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(Journal of Leukocyte Biology. 2000;68:641-649.)
© 2000 by Society for Leukocyte Biology

{alpha}Mß2 (CD11b/CD18, Mac-1) integrin activation by a unique monoclonal antibody to {alpha}M I domain that is divalent cation-sensitive

Randal P. Orchekowski*, Janet Plescia{dagger}, Dario C. Altieri{dagger} and Mary Lynn Bajt*

* Cell and Molecular Biology, Pharmacia Corporation, Kalamazoo, Michigan; and
{dagger} Department of Pathology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The ß2 (CD18) leukocyte integrins play a key role in normal and inflammatory immune responses. In resting leukocytes, these receptors do not bind ligands. However, when leukocytes are exposed to an appropriate agonist, high-affinity ligand binding is achieved, presumably as a result of conformational changes in the integrin. In this study, we describe a novel monoclonal antibody, mAb 6C1, directed against the {alpha}M subunit, which directly induces adhesion of {alpha}Mß2-transfected CHO cells to fibrinogen, ICAM-1, and iC3b. Induction of binding could also be accomplished by monovalent Fab fragments of mAb 6C1 at concentrations similar to that observed with intact IgG, demonstrating stimulation of adhesion was not because of receptor cross-linking at the cell surface. The binding of mAb 6C1 induces conformational changes in the receptor, as evidenced by the expression of an "activation reporter" epitope recognized by mAb 24. The binding of mAb 6C1 is modulated by divalent cations. Mn2+ promoted high levels of 6C1 binding, and Mg2+ supported low levels of binding, however Ca2+ failed to support binding. A unique distinction of mAb 6C1 is localization of its epitope to the {alpha}M I domain. The {alpha}M I domain is essential for ligand binding, can directly bind divalent cations, and participates in the regulation of {alpha}Mß2 ligand-binding affinity. Thus, these studies have identified a novel {alpha}M I domain activation epitope of {alpha}Mß2 and support the idea that the I domain modulates the activational state of the ß2 integrins.

Key Words: adhesion • active conformation • inflammatory immune response


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The integrins are a family of adhesion molecules that mediate cell-cell and cell-extracellular matrix interactions in a variety of biological processes [1 ]. The ß2 (CD18) leukocyte integrin subfamily is essential for regulation of normal and inflammatory immune responses [2 , 3 ]. The importance of the ß2 integrins is exemplified by the hereditary disease, leukocyte adhesion deficiency (LAD) [4 , 5 ]. Because of an absence of cell-surface expression of the ß2 integrins, LAD patients suffer from profound immunodeficiency, resulting in reoccurring life-threatening infections. As is common to all integrins, the ß2 integrins are heterodimeric glycoproteins consisting of a common ß subunit noncovalently associated with a unique {alpha} subunit [6 ]. There are four ß2 integrins: {alpha}Lß2 [CD11a/CD18, lymphocyte function-associated antigen-1 (LFA-1)], {alpha}Mß2 (CD11b/CD18, Mac-1, CR3), {alpha}Xß2 (CD11c/CD18, p150,95), and {alpha}dß2 (CD11d/CD18) [6 , 7 ]. All four {alpha} subunits contain a 200 amino acid-inserted or "I" domain. The I domain is also present in five other integrin {alpha} subunits: {alpha}1 of {alpha}1ß1[8 ], {alpha}2 of {alpha}2ß1 [9 ], {alpha}10 of {alpha}10ß1[10 ], {alpha}11 of {alpha}11ß1 [11 ], and {alpha}E of {alpha}Eß7 [12 ]. The I domain plays a significant role in ligand binding [13 ]. In addition, crystal structures of two of the ß2 integrins, {alpha}Lß2 and {alpha}Mß2, have identified a cation-binding site, referred to as the MIDAS motif [14 15 16 ]. Because binding ligands to all integrins requires divalent cations, this suggests that the I domain may influence activation as well as ligand binding.

{alpha}Mß2 is the major leukocyte integrin expressed on neutrophils and mediates a diverse range of biological functions, including phagocytosis of opsonized particles [17 ], adherence to the endothelium [18 , 19 ], neutrophil homotypic aggregation, and chemotaxis [20 ]. {alpha}Mß2 recognizes a multiplicity of protein ligands, including intercellular adhesion molecule-1 (ICAM-1) [21 ], complement C3 fragment iC3b [17 , 20 ], and the coagulation proteins, fibrinogen [22 ] and factor X [23 ]. The diverse functional activities of the leukocyte integrins require that their adhesive interactions be highly regulated [24 , 25 ]. The ß2 integrins are expressed normally in a low-affinity state. Upon activation, the integrins undergo presumptive conformational changes in the extracellular portion of the receptor. These changes regulate access of the ligand to recognition sites in the receptor leading to increased receptor affinity [24 25 26 ]. The increase in ß2 integrin affinity can be accomplished by a variety of different stimuli, including 1) cellular stimulation with chemotactic factors, cytokines, or phorbol esters; 2) the divalent cation Mn2+; 3) cross-linking of functionally related cell-surface receptors; and 4) physiological ligands [24 , 25 ].

