minutes at 37°C before incubation with the primary mAb. After washing, cells were resuspended in 200 µL of the same medium containing FITC-conjugated goat anti-mouse F(ab')2 fragments and incubated for 30 minutes on ice. Flow cytometry was performed on a Becton Dickinson FACScan (Becton Dickinson Immunocytometry Systems). Excitation wavelength was set at 488 nm and emission at 550 nm. Nonspecific fluorescence was determined on cells incubated in the absence of the primary mAb.

Fibrinogen Binding Assay
The binding of soluble FGN to human peripheral blood monocytes was investigated as previously described by Altieri and coworkers20 with modification. 125I-labeled FGN (1.0 µmol/L) was added to ADP-stimulated human monocytes (105) adherent to microtiter wells for 60 minutes at 25°C. Cell-free FGN was removed by washing the adherent human monocytes, and cell-bound FGN was determined by counting an aliquot of the cell lysate. Specific binding was calculated by subtracting the counts obtained in the presence of a 20-fold excess of unlabeled FGN from total binding. The effect of mAbs was determined by preincubating ADP-stimulated monocytes for 30 minutes before the addition of 125I-FGN.


*    Results
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 up arrowMethods
 *Results
down arrowDiscussion
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Interaction of 7E3 With Myelomonocytic Cells
The ability of 7E3 to interact with monocytic cells was first explored by using flow cytometry. Prior reports that 7E3 cross-reacts with Mac-1 on blood monocytes have been cautiously interpreted due to concerns about potential platelet contamination. To exclude the possibility that the responses of monocytes would be influenced by platelet contamination, we have used a monocytic (THP-1) cell line and cell lines (K562 and CHO cells) transfected with human Mac-1. Integrin receptors require regulated and reversible activation, which can be induced by ligand binding and by cellular stimulation with a variety of agonists (eg, ADP, fMLP).46 47 Reporter mAbs are capable of defining this high-affinity or activated state. A small subpopulation of Mac-1, representing 10% to 20% of total Mac-1, is recognized by mAb CBRM1/5.37 CBRM1/5 is directed to an activation-specific neoepitope on Mac-1 and is capable of completely blocking Mac-1–dependent adhesion to FGN or ICAM-1. In contrast, LPM19c, a mAb to the {alpha}M subunit (CD11b) of Mac-1 that also blocks adhesion to FGN and ICAM-1,35 recognizes total Mac-1 expressed. In accordance with the findings of Diamond and Springer37 with neutrophils and monocytes, CBRM1/5 bound to fMLP activated THP-1 cells at a significantly lower level than LPM19c (mean fluorescence 49 versus 207; Fig 1ADown). The fraction of Mac-1–bearing cells displaying the activation epitope recognized by CBRM1/5 ranged from 6% to 24% in THP-1 cells activated with fMLP or ADP. Similar to CBRM1/5, 7E3 bound fMLP-activated THP-1 cells at a lower level than LPM19c (mean fluorescence 19 versus 207; Fig 1ADown). We further examined whether CBRM1/5 and 7E3 recognize distinct epitopes on activated THP-1 monocytic cells. Because c7E3 Fab is a humanized fragment of 7E3 that is not recognized by the FITC-conjugated anti-mouse antibody used in our flow cytometry experiments, we were able to address this question using flow cytometry. c7E3 Fab failed to block CBRM1/5 binding (data not shown), suggesting that these mAbs recognize distinct epitopes.



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  		Figure 1. Interaction of 7E3 antibody with THP-1 and K562 myelomonocytic cells. Binding of 7E3 to THP-1, K562, and CD11b K562 cells was analyzed using immunofluorescence flow cytometry as described in "Methods." Cytokine-primed THP-1 cells were stimulated with fMLP (1 µmol/L) and incubated with the following primary mAbs: LPM19c (anti-CD11b), CBRM1/5 (directed to an activation-specific neoepitope on CD11b), or 7E3. Deletion of primary antibody served as the negative control (A). K562 (unshaded) and CD11b K562 (shaded) cells were stimulated with fMLP and incubated with LPM19c or 7E3 (B). The abscissa represents fluorescence intensity; the ordinate, cell number.

To determine whether 7E3 bound directly to Mac-1 on monocytic cells, we used erythroleukemic K562 cells, which do not express Mac-1 and are capable of being stably transfected.40 7E3 bound to K562 cells transfected with the {alpha} subunit (CD11b) of Mac-1 but not to nontransfected cells, supporting a direct interaction between 7E3 and Mac-1 (Fig 1BUp).

