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Leukocyte Biology and Inflammation Program, Structural Biology Program, Renal Unit, Massachusetts General Hospital, Charlestown, MA 02129, and Department of Medicine, Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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subunit-derived A domain provide a direct
bridge between ligands and the integrin, and it has been proposed that
activation dependency is caused by reorientation of the surrounding
residues relative to the metal ion, forming an optimal binding
interface. To gain more insight into the functional significance of the
protein movements on the MIDAS face, we raised and characterized a
murine mAb 107 directed against the MIDAS face of the A domain from
integrin CD11b. We find that mAb 107 behaves as a ligand mimic. It
binds in a divalent-cation-dependent manner to solvent-exposed residues
on the MIDAS face of CD11b, blocks interaction of 11bA or the
holoreceptor with ligands, and inhibits spreading and phagocytosis by
human neutrophils. However, in contrast to physiologic ligands, mAb 107
preferentially binds to the inactive low-affinity form of the integrin,
suggesting that its antagonistic effects are exerted in part by
stabilizing the receptor in the low-affinity state. These data support
a functional relevance of the protein movements on the MIDAS face and
suggest that stabilizing the A domain in the low-affinity state may
have therapeutic benefit. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3-6
1
ligands derived from Yersinia and Escherichia
coli, respectively (4, 5).
Biochemical, structural, and mutational studies have defined an
subunit-derived, A-type domain (A) as a major
ligand binding site (6). It is present in one-half of all
integrin subunits. Its structure (7, 8, 9, 10, 11, 12, 13)
contains a unique divalent cation coordination site designated metal
ion-dependent adhesion site (MIDAS) located in a shallow groove at the
top of the structure (on the opposite end to where the domain connects
to the rest of the -chain). Two conformations of A were described
that differ in the way the metal ion is coordinated and in the packing
of the C-terminal helix. In the "open" or "liganded" form, the
metal ion is coordinated directly by the side chains of S142, S144, and
T209 and by D140 and D242 through water molecules. A glutamate pseudo
ligand or ligand provided respectively by a neighboring A domain
(7) or an exogenous ligand peptide (14)
completes metal coordination. In the "closed" or "unliganded"
conformation, a third water molecule provides the sixth coordination
site of the metal, replacing the glutamate, and D242 replaces T209 in
directly coordinating the metal ion.
The changes in metal coordination observed in the open and closed forms
of A are associated with significant changes in protein structure,
which affect the topology of the MIDAS face and the ability of the
integrin to bind physiologic ligands. Integrin ligands segregate into
two types with respect to their A binding potential. Physiologic
ligands such as iC3b, fibrinogen, CD54 (ICAM-1), or certain
activation-dependent mAbs are sensitive to the conformational changes
observed in the crystal structure of A (11, 15, 16).
Other ligands, including the hookworm-derived NIF (11),
and several mAbs are conformation insensitive (17). A
model of integrin-ligand interaction has been proposed, where
activation-independent ligand binding is mediated by
conformationally insensitive residues on the MIDAS face
(11); cell activation modifies the topology of MIDAS,
creating an optimal binding interface for ligation of
activation-dependent ligands. The switch from the low- to the
high-affinity state appears to be regulated allosterically; a single
Ile316 to Gly substitution in the
7 tail of the CD11b A domain (11bA) switches
MIDAS coordination at the top of the structure to the open state and
results in a high-affinity A domain (17).
To explore further the general applicability of the above model, we generated a mAb, mAb 107, directed against 11bA and examined its structure-activity profiles. mAb 107 exhibited properties similar to those of CD11b ligands, such as metal dependency and binding to the MIDAS face. mAb 107 blocked ligand binding to 11bA and the holoreceptor as well as spreading and phagocytosis, indicating that, although competitive, mAb 107 has no agonist properties. In contrast to other physiologic ligands or antagonists of CD11b, mAb 107 exhibits preferential binding to the low-affinity form of the domain, suggesting that its mechanism of action involves stabilizing the domain in the inactive state. The significance of these findings in relation to current models of integrin-ligand interactions and their therapeutic potential are discussed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Site-directed mutagenesis was conducted as previously described (18). Each mutation was confirmed by the presence of the introduced restriction site and by direct DNA sequencing (19).