Activation of integrins can be mimicked also by monoclonal antibodies (mAbs) directed against the integrin {alpha} or ß subunit in the absence of "inside-out" signals [27 28 29 30 31 32 33 34 ]. Several antibodies have been shown that induce activation of the leukocyte integrins. Thus far, seven activating antibodies have been identified that bind to epitopes on the {alpha}L (NKI-L16 and MEM-83) or ß2 (KIM127, KIM185, MEM-48, CBR LFA-1/2, and mAb 2D8) subunit [27 , 30 31 32 33 , 35 , 36 ]. In this study, we have generated and characterized a novel {alpha}M subunit-specific antibody, mAb 6C1, that promotes {alpha}Mß2 ligand-binding function. The mAb 6C1 induces adhesion of {alpha}Mß2 expressing cells to multiple ligands, including fibrinogen, ICAM-1, and iC3b. The binding of mAb 6C1 induces a conformational change in {alpha}Mß2, which may contribute to the observed, enhanced, ligand-binding function. Moreover, mAb 6C1 recognizes an epitope induced by the divalent cations Mn2+ or Mg2+ within the I domain of the {alpha}M subunit, further substantiating the involvement of this domain in integrin activation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
mAbs and reagents
The following murine mAbs directed against human antigens were used as purified immunoglobulin G (IgG): mAb 3H5 (anti-{alpha}M) [37 ], mAb 8H1 (anti-ß2) [37 ], mAb LM2/1 (anti-{alpha}M) [38 ], and mAb 8.4A6 (anti-ICAM-1) [37 ]. Murine mAb 24 (anti-{alpha}L, -{alpha}M, -{alpha}X) was used as purified IgG, and characterization was described previously [39 , 40 ]. Fluorescein-conjugated goat (Fab')2 anti-mouse immunoglobulins, heavy- and light-chain, were purchased from Biosource International (Camarillo, CA). All mAbs were used at saturating concentrations as determined by flow cytometry.

Construction, expression, and purification of the glutathione S-transferase fusion protein, containing a 208 amino acid fragment comprising the I domain of {alpha}M (Gly111 to Ala318), were described previously [41 ]. Recombinant {alpha}M I domain was released from the pGEX vector frame by thrombin cleavage [41 ]. Fibrinogen was purchased from Kabi Pharmacia (Franklin, OH) and then depleted of residual fibronectin using gelatin-sepharose (Pharmacia, Piscataway, NJ), according to the manufacturer’s instructions. ICAM-1 was purified from human placental lysate by affinity chromatography as previously described [37 ].

Cells
Stably transfected Chinese hamster ovary (CHO) cell lines were established by electroporation with {alpha}L [42 ] or {alpha}M [43 44 45 ] cloned into the expression vector pCDM8 (Invitrogen, San Diego, CA) and ß2 [46 ], cloned into the expression vector pcDNA1/neo (Invitrogen). Cells were placed into selection media containing 700 µg/ml G418 (Geneticin; Gibco-BRL, Grand Island, NY) for two weeks, 48 h after electroporation. Resistant colonies were isolated, and positive colonies were identified by flow cytometry using subunit-specific antibodies. Clonal cell lines were established by single cell sorting in an Epics 753 Coulter instrument (Coulter Cytometry, Hialeah, FL). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Irvine Scientific, Santa Ana, CA), supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT), 2 mM l-glutamine (Irvine Scientific), 1% nonessential amino acids (Sigma Chemical Co., St. Louis, MO), 100 units/ml penicillin G, 100 µg/ml streptomycin sulfate (Irvine Scientific), and 700 µg/ml G418.

COS-7 cells [a monkey kidney fibroblastoid cell line from American Type Culture Collection (ATCC), Rockville, MD] were maintained in DMEM supplemented with 10% FBS, 2 mM l-glutamine, 1% nonessential amino acids, 100 units/ml penicillin G, and 100 µg/ml streptomycin sulfate. COS-7 cells were transiently transfected by electroporation with the wild-type {alpha}M or the mutant {alpha}M subunit with the wild-type ß2 subunit. Construction of the mutant {alpha}M constructs {alpha}M(D140A), {alpha}M(D142A), {alpha}M(D140GS/A140GA), {alpha}M(S144A), {alpha}M(L250A), and {alpha}M(N224A) was described previously [47 ]. Cells were evaluated for surface expression 48 h after electroporation.

Baby hamster kidney cells (BHK/VP16) stably expressing the glycosyl phosphatidylinositol (GPI)-anchored form of the {alpha}L I domain [48 ] were kindly provided by M. L. Dustin (Washington University School of Medicine, St. Louis, MO). BHK/VP16 cells were maintained in DMEM supplemented with 10% FBS, 2 mM l-glutamine, 1 mM Na pyruvate, 50 units/ml penicillin G, 50 µg/ml streptomycin sulfate, and 452 units/ml hygromycin.