7E3 and c7E3 Fab Antibodies Inhibit Mac-1–Mediated Adhesion to FGN and ICAM-1
The functional relevance of 7E3's cross-reaction with Mac-1 was investigated by exploring the effect of 7E3 or c7E3 Fab on cellular adhesion to FGN and ICAM-1. The agonist fMLP increased adhesion of monocytic THP-1 cells to FGN-coated wells >10-fold (Fig 2ADown and 2BDown). LPM19c, an anti-CD11b mAb that blocks FGN binding to Mac-1,35 inhibited FGN adhesion by 82% (P<.01), indicating that THP-1 cell adhesion is Mac-1 dependent. 7E3 and c7E3 Fab blocked adhesion to FGN by 80% and 78% (P<.01), respectively. 10E5, an anti-IIb/IIIa mAb that does not cross-react with Mac-1,45 had minimal effect. A dose response for the inhibition of monocytic cell adhesion by 7E3 is depicted (Fig 2CDown), indicating that the IC50 is {approx}10 µg/mL.



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  		Figure 2. Inhibition of THP-1 monocytic cell adhesion to fibrinogen by 7E3 and anti-CD11b antibodies. The effect of 7E3/c7E3 Fab and anti-CD11b (LPM19c) on Mac-1–mediated adhesion to FGN-coated microtiter wells was investigated in THP-1 monocytic cells. TGF-ß1/1,2-(OH)2 vitamin D3 THP-1 cells were stimulated with fMLP (1 µmol/L), incubated with 7E3, c7E3 Fab, LPM19c, or control (10E5) mAbs (20 µg/mL), and then added to FGN-coated wells for 2 hours at 37°C. Adherent cells were fixed and stained for quantification (A) or photography (B). Adhesion is expressed relative to adhesion stimulated by fMLP in the absence of mAb treatment. A dose response for inhibition by 7E3 (0 to 20 µg/mL) is depicted (C). Values represent mean±SD (n=3); *P<.01.

To confirm the ability of 7E3 to inhibit Mac-1 function and establish that inhibition by 7E3 or c7E3 Fab required Mac-1 expression, we also investigated CHO cells transfected with human Mac-1. CHO cells, which are devoid of Fc receptors, were selected for these experiments because our soluble ICAM-1 preparation is a recombinant fusion protein comprising the extracellular domain of ICAM-1 linked to the constant region of human IgG1. Mac-1 CHO cells adhered to FGN after activation with the KIM 127 mAb (Fig 3Down). Adhesion by Mac-1 CHO cells was inhibited by LPM19c (95±2% inhibition), confirming the role of Mac-1 in this adhesive pathway. Further evidence that Mac-1 expression is necessary to promote the adhesion of CHO cells to FGN is supported by lack of demonstrable adhesion, under these conditions, of nontransfected DG44 CHO cells (data not shown) and CHO cells transfected with a distinct but closely related ß2 integrin, p150,95 (CD11c/CD18) (Fig 3Down). While p150,95 has been shown to mediate adhesion of fMLP-activated neutrophils48 and phorbol ester–activated B lymphocytes49 to FGN-coated surfaces, we were unable to demonstrate significant adhesion of KIM 127–stimulated p150,95 CHO cells to FGN in this experimental system. This lack of adhesion of p150,95 CHO cells may reflect cell-type–specific differences (transfected versus nontransfected cells) or different activation requirements in transfected cells.



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  		Figure 3. Adhesion to FGN of CHO cells transfected with human Mac-1 (CD11b/CD18) or p150,95 (CD11c/CD18). The ability of CHO cells transfected with human Mac-1 (Mac-1 CHO) or p150,95 (p150,95 CHO) to adhere to FGN-coated wells was examined as outlined in "Methods." CHO cells were stimulated with KIM 127 (10 µg/mL), incubated with LPM19c mAb (20 µg/mL), and then added to FGN-coated wells for 2 hours at 37°C. Adherent cells were washed, fixed, and stained with Giemsa for quantification. Values represent mean±SD (n=3).

We next tested whether 7E3 would inhibit Mac-1 binding to ICAM-1, an adhesion molecule that facilitates the adhesion of monocytes and neutrophils to the endothelium.19 KIM 127 stimulated adhesion of Mac-1 CHO cells to ICAM-1 (Fig 4ADown and 4BDown). LPM19c blocked adhesion to ICAM-1 by 86% (P<.01), indicating that transfected CHO cell adhesion to ICAM-1 is Mac-1 dependent. 7E3 and c7E3 Fab reduced adhesion to ICAM-1 by 62% and 63%, respectively (P<.05), while 10E5 had no effect.