Protein purification
Wild-type (WT) recombinant 11bA spanning residues Gly111-Gly321 (11bA111–321), Glu123-Gly321 (11bA123–321), and Glu123-K315 (11bA123–315);11bA mutants D140GS/AGA and D242A; and the CD11a A-domain (11aA) were expressed as GST fusion proteins in E. coli as described previously (6, 17, 20). 11bA123–321 and 11bA123–315 have been shown to represent respectively the low- and high-affinity states of 11bA based on biochemical and crystallographic studies (17). The fusion proteins were purified by affinity chromatography and cleaved with thrombin to release the recombinant A domains. WT and mutant 11bA were further purified by ion exchange chromatography on a Mono S HR5/5 column (Pharmacia, Uppsala, Sweden) using the fast protein liquid chromatography system (Pharmacia). Analysis of each of the purified proteins on 12% SDS-PAGE revealed a single band after staining with Coomassie blue (data not shown).
Generation of mAb 107
mAb 107 was produced using established procedures (21). BALB/c mice (Charles River Breeding Laboratories, Wilmington, MA) were immunized i.v. with 50 µg of 11bA111–321 in CFA (Life Technologies, Gaithersburg, MD). Animals were boosted 2 and 4 wk later with the same dose of Ag in IFA given i.p. A final i.v. boost was given 3 days before sacrifice. The mouse myeloma NS1 cell line was used as the fusion partner. Hybridoma supernatants were screened by ELISA. The initial clone 107 was subcloned three times, and the Ab belonged to the IgG1 subclass.
Other reagents
Restriction and modification enzymes were purchased from New England Biolabs (Beverly, MA), Roche (Indianapolis, IN), or Life Technologies (Rockville, MD). The anti-CD11b mAbs 44a and 904, the anti-CD11a mAb TS1/22, and anti-CD18 mAb TS1/18 have been previously described (22, 23, 24, 25). The anti-CD11a mAb L-1 (26) and mouse IgG1 (Sigma-Aldrich, St. Louis, MO) were used as controls. Purified fibrinogen was a gift from Dr. J. Ylanne (University of Helsinki, Helsinki, Finland). Soluble human CD54 D1-5-Fc, a recombinant fusion protein comprising the five extracellular domains of human CD54 linked to the constant region of human IgG1, was a gift from Dr. L. B. Klickstein (Brigham and Women’s Hospital, Boston, MA).
COS and CHO cell transfections
COS M7 simian fibroblastoid cells at 60–70% confluence were transfected with supercoiled cDNAs encoding CD11b or CD11a and CD18 as previously described (6, 27). Transfected COS cells were grown for 24 h in IMDM (BioWhittaker, Walkersville, MD) supplemented with 10% FBS, 2 mM glutamine, 50 IU/ml penicillin, and streptomycin at 37°C. Cells were then washed, detached with 0.1% trypsin-EDTA, and seeded in replicates onto 24- or 48-well plates (Costar, Cambridge, MA). Confluent monolayers in 24- or 48-well plates were then used for cell surface Ag quantification and Ab binding studies. Formation of CD11b and CD18 heterodimers was confirmed by immunoprecipitation of the CD11b subunit, followed by Western blotting using an anti-CD18 Ab as previously described (6).
The Chinese hamster ovary (CHO)-K1 cell line was maintained in
Ham’s F-12 nutrient mixture (Life Technologies) supplemented with 10%
FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Transfection of
CHO-K1 cells with WT CD11b and CD18 cDNA in pcDNA3/Neo and 
H3M
plasmids, respectively, was performed using the calcium phosphate
precipitation method as previously described (28). After
48 h, the medium was replaced with fresh medium containing 1 mg/ml
G418. The G418-resistant cell population was analyzed for CD11b/CD18
expression using a FACScan flow cytometer (BD Biosciences, Franklin
Lakes, NJ), and the CD11b and CD18 double-positive cells were enriched
by cell sorting. CHO cells expressing CD11b/CD18 were then cloned by
limiting dilution. CHO cells transfected with pcDNA3/Neo alone were
made and used as a negative control.