Generation of mAb 6C1
{alpha}Mß2 was purified from polymorphonuclear leukocytes by mAb LM2/1 immunoaffinity chromatography as previously described [21 ]. Mice (BALB/c) were immunized with purified {alpha}Mß2 (20 µg) subcutaneously, which was followed by three intraperitonial injections of purified {alpha}Mß2 (40 µg/immunization) at 1-week intervals. The immunogen was mixed with Freund’s complete adjuvant (Difco, Detroit, MI) for the primary immunization and incomplete adjuvant for subsequent injections. Four days before fusion, animals received an intrasplenic boost of 20 µg of purified {alpha}Mß2. Hybridomas were generated by fusion of spleen cells to the murine myeloma cell line P3X62Ag8.653 (CRL 1580; ATCC) maintained in selection media (hypoxanthine-aminopterin-thymidine). The protocol for fusion and maintenance of the cells has been previously described [49 ]. Primary hybridoma supernatants were screened by enzyme-linked immunosorbent assay (ELISA) for reactivity with {alpha}Mß2-transfected CHO cells. Positive hybridomas were tested for their ability to enhance the adhesion of {alpha}Mß2-transfected CHO cells to immobilized fibrinogen and iC3b. Certain antibodies were subcloned twice by limiting dilution. Of these, one clone, designated mAb 6C1, was produced as ascites and purified on protein A-sepharose (Pierce Chemical Co., Rockford, IL), according to the manufacturer’s instructions. Fab fragments were prepared by digestion of the antibody with immobilized papain using the ImmunoPure Fab Preparation Kit (Pierce). Fc fragments and undigested IgG were removed by protein A chromatography. The purity of the Fab fragments was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and nonreducing conditions and visualized by silver staining. The reduced Fab fragments migrated as a single band at 25 kD. There were no detectable IgG heavy or light chains, Fc fragments, or intact IgG present. Isotyping of mAb 6C1 was determined using a murine immunoglobulin isotyping kit (Biosource International) according to the manufacturer’s instructions.

ELISA
Purified I domain of {alpha}M was immobilized on Immunlon II plates (Dynatech Laboratories, Chantilly, VA) by the addition of 50 µl/well of 10 µg/ml {alpha}M I domain in 100 mM NaHCO3, pH 8.2, at 4°C overnight. Alternatively, CHO cells expressing {alpha}Mß2 were seeded in 96-well, flat-bottom, tissue culture-treated plates (Corning Costar, Cambridge, MA) and grown to confluence. Culture media was removed, and the cells were fixed by addition of 1% paraformaldehyde (Fisher Scientific, Itasca, IL) in Dulbecco’s phosphate-buffered saline (PBS; Gibco Labs, Gaithersburg, MD) for 12–18 h at 4°C. After two washes with PBS containing 0.05% Tween 20 (PBST), the remaining binding sites were blocked with 3% bovine serum albumin (BSA; Sigma) for 2 h at ambient temperature. Wells were washed with PBST three times, and 100 µl primary hybridoma supernatant or purified IgG was added per well. Following incubation for 60 min at 37°C, the plates were washed three times with PBST, and goat anti-mouse alkaline phosphatase-conjugated antibody (Zymed Laboratories, South San Francisco, CA) diluted with PBS containing 1% BSA was added. Plates were incubated for 1 h at 37°C, washed three time with PBST, and bound antibody was detected with 2 mg/ml p-nitrophenyl phosphate (PNPP) (Sigma) in 50 mM diethanolamine, pH 9.5, 0.06 N HCl, 5 mM MgCl2, and 0.02% NaN3. Plates were read at an absorbance of 405 nm with a ThermoMax multiwell plate reader (Molecular Devices, Menlo Park, CA).

Adhesion assay
Flat-bottom, 96-well microtiter plates (Immulon 2; Dynatech Laboratories) were coated with fibrinogen (100 µg/ml), diluted in 0.1 M NaHCO3 overnight at 4°C, pH 8.0. Alternatively, plates were coated with human-purified placental ICAM-1 or iC3b as previously described [37 ]. The remaining binding sites on the plastic were blocked with 1% gelatin in PBS for 30 min at ambient temperature. Transfected CHO cells were detached from culture plates with nonenzymatic cell dissociation solution (Sigma) and 0.01% 1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (TPCK-trypsin; Worthington Biochemical, Freehold, NJ). Cells were washed in the presence of 0.1% soybean trypsin inhibitor (Sigma) and resuspended in modified Tyrode’s buffer (138 mM NaCl, 12 mM NaHCO3, 2.5 mM KCl, 5 mM HEPES, 0.1% glucose, 0.1% BSA, 1 mM MgCl2, and 1 mM CaCl2). Transfected CHO cells were fluorescently labeled with 2'7'-bis-(carboxyethyl)-5(6)-carboxyfluorescien (Molecular Probes, Eugene, OR). A 100 µl aliquot of 8 x 104 cells was plated in triplicate to the microtiter wells. Cells were incubated in the presence or absence of the activating mAb 6C1, or blocking mAbs 3H5 (anti-{alpha}M) or 8H1 (anti-ß2) at a final concentration of 50 µg/ml (except as indicated) for 30 min before addition to the plate. To inhibit adhesion to ICAM-1-coated wells, 50 µl mAb 8.4A6 (anti-ICAM-1; 50 µg/ml) was added to the wells 30 min before addition of cells to the wells. Following incubation for 30 min at 37°C, the plates were washed five times with modified Tyrode’s buffer. Fluorescence was quantitated in the wells using a Pandex fluorescence concentration analyzer (Baxter Healthcare, Mundelein, IL).