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  		Figure 4. Inhibition of Mac-1 CHO cell adhesion to ICAM-1 by 7E3 and anti-CD11b antibodies. The effect of 7E3/c7E3 Fab and anti-CD11b (LPM19c) on adhesion to ICAM-1–coated microtiter wells was investigated in Mac-1 CHO cells. Mac-1 CHO cells were stimulated with KIM 127 (10 µg/mL), incubated with 7E3, c7E3 Fab, LPM19c, or control (10E5) mAbs (20 µg/mL) and then added to ICAM-1–coated wells for 2 hours at 37°C. Adherent cells were fixed and stained for quantification (A) or photography (B). Adhesion is expressed relative to adhesion stimulated by KIM 127 in the absence of mAb treatment. Values represent mean±SD (n=3); *P<.05.

7E3 and c7E3 Fab Antibodies Inhibit Mac-1–Mediated Soluble FGN Binding by Human Peripheral Blood Monocytes
To further evaluate the functional relevance of the interaction between 7E3 and Mac-1, we investigated whether 7E3 and c7E3 Fab are capable of modulating Mac-1 function in freshly isolated human peripheral blood monocytes. ADP-stimulated human monocytes bound soluble FGN ({approx}175 000 FGN molecules per cell). LPM19c inhibited FGN binding to human monocytes by 93% (TableDown), confirming that FGN binding to human monocytes is Mac-1 dependent. Both 7E3 and c7E3 Fab significantly inhibited soluble FGN binding to human peripheral blood monocytes. Lack of inhibition of FGN binding by 10E5, an anti-IIb/IIIa mAb that blocks FGN binding to IIb/IIIa but not to Mac-1,45 not only confirms the specificity of 7E3 but also suggests that platelet contamination of peripheral blood monocytes is unlikely to account for soluble FGN binding to human monocytes.


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  		Table 1. Inhibition of Soluble Fibrinogen Binding to Human Peripheral Blood Monocytes by 7E3


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
This study demonstrates that c7E3 Fab or 7E3 directed against platelet IIb/IIIa also binds to Mac-1–expressing monocytic and transfected cells and thereby inhibits Mac-1–dependent adhesion to fibrin(ogen) and ICAM-1. mAbs that cross-react with multiple integrins have been described. Those reported include mAb 25E11, which cross-reacts with platelet IIb/IIIa and monocyte Mac-1,50 and mAb 24, which detects an epitope within the cation binding domain common to the {alpha} subunit of the leukocyte integrins LFA-1 (CD11a), Mac-1 (CD11b), and p150,95 (CD11c).51 Altieri and Edgington16 have previously shown that 7E3 cross-reacts with monocyte Mac-1, recognizing an activation-dependent neoepitope induced by agonists such as ADP and fMLP, which induce transients in cytosolic Ca2+. However, reports that 7E3 cross-reacts with Mac-1 have been cautiously interpreted owing to concerns about potential platelet contamination.39 We have addressed this concern by using a monocytic cell line and cells transfected with human Mac-1. 7E3 bound to K562 cells transfected with the {alpha} subunit (CD11b) of Mac-1 but not to nontransfected cells, supporting a direct interaction between 7E3 and Mac-1. The demonstration of 7E3 binding to purified ligand binding or I domain of CD11b reported previously by Zhou and colleagues52 provides additional compelling evidence for a direct interaction between 7E3 and Mac-1. These prior studies have explored the functional consequence of the cross-reaction of 7E3 with Mac-1 by examining soluble ligand (ie, factor X) binding, which was largely inhibited by 7E3 (75% to 88%).16 The current study now shows that 7E3 blocks Mac-1–dependent adhesion to fibrin(ogen) and ICAM-1, ligands abundant in the acutely injured vessel wall.53 A dose response for the inhibition of monocytic cell adhesion by 7E3 demonstrated an IC50 of {approx}10 µg/mL (67 nmol/L). This is in close agreement with the reported Kd ({approx}150 nmol/L) of 7E3 for binding to Mac-1–bearing monocytes.16 It is important to note that 7E3 apparently binds to Mac-1 with lower affinity than IIb/IIIa (Kd, 3.4 nmol/L).38 Pharmacokinetic data indicate that bolus infusion of c7E3 Fab in cynomolgus monkeys resulted in a peak plasma concentration of {approx}2.5 µg/mL (54 nmol/L, assuming molecular weight of c7E3 Fab of 48 kD).39 Thus, the relative importance of 7E3-mediated inhibition of IIb/IIIa versus Mac-1 in vivo is currently unknown.