Preparation of complement EiC3b and AP-conjugated iC3b
Sheep erythrocytes coated with complement iC3b were prepared as previously described (6). iC3b-coated erythrocytes (EiC3b; 1.5 x 108 cells/ml) were surface labeled with biotin by incubating the cells with sulfo-NHS-biotin at 0.5 mg/ml (Pierce, Rockford, IL) for 30 min at 4°C. Cells were then washed and resuspended to the original concentration for use in the binding studies. Fluid phase human iC3b was purified from fresh human serum by affinity chromatography and coupled with alkaline phosphatase (AP) as described previously (11, 29).
Binding of Abs and iC3b to purified WT and mutant 11bA
Ab binding to WT or mutant forms of 11bA were assessed in microtiter plates or on Immobilon-P membranes. For the former, 2 µg of WT or mutant 11bA in 50 µl of 25 mM Tris-HCl (pH 8) and 150 mM NaCl (TBS) were placed in triplicate in 96-well plates overnight at 4°C. Wells were washed and blocked with 1% BSA for 2 h at room temperature and then used for the different binding assays. For binding studies, mAbs (at 10 µg/ml, diluted in TBS containing 1 mM MgCl2, 1 mM CaCl2, and 0.1% gelatin) were added to each well and incubated for 1 h at room temperature. After washing, a second incubation with AP-coupled secondary Ab was conducted. Color reaction was then developed by adding 1 mg/ml p-nitro phenol phosphate and was quantified by measuring the absorbance at 405 nm using a plate reader. In experiments in which the effects of divalent cations on mAb 107 binding to 11bA were examined, the 11bA-coated wells were washed first with 5 mM EDTA and then with TBS/0.1% gelatin alone or containing 1 mM Mg2+, 1 mM Ca2+, 1 mM Mn2+, 5 mM EGTA, 5 mM EDTA, or 5 mM EGTA plus 1 mM Mg2+ or Mn2+ before mAb 107 was added. For slot-blotting analysis, 1 µg of WT or 11bA mutant proteins were blotted onto Immobilon-P paper in duplicate as previously described (18), followed by blocking with BSA. The reactivity of the slot-blotted 11bA with anti-CD11b Abs was assessed by incubating membranes with mAb 107 (at 2 µg/ml final concentration) or the polyclonal anti-CD11b Ab (at 4 µg/ml, final concentration) in Tris-NaCl buffer, pH 7.4, containing 1 mM MgCl2, 1 mM CaCl2, and 1% BSA for 1 h at 4°C. Membranes were washed and reprobed with HRP-coupled secondary Ab (Sigma-Aldrich) for an additional hour. Membranes were then washed and developed using the ECL system from Amersham (Arlington Heights, IL).
For iC3b binding studies, biotinylated EiC3b (3 x 106 cells in 50 µl of Veronal-buffered saline, pH 7.4, containing 1 mM MgCl2, 1 mM CaCl2, and 0.1% gelatin) were added to each well in the presence or the absence of different mAbs or inhibitors. The reactants were briefly spun down and incubated for 15 min at 37°C. After gentle washing, bound EiC3b were fixed with 1% glutaraldehyde, blocked with BSA, and treated with streptavidin-AP conjugate and p-nitro phenol phosphate. Bound EiC3b was quantified by measuring the absorbance at 405 nm. In experiments in which purified iC3b was used, 40 nM AP-conjugated iC3b in TBS containing 1 mM MgCl2, 1 mM CaCl2, and 0.1% gelatin was added to each well and incubated for 1 h at 37°C, and binding was quantified.
Flow cytometry
Human neutrophils were isolated as previously described (30). The purified neutrophils were washed and resuspended to 107 cells/ml in RPMI 1640 medium (Life Technologies) containing 1 mM Mg2+ plus 1 mM Ca2+, or 5 mM EDTA. To 100 µl of cell suspension, mAb 44a or 107 was added to a final concentration of 10 µg/ml and incubated for 20 min at room temperature. The cells were washed three times and incubated with FITC-conjugated goat anti-mouse IgG for another 20 min. After washing, the cells were fixed with 1% formaldehyde in PBS and analyzed on a BD Biosciences FACScan flow cytometer.