Flow cytometric analysis
Flow cytometric analysis was carried out as previously described or with modification for mAb 24 epitope expression [47 ]. Briefly, cells were harvested as described above and washed twice in chelex-treated tris-buffered saline (TBS; 50 mM Tris-HCl, pH 7.4, 150 mM NaCl). A 50 µl aliquot of cells (1x107 cells/ml) was pelleted in V-bottom 96-well plates (Corning Costar). Cells were resuspended with 50 µl mAb 6C1 or control IgG at the indicated concentrations in TBS containing 1 mM CaCl2 and 1 mM MgCl2. Alternatively, cells were incubated with TBS containing 0.5 mM MnCl2, 1 mM CaCl2, and 1 mM MgCl2 or 10 mM ethylenediamine tetraacetate (EDTA). Incubations were performed at ambient temperature for 30 min. Cells were washed with TBS containing the indicated cations and incubated for 30 min with 50 µl fluorescein-conjugated mAb 24 (20 µg/ml). Cells were then washed with TBS containing the indicated cations, and antibody binding to cells was analyzed by flow cytometry on a FACScan (Beckman Instruments, Fullerton, CA).

Antibody-binding assay
Transfected CHO cells were harvested as described above, washed twice in chelex-treated TBS, and resuspended in TBS containing 1 mM MgCl2, 1 mM CaCl2, 0.5 mM MnCl2, 1 mM MgCl2, and CaCl2 or 10 mM EDTA. Cells (1x106) were incubated with 125I-mAb 6C1 at the indicated concentrations. After 30 min, bound 125I-mAb 6C1 was separated from free 125I-mAb 6C1 by centrifugation through 0.3 ml of 20% sucrose in a Beckman Microfuge B, and the amount of 125I-mAb 6C1 associated with the cell pellet was determined by scintillation spectrometry. Nonsaturable binding of 125I-mAb 6C1 was measured in the presence of a 10-fold excess of unlabeled mAb 6C1. Data were fit to equilibrium-binding models by the nonlinear least-squares, curve-fitting MULTI program [50 ]. The molecules of mAb 6C1 bound per cell were calculated from the specific activity of the 125I-mAb 6C1 and by using a molecular weight of 150,000. The total number of {alpha}Mß2 receptors on CHO cells was determined to be ~500,000/cell, as calculated from the specific activity of the 125I-mAb LM2/1 (anti-{alpha}M) and by using the molecular weight of 150,000.

Surface iodination, immunoprecipitation, and SDS-PAGE
{alpha}Mß2-transfected CHO cells were harvested with 3.5 mM EDTA, washed twice in TBS, and resuspended in 1 ml TBS containing 1 mM CaCl2 and 1 mM MgCl2. Cells were surface-radioiodinated by the lactoperoxidase-glucose oxidase method and immunoprecipitated with antibody as previously described [47 , 51 ]. Precipitated proteins were resolved by 7.5% SDS-PAGE under reducing conditions. Gels were dried and visualized by autoradiography.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
mAb 6C1 induces adhesion of {alpha}Mß2-transfected CHO cells to immobilized Fg, ICAM-1, and iC3b
Antigen-sensitized splenocytes from mice immunized with purified {alpha}Mß2 were fused with myeloma cells to produce hybridomas. Hybridoma supernatants were screened initially for reactivity to {alpha}Mß2-transfected CHO cells in a whole-cell ELISA. Antibodies reacting in this assay were further screened for their ability to enhance adhesion of {alpha}Mß2-transfected CHO cells to immobilized fibrinogen. One antibody, designated mAb 6C1, which induced adhesion to fibrinogen, was identified (Fig. 1A ). In the absence of any added stimulation, {alpha}Mß2-transfected CHO cells did not adhere to immobilized fibrinogen. However, following incubation with mAb 6C1, there was a marked increase in adhesion to fibrinogen in a dose-dependent manner. Maximal adhesion was observed at 100 µg/ml, and half-maximal adhesion was observed at 10 µg/ml. This adhesion was inhibited completely by the addition of the blocking anti-ß2 mAb 8H1 (Fig. 1A) or anti-{alpha}M mAb 3H5 (unpublished results), consistent with mediation by {alpha}Mß2.



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  		Figure 1. mAb 6C1 induces adhesion of recombinant {alpha}Mß2 expressed on CHO cells to immobilized ligand. Fluorescently labeled {alpha}Mß2-transfected CHO cells were pretreated with the indicated concentrations of mAb 6C1 in the presence or absence of blocking anti-ß2 antibody 8H1. (C) Alternatively, fluorescently labeled {alpha}Mß2-transfected CHO cells were pretreated with whole IgG (60 µg/ml) or Fab fragments (180 µg/ml) of mAb 6C1 in the presence or absence of blocking anti-{alpha}M antibody 3H5. Fab fragments of mAb 6C1 were prepared as described in Materials and Methods. Cells were then allowed to attach (in the presence of 1 mM Mg2+ and 1 mM Ca2+) to microtiter wells coated with fibrinogen (A, C), ICAM-1, or iC3b (B). Unbound cells were removed, and adherent cells were quantitated by fluorescence using a Pandex fluorescence concentration analyzer. Results are representative of three experiments. The maximum level of fluorescence obtained with each ligand corresponds to 60–70% of cell attachment, where 100% equals the total number of cells bound to wells coated with the anti-ß2 mAb 8H1. Each point or bar represents the mean, and the vertical line is the SD of triplicate determinations.