The avidity of integrins may be rapidly and reversibly altered from a latent ("inactive") state to a high-affinity ("active") state without changes in receptor number. Receptor activation, most likely secondary to conformational change(s), increases ligand affinity of ß1,54 ß2,36 and ß355 integrins. For example, agonist stimulation of platelets induces fibrinogen binding to activated IIb/IIIa that is blocked by mAb 7E3.38 Coller and coworkers38 have demonstrated that 7E3 recognizes an activation-dependent change in the conformation of IIb/IIIa, because resting platelets bind 7E3 more slowly than stimulated platelets. In the case of Mac-1, specific agonists (eg, ADP, fMLP) stimulating mAbs (eg, KIM 127 and 185)36 56 and Mn2+57 are capable of directly activating Mac-1 from a low- to high-affinity ligand-binding state by inducing Ca2+ transients or by binding to the ß2 subunit or divalent-cation domains of the {alpha}M subunit, respectively. Analagous to the interaction between 7E3 and platelet IIb/IIIa, CBRM1/5 is directed to an activation-specific epitope on leukocyte Mac-1 and is capable of completely blocking Mac-1–dependent adhesion.37 Our data suggest the possibility that 7E3 also binds to activated Mac-1. 7E3 bound to CD11b K562 cells but not nontransfected K562 cells and identified a subpopulation of Mac-1 by flow cytometry that is qualitatively similar to the subpopulation identified by CBRM1/5. However, these mAbs apparently recognize distinct activation epitopes, because c7E3 Fab, a humanized Fab fragment of intact 7E3, failed to block CBRM1/5 binding as assessed by flow cytometry.

The early response to vascular injury is characterized by migration of platelets and inflammatory cells, including monocytes, to the injured vessel wall.58 59 Therefore, the ability of 7E3 to inhibit not only IIb/IIIa-mediated platelet aggregation but also Mac-1–mediated monocyte adhesion may contribute to the process of vessel wall passivation observed clinically in the EPIC Trial. Among the earliest cells recruited into experimental vascular lesions induced in animals and spontaneous atherosclerosis in human arteries,28 29 monocytes serve as markers, initiators, and promoters of vascular injury. Balloon injury of the vessel wall is associated with a marked increase in the expression and secretion of monocyte chemoattractant protein-1.60 Pathological examination of human arteries and saphenous veins after angioplasty or stent placement has revealed monocytes adherent to and within vessel walls.61 There is emerging evidence implicating infiltrating monocytes in the pathogenesis of neointimal hyperplasia after mechanical arterial injury. The activation status of circulating monocytes62 and expression of Mac-163 at the time of angioplasty have been reported to predict later restenosis, and recently, Rogers and coworkers33 have demonstrated that the number of adherent and infiltrating monocytes exquisitely correlated with the extent of neointimal thickening and proliferation after arterial injury. Libby and coworkers7 have proposed a cascade model for restenosis, in which local inflammatory activation of endothelial cells, smooth muscle cells, and leukocytes occurs in a predictable sequence driven by ongoing autocrine and paracrine signals that persist after the original injury and contribute to later phases of intimal thickening. The precise mechanisms underlying the linkage between monocyte adhesion/infiltration and neointimal hyperplasia in this cascade model remain to be elucidated but may involve the elaboration of cytokines and growth factors chemotactic and mitogenic for smooth muscle cells.30 31 32

Therefore, we speculate that the cross-reaction of 7E3 or c7E3 Fab with Mac-1 may play an additional role in inducing passivity of the vessel wall by two mechanisms: (1) blocking the adhesion of monocytes to ICAM-1 and fibrin(ogen) and (2) decreasing thrombus deposition at the site of arterial injury by inhibiting the binding of factor X and its activation to factor Xa, as previously shown by Altieri and coworkers.23 Other platelet IIb/IIIa receptor inhibitors under active clinical investigation (ie, lamifiban, tirofiban, and xemlofiban) have varying degrees of specificity with the target (ie, IIb/IIIa) and homologous (ie, {alpha}vß3, Mac-1) integrins.64 The clinical relevance of variable IIb/IIIa, Mac-1, and {alpha}vß3 blockade and its resultant effects on acute ischemic complications and restenosis after percutaneous transluminal coronary angiography remain to be determined.


*    Selected Abbreviations and Acronyms
 
FGN = fibrinogen
ICAM-1 = intercellular adhesion molecule-1
mAb = monoclonal antibody
TGF-ß1 = transforming growth factor-ß1


*    Acknowledgments
 
This work was supported by National Institutes of Health grants HL02768 (to Dr Simon) and HL03104 87 (to Dr Rogers) and was aided by grant No. 13-526-945 from the American Heart Association, Massachusetts Affiliate, Inc (to Dr Simon). The authors thank Dr Barry Coller for providing 7E3, c7E3 Fab, and 10E5; Dr M. Uskokvic for providing 1,25-(OH)2 vitamin D3; Dr Timothy A. Springer for providing TS1/18, CBRM1/5, and transfected CHO cells; and Dr Martyn Robinson for providing KIM 127.

Received September 30, 1996; accepted December 3, 1996.


*    References
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