Adhesion of CD11b/CD18-transfected CHO cells to different CD11b ligands
Purified iC3b, fibrinogen, and soluble human CD54-Fc were diluted to 10 µg/ml, and 50 µl of each diluted protein was placed in triplicate in 96-well plates. After incubating overnight at 4°C, the plates were washed and blocked with BSA. Fifty microliters of CD11b/CD18-expressing CHO cells (2 x 106 cells/ml in Ham’s F-12 nutrient mixture with 1 mM MgCl2, 1 mM CaCl2, and 0.1% BSA) were added to each well in the presence or the absence of different mAbs or inhibitors and incubated for 30 min at 37°C. After gentle washing, bound cells were quantified by detecting the cellular acid phosphatase level. Binding was normalized to the percentage of binding obtained without any inhibitor.
Phagocytosis and cell spreading assays
Phagocytosis of serum opsonized Oil Red O (ORO) particles was performed as previously described (31). Briefly, purified human neutrophils were preincubated with mAb 44a or 107 ascites (final dilution, 1/200) for 10 min at room temperature. The neutrophils and ORO were prewarmed to 37°C for 2 min before they were mixed and then were incubated for 5 min at 37°C with shaking. The reaction was stopped by adding 1 ml of ice-cold PBS containing 1 mM N-ethyl-malemide. After washing, the cell pellet was examined visually and then solubilized with 0.5 ml dioxan, and the amount of ORO in the extract was quantified by measuring absorption at 525 nm. Specific uptake of ORO was determined by subtracting the background (ORO uptake in the presence of 1 mM N-ethyl-malemide). Phagocytosis was normalized to the percentage of phagocytosis obtained without Ab.
For the cell spreading assay, gelatin (1% in RPMI 1640) was coated on 48-well plates overnight at 4°C. Purified neutrophils (5 x 106 cells/ml in RPMI 1640), labeled with 5-(and 6)-carboxyfluorescein (Molecular Probes, Eugene, OR), were added to gelatin-coated plates with or without 100 ng/ml PMA or 10 µg/ml mAbs 44a, 107, or L-1 and incubated for 10 min at 37°C. Unbound neutrophils were removed by gentle washing, and attached cells were solubilized with 1% SDS containing 0.2 N NaOH. Fluorescence was quantified (excitatory wavelength, 490 nm; emission wavelength, 300 nm) using an SLM 8000 fluorometer (SLM Instruments, Urbana, IL).
Binding of mAb 107 to WT or mutant CD11b/CD18-transfected COS cells
Binding of mAb 107 to COS cells expressing WT and coded mutant forms of CD11b/CD18 was assessed as previously described (18). Briefly, triplicate wells (from 48-well plates) containing confluent transfected COS cells were incubated with mAb 107 at 2 µg/ml in TBS containing 1 mM MgCl2, 1 mM CaCl2, 1% BSA, and 0.02% sodium azide for 1 h at 4°C. Cells were then washed and incubated with 125I-labeled goat anti-mouse Ig (NEN, Boston, MA) under similar conditions. After washing, cells were solubilized with 1% SDS/0.2 N NaOH, and the extracts were counted. Specific binding was obtained by subtracting background binding to mock-transfected COS cells (usually <5% of total binding). The binding data from three independent experiments were pooled and expressed as histograms representing the mean ± SEM before the mutants were decoded. Binding was normalized for the percentage of binding obtained with WT. Binding of the anti-CD11b mAb OKM10 (which binds outside of 11bA) to COS cells was assessed in the same way to assure that comparable levels of expression were obtained for all mutants (data not shown).