 
Next, we examined the ability of mAb 6C1 to support {alpha}Mß2-transfected CHO cells’ adhesion to immunopurified ICAM-1 or to the complement C3 fragment iC3b, two other known ligands for {alpha}Mß2. {alpha}Mß2-transfected CHO cells displayed a low level of adherence to immobilized ICAM-1 (Fig. 1B) . In the presence of mAb 6C1, there was a 2.5-fold increase in adherence. Adhesion was specific because it could be inhibited by anti-ß2 mAb 8H1 (Fig. 1B) or anti-{alpha}M mAb 3H5 (unpublished results). In contrast to immobilized fibrinogen and ICAM-1, {alpha}Mß2-transfected CHO cells adhere to immobilized iC3b under the coating conditions used. However, mAb 6C1 induced a further increase in binding of {alpha}Mß2-transfected CHO cells to iC3b. Adhesion to iC3b in the presence or absence of mAb 6C1 was completely inhibited by the blocking antibody mAb 8H1 to ß2 (Fig. 1B) or mAb 3H5 to {alpha}M (unpublished results). Taken together, these results demonstrate that mAb 6C1 promotes adhesion of {alpha}Mß2-transfected CHO cells to multiple ligands. Importantly, monovalent Fab fragments of mAb 6C1 were equivalent to whole, intact IgG in their ability to induce {alpha}Mß2-transfected CHO-cell adhesion (Fig. 1C) .

mAb 6C1 recognizes an epitope on {alpha}M
To identify the subunit specificity of mAb 6C1, immunoprecipitates of detergent-lysed 125I-labeled CHO cells, expressing recombinant {alpha}Mß2or {alpha}Lß2and 125I-labeled human neutrophils, were analyzed by SDS-PAGE (Fig. 2A ). The mAb 6C1 immunoprecipitated {alpha}M and ß2 in the stably transfected {alpha}Mß2 cells only, analogously to the control anti-{alpha}M mAb 3H5. In contrast, an antibody directed against {alpha}L, mAb TS1/22, precipitated {alpha}L and ß2 in the stably transfected {alpha}Lß2 cells only. The anti-ß2 mAb 8H1 precipitated bands concordant with {alpha}Lß2 and {alpha}Mß2 complexes in the {alpha}Lß2- and {alpha}Mß2-transfected cell lines, respectively. In addition, the mAb 6C1 immunoprecipitated {alpha}M and ß2in human neutrophils similarly to the control anti-{alpha}M mAb 3H5 and anti-ß2mAb 8H1 antibodies. Analysis by immunoblots of purified {alpha}Mß2from neutrophils revealed that mAb 6C1 did not recognize the {alpha}M subunit under reduced or nonreduced conditions (unpublished results).



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  		Figure 2. mAb 6C1 recognizes an epitope on the {alpha}M subunit. (A) Detergent lysates from surface-iodinated {alpha}Lß2- or {alpha}Mß2-transfected CHO cells and human neutrophils were immunoprecipitated with mAbs 8H1 (anti-ß2, TS2/4 (anti-{alpha}L), 6C1, or LM2/1 (anti-{alpha}M). The precipitated proteins were resolved by electrophoresis on 7.5% SDS-PAGE under reducing conditions and detected by autoradiography. Molecular mass markers are shown on the right in kDa. (B) The binding of mAb 6C1 to {alpha}Mß2- or {alpha}Lß2-transfected CHO cells was examined by flow cytometry. {alpha}Mß2- (top panels) or {alpha}Lß2- (bottom panels) transfected CHO cells were incubated with control IgG antibody (open peaks) or subunit-specific antibodies (solid peaks), anti-ß2 (mAb 8H1), anti-{alpha}M (mAb 3H5), anti-{alpha}L (mAb TS1/22), or mAb 6C1 (in the presence of 1 mM Mg2+ and 1 mM Ca2+). Cells were washed, stained with fluorescein-conjugated goat anti-mouse IgG, and analyzed by flow cytometry. Results are depicted as histograms with the log of fluorescence on the abscissa and the cell number on the ordinate.

 
The subunit specificity of mAb 6C1 was determined further by flow cytometry using CHO cells expressing recombinant {alpha}Mß2or {alpha}Lß2(Fig. 2B) . The mAb 6C1 bound to the {alpha}Mß2-transfected CHO cells only, similar to the control anti-{alpha}M mAb 3H5; the anti-{alpha}L mAb TS1/22 bound to the {alpha}Lß2-transfected CHO cells only. In contrast, both CHO-cell transfectants bound to the control anti-ß2 mAb 8H1. These data support our previous results [52 ] demonstrating mAb 6C1 binding to COS-7 cells transiently transfected with the {alpha}M subunit alone or {alpha}M cotransfected with ß2. In contrast, mAb 6C1 did not bind to cells transiently transfected with the ß2 subunit alone. Taken together, these results demonstrate that the mAb 6C1 epitope is located within the {alpha}M subunit.

mAb 6C1 induces a conformational change in {alpha}Mß2
To determine whether mAb 6C1 induces a conformational change in the {alpha}Mß2receptor, we examined the expression of the "activation-dependent" conformational epitope recognized by mAb 24 by flow cytometry. The mAb 24 recognizes a common epitope on all three {alpha} subunits of the ß2 integrins [39 , 40 ]. Expression of mAb 24 is associated with the Mn2+- or Mg2+-occupied form of the ß2 integrins and coincides with an increase in receptor activity. In the absence of stimulation, fluorescein isothiocyanate (FITC)-conjugated mAb 24 bound minimally to {alpha}Mß2-transfected CHO cells (Fig. 3 ). The activating antibody mAb 6C1 induced mAb 24 expression in a dose-dependent manner. A control anti-ß2antibody had no effect on mAb 24 epitope expression. These results suggest that the mAb 6C1 induces a functionally active {alpha}Mß2 integrin conformation recognized by mAb 24.



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  		Figure 3. mAb 6C1 induces expression of the activation-dependent neoepitope recognized by mAb 24 on {alpha}Mß2-transfected CHO cells. The binding of mAb 24 to {alpha}Mß2-transfected CHO cells was examined by flow cytometry. Cells were incubated with FITC-conjugated mAb 24 in the absence or presence of the indicated concentrations of mAb 6C1 in buffer containing 1 mM Mg2+ and 1 mM Ca2+. Cells were then washed and analyzed on a FACScan flow cytometer. Results are representative of three experiments.

 
mAb 6C1 recognizes a divalent cation-dependent epitope on {alpha}M
Because of the importance of divalent cations in modulating the {alpha}Mß2function, the expression of the mAb 6C1 epitope on {alpha}Mß2-transfected CHO cells under defined divalent cation conditions was examined by flow cytometry (Fig. 4 ). The mAb 6C1 binding was maximal in the presence of 0.1 mM Mn2+ and lower in the presence of 1.0 mM Mg2+, while 1.0 mM Ca2+ and 10 mM EDTA failed completely to support binding. The mAbs 24 and 3H5, which recognize cation-dependent and -independent epitopes, were used as positive and negative controls, respectively. The divalent cation regulation of the mAb 6C1 binding was confirmed in whole-cell binding assays (Table 1 ). Analysis of isotherms of equilibrium binding of mAb 6C1 in the presence of 0.5 mM Mn2+ to {alpha}Mß2-transfected cells indicated Ka = 1.01 ± 0.06 x 107M-1 (Kd=99 nM). In the presence of 1.0 mM Mg2+, mAb 6C1 binding affinity decreased to Ka = 3.99 ± 0.66 x 106M-1 (Kd=255 nM), while 1.0 mM Ca2+ and 1.0 mM Mg2+ or 1.0 mM Ca2+ alone failed to support binding. Thus, the expression of the mAb 6C1 epitope is cation-dependent.



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  		Figure 4. Expression of mAb 6C1 epitope is divalent cation-dependent. {alpha}Mß2-transfected CHO cells were incubated with fluorescein-conjugated antibodies mAb 6C1 or anti-{alpha}M antibody 3H5 in buffer containing 1mM Ca2+, 1 mM Mg2+, 0.1 mM Mn2+, or 10 mM EDTA. Cells were washed and analyzed by flow cytometry. Results are representative of three experiments and depicted as histograms with the log of fluorescence on the abscissa and the cell number on the ordinate.

 

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  		Table 1. 125I-6C1 binding affinities to recombinant {alpha}Mß2-expressing CHO cells

 
A number of potential divalent cation-binding sites have been identified within {alpha}Mß2. These divalent cation-binding site(s) are important regulators of {alpha}Mß2 ligand-binding function. The I domain of {alpha}M contains a cation-binding site, which enables it to form two different conformations dependent on the divalent cation coordination [14 , 15 ]. Furthermore, we have demonstrated previously that alanine substitution of residues, which coordinate cation binding D140A, S142A, D140GS-A140GA, and S144A within the {alpha}M I domain, modulates a divalent cation conformation recognized by mAb 24 [47 ]. Consequently, the effects of the {alpha}M I domain mutations, which disrupt the interaction between bound divalent cation and {alpha}Mß2 on mAb 6C1 expression, were examined by flow cytometry in transiently transfected COS cells (Fig. 5 ). In the presence of 0.5 mM Mn2+, cells expressing wild-type {alpha}Mß2 stained brightly with mAb 6C1 when compared with the anti-{alpha}M control antibody 2LPM19c. In contrast, mAb 6C1 binding in the presence of 0.5 mM Mn2+ to cells expressing {alpha}M(D140A)ß2 or {alpha}M(S142A)ß2 was reduced considerably when compared with the anti-{alpha}M control antibody 2LPM19c. However, cells expressing {alpha}M(S144A)ß2 resulted in a modest decrease of mAb 6C1 binding. In addition, an I domain mutation, which did not affect mAb 24 expression {alpha}M(N224A), did not alter mAb 6C1 binding. These data demonstrate that the expression of the mAb 6C1 epitope is dependent on divalent cation-binding interactions within the I domain and {alpha}Mß2.



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  		Figure 5. Expression of mAb 6C1 epitope on recombinant wild-type or mutant {alpha}Mß2 receptors. The binding of mAb 6C1 to COS-7 cells transfected with wild-type or mutant {alpha}Mß2 receptors was examined by flow cytometry. Transfected cells were incubated with mAb 2LPM19c or mAb 6C1 in the presence of 0.1 mM MnCl2 for 30 min at room temperature. Cells were washed, stained with fluorescein-conjugated goat F(ab')2 anti-mouse Igs for 30 min, and analyzed. Results are representative of three experiments and depicted as histograms with the log of fluorescence on the abscissa and the cell number on the ordinate.