BIAcore analysis
The kinetic parameters (apparent association and dissociation rate constants, Kon and Koff, respectively) and the apparent equilibrium constants (Kd) for mAb 107–11bA123–321 and mAb 107–11bA123–315 interactions were measured using surface plasmon resonance on a BIAcore instrument (BIAcore, Uppsala, Sweden). The biosensor device was used in accordance with the manufacturer’s instructions. Briefly, mAb 107 was covalently coupled via primary amine groups to the dextran matrix of a CM5 sensor chip (BIAcore). Chip surface treated in the same way, but without mAb, was used as the control. Preparations of 11bA were flowed over the chip at 20 µl/min. TBS containing 1 mM MgCl2, 1 mM CaCl2, and 0.005% P20 (BIAcore) was used as running buffer throughout. HCl (10 mM) was used to remove the bound protein and to regenerate the surface for additional binding experiments. Binding was measured as a function of time. The binding data (after subtracting the background binding to control surface) were analyzed by the linear transformation method to obtain the kinetic constants (32).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Of 172 clones obtained from mice immunized with human 11bA,
supernatant from 12 reacted with the protein in ELISA. One of these
clones, named 107, was chosen because of its ability to bind to 11bA in
a divalent cation-dependent manner; binding was abolished by EDTA. mAb
107 bound to the 11bA, but not 11aA (Fig. 1
A), and its epitope was
expressed in the recombinant human CD11b but not CD11a heterodimer
(Fig. 1B). The CD18-reactive mAb, shared by both CD11b and
CD11a, reacted with both heterodimers, indicating that loss of binding
to CD11a is not caused by lack of expression of this integrin.
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C). mAb
107-11bA interaction occurred in the presence of
Mn2+ or Mg2+ as expected,
but interestingly also when Ca2+ alone was used
(Fig. 2A). Both EDTA as well
as the Ca2+ chelator EGTA abolished this
interaction, again confirming a requirement for
Ca2+. The presence of either
Mg2+ or Mn2+ without
Ca2+ supported this interaction only partially
(Fig. 2A). Optimal interaction between 11bA and mAb 107
required micromolar concentrations of Ca2+ (Fig. 2B). However, BIAcore studies failed to show specific
binding of Ca2+ to mAb 107 alone (data not
shown). Taken together, these data suggest that
Ca2+ may bind at the interface between 11bA and
the mAb 107 and is required for the stability of this interaction.
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mAb 107 also bound to human neutrophils as assessed by FACS
analysis (Fig. 3). Binding was again
abolished by inclusion of EDTA in the reaction mixture (Fig. 3). In
contrast, binding of 44a, another mAb directed against 11bA
(6) (Fig. 1A), was unaffected by the presence
of EDTA (Fig. 3B). Immunoprecipitation studies from
detergent extracts of human neutrophils using mAb 107 yielded a
heterodimer of 
160 and 94 kDa following nonreduced SDS-PAGE and
Western blotting with CD11b/CD18 subunit-specific Abs (not shown),
confirming that the reactivity observed in whole cells is directed
against CD11b/CD18.
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We next determined the functional profile of mAb 107 by assessing
its effect on the binding of several physiologic ligands to CD11b/CD18.
We found that the divalent cation-dependent interaction of solid or
fluid phase iC3b with 11bA was completely blocked in the presence of
mAb 107 (Fig. 4, A and
B). 44a and 904, two other mAbs to 11bA, blocked binding of
solid phase iC3b to 11bA but were less effective in blocking binding of
fluid phase iC3b. The anti-CD11a mAb L-1 had no inhibitory
activity, as expected. The blocking effect of mAb 107 on ligand binding
was also observed when binding assays were conducted using recombinant
CD11b/CD18 stably expressed on CHO cells. iC3b, fibrinogen, and CD54-Fc
bound to these cells specifically and in a divalent cation-dependent
manner (Fig. 5). mAb 107 completely
blocked binding of all three physiologic ligands to the recombinant
receptor, an effect also produced by mAb 44a (Fig. 5, A–C).
L-1 and mouse IgG1 had no inhibitory effect. Thus, mAb 107 interferes
with binding of physiologic ligands to the isolated A domain as well as
to the heterodimeric receptor.
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We next assessed the effects of mAb 107 on known post-ligand
binding cellular events mediated by CD11b/CD18 in normal human
neutrophils. We found that mAb 107 completely blocked phagocytosis of
serum-opsonized ORO particles (Fig. 6A) as well as neutrophil
spreading on gelatin-coated surfaces (Fig. 6B). Half-maximal
inhibition in the latter case was observed at approximately 5 µg/ml
(33 nM).