 
The mAb 6C1 epitope is located within the I domain of {alpha}M
The I domain has been implicated in the regulation of activation-dependent, integrin-binding function. Discrete sites, which modulate the adhesive activity of {alpha}Mß2 within the I domain of {alpha}M, have been identified. Furthermore, a novel {alpha}Lß2 activation epitope recognized by mAb 83, which is located within the I domain of {alpha}L, has also been identified [32 ]. To determine whether the mAb 6C1 activation epitope was located within the I domain of {alpha}M, mAb 6C1 was tested for its ability to recognize a purified I domain in an ELISA assay. The {alpha}M I domain was isolated and purified from a glutathione S-transferase fusion protein as previously described [41 ]. The recombinant {alpha}M I domain was recognized specifically by anti-{alpha}M antibodies LM2/1 and 3H5 (Fig. 6A ; and unpublished results). Similarly, mAb 6C1 reacted with the I domain protein. In contrast, the anti-ß2 mAb TS1/18 and anti-{alpha}M antibody mAb 24 did not recognize the {alpha}M I domain. None of the antibodies tested reacted with BSA. No differences in the binding of mAb 6C1 to the {alpha}M I domain were detected using different cation conditions in a way that mirrors the effects of cation binding of mAb 6C1 to recombinant {alpha}Mß2 expressed on CHO cells (unpublished results). It is possible that once the {alpha}M I domain is immobilized, it is unable to undergo further cation-dependent conformational changes.



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  		Figure 6. mAb 6C1 recognizes an epitope within the {alpha}M I domain. (A) Recombinant {alpha}M I domain (10 µg/ml) or BSA (1 mg/ml) in 100 mM NaHCO3, pH 8.2, was immobilized on Immunlon II plates at 4°C overnight. After washing with PBS (no cations) containing 0.05% Tween 20, the remaining binding sites were blocked with 1% BSA for 2 h at ambient temperature. Following extensive washing, 50 µl of primary antibody (50 (µg/ml) was added per well for 60 min at 37°C. Plates were washed extensively, and goat anti-mouse alkaline phosphotase diluted in PBS containing 1% BSA was added. Plates were incubated for 1 h at ambient temperature, and bound antibody was detected with PNPP. Plates were read at an absorbance of 405 nm with a ThermoMax multiwell plate reader. Results are representative of three experiments. Each bar represents the mean and the vertical line is the SD of triplicate determinations. (B) The binding of mAb 6C1 to the {alpha}L I domain expressed on BHK/VP16 cells was examined by flow cytometry. The {alpha}L I domain-transfected cells were incubated with anti-ß2 mAb 8H1, anti-{alpha}LmAb TS1/22, anti-{alpha}LmAb 24, or mAb 6C1 (in the presence of 1 mM Mg2+ and 1 mM Ca2+). Cells were washed, stained with fluorescein-conjugated goat anti-mouse IgG, and analyzed by flow cytometry. Results are representative of three experiments.

 
As an additional, comparable control to the {alpha}M I domain, the mAb 6C1 was tested for its ability to recognize BHK cells expressing the GPI-anchored form of the {alpha}L I domain [48 ], as determined by flow cytometry (Fig. 6B) . The {alpha}L I domain was recognized specifically by the anti-{alpha}L mAb TS1/22. In contrast, mAb 6C1 did not recognize the {alpha}L I domain. Similarly, the anti-ß2 mAb TS1/18 and anti-{alpha}L mAb 24 did not react with {alpha}L I domain-expressing cells. Thus, the mAb 6C1 activation epitope is located with the I domain of {alpha}Mß2.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The major findings in this work are as follows: 1) We have generated and characterized a novel {alpha}M-specific antibody, mAb 6C1, which induces {alpha}Mß2-binding directly to fibrinogen, ICAM-1, and iC3b. 2) The mAb 6C1 induces expression of the mAb 24 "activation dependent" conformational epitope. 3) The epitope recognized by mAb 6C1 is regulated by divalent cations. 4) The mAb 6C1 epitope is located within the I domain of the {alpha}M subunit. Thus, these studies have identified a novel {alpha}M activation epitope within the I domain of {alpha}Mß2 and further suggest the involvement of this domain in regulating ß2 integrin-ligand interactions.

With the exception of iC3b, {alpha}Mß2-expressing cells are competent to bind ligands only following cellular activation [53 ]. {alpha}Mß2-transfected CHO cells are in a low-affinity state, as determined by the absence of binding to immobilized fibrinogen or ICAM-1. Using this information, we developed a screening strategy to identify antibodies that stimulate {alpha}Mß2-adhesive interactions. mAb 6C1 was identified by its ability to enhance {alpha}Mß2-adhesive interactions with fibrinogen and ICAM-1. Although {alpha}Mß2-transfected CHO cells adhere to iC3b, mAb 6C1 enhanced this interaction further as well. Binding of {alpha}Mß2-transfected CHO cells induced by mAb 6C1 was dose-dependent and inhibited by blocking mAbs to {alpha}M and ß2. The ability of Fab fragments to reproduce the activating properties of mAb 6C1 excludes sufficiently the possibility that these events are mediated by cross-bridging of cell-surface receptors.

The mAb 6C1 promotes {alpha}Mß2-dependent adhesive interactions by inducing a conformational change directly in the receptor upon its binding. This conclusion is supported by the fact that mAb 6C1 binding induced expression of the "activation dependent" conformational epitope recognized by mAb 24. Expression of mAb 24 epitope is associated with the active conformation of the ß2 integrins and increased affinity for their ligands [39 , 40 ]. Therefore, the ability of mAb 6C1 to promote adhesive interactions may be a result of direct modulation of the affinity of ß2 integrin receptors for their ligands. Similarly, other antibodies that promote ligand binding have been shown to induce expression of activation-dependent epitopes also; anti-{alpha}L mAb MEM-83 induces mAb 24 binding [32 ], anti-ß2 mAb KIM 185 induces KIM 127 epitope expression [31 ], and anti-ß3 mAbs P41 and 62 induce PAC-1 binding [54 ].

A third characteristic pertinent to the recently proposed ligand-binding model [55 ] is that the binding of mAb 6C1 is modulated by divalent cations. The degree of binding relies on the composition of divalent cations in the buffer. Mn2+ promoted high levels of 6C1 binding, and Mg2+ supported low levels of binding, however Ca2+ failed to support binding totally. In addition, mutations, which disrupt the interaction between bound divalent cation and {alpha}Mß2 within the {alpha}M I domain, affected severely the expression of the mAb 6C1 epitope. Divalent cation-binding site(s) have been proposed to participate directly in a ternary complex among cation, ligand, and receptor [26 , 55 , 56 ]. Binding of ligand causes displacement of cation from the interactive site(s) within the receptor, subsequently stabilizing the receptor-ligand interaction. Because mAb 6C1 defines a functionally important integrin epitope, which is involved in receptor activation and is sensitive to divalent cations, the epitope may participate in stabilizing this favorable ligand-binding conformation.

An unusual attribute of mAb 6C1 is its low affinity of recognition. A Kd of 100 nM in the presence of Mn2+ and micromolar in the presence of physiological cations opens intriguing questions as to its mechanism(s) of receptor activation. Exactly how mAb 6C1 contributes to the enhanced ligand-binding activity of {alpha}Mß2 is currently not known. One possibility is that mAb 6C1 may upregulate ligand binding by directly binding to a nonactivated integrin conformation, resulting in the receptor transition to an activated state. Furthermore, the mAb 6C1 may detect a conformation-sensitive epitope on the {alpha}M I domain that is induced not only following cation but also ligand-occupancy, ligand-induced binding sites (LIBS), further stabilizing the conformation [56 ]. Alternatively, mAb 6C1 may bind to a subpopulation of {alpha}Mß2 receptors in a particularly favorable conformation, which leads to their transition to a high-affinity state. This hypothesis is consistant with a model in which the integrin population exists in an equilibria among a complex series of multiple activation states. A portion of the integrin population always has the capablility of existing transiently in a high-affinity conformation. The mAb 6C1 may bind to this subpopulation, shifting the equilibrium of the integrin population toward a more active conformational state. For example, the mAb CBRM1/5 recognizes only a subpopulation of {alpha}Mß2 molecules (10–30%) on activated neutrophils [57 , 58 ]. However, this subpopulation of {alpha}Mß2 molecules mediates at least 90% of the adhesion activity of {alpha}Mß2. The mAb CBRM1/5, like mAb 6C1, recognizes the {alpha}M I domain itself. However, unlike mAb 6C1, it does not induce activation.

Another unique feature of the mAb 6C1 epitope is its localization to the {alpha}M I domain. Unfortunately, we were unable to map precisely within the {alpha}M I domain mAb 6C1 epitope using conventional approaches such as peptide mapping or binding to synthesized I domain subregions. In all likelihood, the epitope is sensitive to conformational constraints. This is supported by the observation that mAb 6C1 failed to recognize a denatured, nonreduced, or reduced receptor, as determined by Western analysis. To date, only one other previously defined activation antibody has been localized to an I domain, MEM83, which recognizes {alpha}L [32 ]. However, in contrast to mAb 6C1, the MEM83 epitope is not regulated by divalent cations. Therefore, mAb 6C1 epitope is distinct from those recognized by all other known activating antibodies.

The I domain has shown to be essential for {alpha}Mß2 ligand binding by the localization of blocking antibody epitopes and by direct ligand binding to the isolated I domain [13 , 41 ]. The {alpha}M I domain binds cations also [14 , 15 ]. Moreover, localization of the mAb 6C1 activation epitope to the {alpha}M I domain indicates that this domain modulates the ligand-binding conformation state of {alpha}Mß2. This concept is supported by the crystal structures of the {alpha}M I domain, showing the structural flexibility of two distinct conformational states dependent on the cation coordination [15 ]. Furthermore, a discrete site, which modulates the activational state of {alpha}Mß2 within the {alpha}M I domain, has been identified [59 ].

In summary, we have described a mAb directed against the {alpha}M I domain that promotes the adhesion of {alpha}Mß2 to multiple ligands, including iC3b, fibrinogen, and ICAM-1. The anti-{alpha}M I domain epitope is unique in its ability to regulate ligand binding and is modulated by divalent cations. Binding of mAb 6C1, as well as other activating antibodies, induces conformational changes directly or stabilizes active conformations of the integrin receptor favorable for ligand binding. Further characterization of these activation epitopes may prove to be an indispensable approach in understanding further the multiple conformational changes in the ß2 integrins that lead to alteration in ligand-binding affinities.


   FOOTNOTES
 
Correspondence at current address: Mary Lynn Bajt, Ph.D., University of Arkansas for Medical Sciences, Dept. of Pharmacology & Toxicology, 4301 W. Markham St. (Mailslot 638), Little Rock, AR 72205-7199. E-mail: Bajtjaeschkemaryl{at}exchange.uams.edu

Received May 20, 1999; revised May 18, 2000; accepted May 23, 2000.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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