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We next investigated the mechanism of inhibition of ligand binding
by mAb 107. The binding sites for the CD11b/CD18 ligands iC3b, FB, and
CD54 and the natural antagonist NIF are located on the MIDAS face of
11bA (2, 6, 18, 34). The metal dependency of mAb 107
binding to 11bA and its blocking effects on the binding of several
ligands suggested that it may also bind to the MIDAS face and may thus
act as a competitive inhibitor. We tested this hypothesis by mapping
the binding site for mAb 107 in CD11b/CD18 using a panel of mutant
receptors previously used to identify the binding site for NIF
(18) and iC3b (11). None of the mutations
introduced into full-length CD11b that involved solvent-accessible
residues covering 11bA affected normal expression of the heterodimer in
COS cells (11). Among the mutants, G143/M, E178E179/AA,
R208/L, and F246/K receptors completely abolished mAb 107 binding to
CD11b/CD18 (Fig. 7A). This
lack of reactivity was also demonstrated in direct binding studies to
the respective 11bA mutants, each slot-blotted onto Immobilon paper
(Fig. 7B). mAb 107 did not bind to the metal ion-defective
receptors D140GS/AGA and D242/A (Fig. 7A) or the respective
mutant A domains (Fig. 7B), in agreement with a requirement
for an intact MIDAS motif. G143, E178E179, R208, and F246 are all
located on the MIDAS face of 11bA in its open and closed states (Fig. 7C). G143, E178E, and R208 have been previously shown to be
involved in the binding of both iC3b and NIF to 11bA (11, 18). F246, in contrast, has been shown to be involved in iC3b,
but not NIF, binding (11, 18).
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G143, E178E179, and R208 are conformationally insensitive residues
(i.e., the respective C atom moves by 1 Å relative to the metal
ion in the two conformations (11, 17); see also Fig. 7C). F246, in contrast, is conformationally sensitive; its
C moves away (by 2.5 Å) from the metal ion when the domain is in
its low-affinity closed conformation (Fig. 7C). This finding
suggests that the mAb 107-11bA interaction is sensitive to the
activation state of the domain. To test the validity of this
hypothesis, we measured the kinetics of binding of mAb 107 to the
purified high and low-affinity forms of 11bA using BIAcore. Binding of
a range of 11bA concentrations (25–1600 nM) of either form to mAb
107-coated chips was measured. The apparent binding constants were
analyzed by the linear transformation method (32). The
reaction on-rate was significantly faster to the closed form, resulting
in an 7-fold higher affinity to this form vs the open form (Table I). Thus, mAb preferentially binds to the
low-affinity state of the receptor.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Binding of physiologic ligands to integrin CD11b/CD18 requires the
MIDAS face of its CD11b A domain, which provides the major ligand
binding interface. Occupancy of MIDAS by an Mg2+
or a Mn2+ ion is essential, because the metal ion
provides a direct bridge to a key acidic residue of the ligand, with
the balance of the binding energy and specificity provided by other
residues surrounding MIDAS (14). The requirement for a
metal-occupied MIDAS for the binding of mAb 107 to 11bA is established
by the loss of binding observed when MIDAS is mutated (D242A or
D140GS/AGA mutants) or when the metal ion is chelated by EDTA. In
addition to an intact MIDAS, the mAb 107-11bA interaction also has an
independent requirement for micromolar concentrations of
Ca2+. This effect is detected only when both
proteins are present (Fig. 2B) and not when either one is
absent; no direct binding of Ca2+ to 11bA or mAb
107 is detected using titration calorimetry (33) (data not
shown). These findings favor the possibility that a
Ca2+ ion binding site that forms at the 11bA/mAb
interface and is distinct from MIDAS is required to stabilize the
interaction between the two proteins.
The epitope of mAb 107 mapped to the MIDAS face of 11bA. The five amino
acids that appear to be involved in mAb 107 binding are found in close
proximity to the metal ion (Fig. 7C). Four of these five
residues (G143, E178E179, and R208) are also involved in the
binding of NIF and iC3b (11, 18); the relative C
positions of these residues relative to the metal ion change very
little (1 Å) in the high- and low-affinity states of the domain
(11). The fifth residue involved in mAb 107 binding, F246,
undergoes significant change relative to the metal ion in the two 11bA
conformations. This conformational sensitivity may account for the
7-fold reduction in affinity of mAb 107 to the high-affinity open form
of the domain. Previous studies suggested that the low- and
high-affinity forms of 11bA exist in an equilibrium in solution in the
absence of a physiologic ligand (11, 17), with added
ligands favoring the open form (14). The present data
provide support for the functional relevance of the protein movements
observed at the MIDAS face in the crystal structures of the two
conformations of 11bA and suggest that stabilizing the inactive state
may contribute to the antagonistic effects of mAb 107.
Most function-blocking mAbs directed against integrins appear to act allosterically by stabilizing the receptor in a low-affinity state and/or by preventing a conformational switch to the active state (17, 35). They may also act by binding to the ligand-occupied state and preventing ligand-induced cellular events (36, 37). The overlap of the binding interface for mAb 107 with that of physiologic ligands suggests that in addition to potentially stabilizing the integrin in the low-affinity state, mAb 107 also acts as a competitive inhibitor of ligand binding, since it clearly shares the binding interface with physiologic ligands. In contrast to some other mAbs to CD11b that induce downstream signals even in their monovalent state upon receptor binding (38), mAb 107 does not display such agonistic effects directly, inasmuch as it blocks cell spreading, a CD11b/CD18-dependent cellular response (22, 23). This is consistent with the interpretation that mAb 107 acts by stabilizing the integrin in the low-affinity state. mAb 107 shares this property with mAb 44a (17). mAb 107 has the added advantage, not shared by mAb 44a, of preferentially binding to the low-affinity state of the integrin through engagement of MIDAS, thus potentially blocking the binding of activation-independent ligands such as NIF. CD11b/CD18 mediates several forms of tissue injury in experimental animal models such as hemorrhagic shock, heart attacks, and stroke (examples of ischemia-reperfusion injury), and immune complex nephritis (reviewed in Refs. 39 and 40). The above properties suggest that mAb 107 may be a useful therapeutic agent.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. M. Amin Arnaout, Renal Unit, Massachusetts General Hospital, 149 13th Street, 8th Floor, Charlestown, MA 02129. E-mail address: arnaout{at}receptor.mgh.harvard.edu
3 Abbreviations used in this paper: NIF, neutrophil inhibitory factor; AP, alkaline phosphatase; MIDAS, metal ion-dependent adhesion site; ORO, Oil Red O; EiC3b, iC3b-coated erythrocyte; WT, wild type; CHO, Chinese hamster ovary; 11bA, the CD11b A domain.
Received for publication August 29, 2001. Accepted for publication November 6, 2001.
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2 integrin CR3 (CD11b/CD18) is a receptor for the hookworm-derived neutrophil adhesion inhibitor NIF. J. Cell Biol. 127:2081.1 chain integrins are receptors for invasin, a protein that promotes bacterial penetration into mammalian cells. Cell 60:861.[Medline]
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2 integrin CR3 (CD11b/CD18). Proc. Natl. Acad. Sci. USA 91:10680. subunits and a common subunit: the lymphocyte function associated antigen (LFA-1), the C3bi complement receptor (OKM1/Mac-1), and the p150,95 molecule. J. Exp. Med. 158:1785.2 integrins. Methods Mol. Biol. 129:105.[Medline]
M2 (CD11b/CD18, Mac-1) integrin activation by a unique monoclonal antibody to M I domain that is divalent cation-sensitive. J. Leukocyte Biol. 68:641.This article has been cited by other articles:
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 V. Gupta, A. Gylling, J. L. Alonso, T. Sugimori, P. Ianakiev, J.-P. Xiong, and M. Amin Arnaout The {beta}-tail domain ({beta}TD) regulates physiologic ligand binding to integrin CD11b/CD18 Blood, April 15, 2007; 109(8): 3513 - 3520. [Abstract] [Full Text] [PDF]
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A. Funaro, E. Ortolan, B. Ferranti, L. Gargiulo, R. Notaro, L. Luzzatto, and F. Malavasi CD157 is an important mediator of neutrophil adhesion and migration Blood, December 15, 2004; 104(13): 4269 - 4278. [Abstract] [Full Text] [PDF]
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 K. Ajroud, T. Sugimori, W. H. Goldmann, D. M. Fathallah, J.-P. Xiong, and M. A. Arnaout Binding Affinity of Metal Ions to the CD11b A-domain Is Regulated by Integrin Activation and Ligands J. Biol. Chem., June 11, 2004; 279(24): 25483 - 25488. [Abstract] [Full Text] [PDF]
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