165
Two Independent Sets of Monoclonal Antibodies
Define Neoepitopes Linked to Soluble Ligand
Binding and Leukocyte Adhesion
Functions of Activated
aMf32
Gabriella S. Elemer, Thomas S. Edgington
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Abstract The integrin aM!3 mediates a variety of events,
adhesive, phagocytic, and inflammatory. Evidence has suggested that the functional events may be mediated by the
"activated" conformational forms of aM3 produced by appropriate stimulation of myeloid and monocytic lineage. The activation of aMA may be associated with new epitopes on aM/32,
sites that may be related to the acquired receptor functions.
Monoclonal antibodies were produced that preferentially bind
neoepitopes expressed by activated aMJ. These anti-neo antibodies each inhibited three activation-associated specific receptor aMA functions, though to different extents. One set of
anti-neo antibodies inhibited in a concordant manner the
binding of factor X and of fibrinogen by >90%, abolished the
aMI-initiated cellular coagulant response, and inhibited monocyte adhesion to unstimulated endothelial monolayers. A sec-
ond set of anti-neo antibodies only diminished factor X and
fibrinogen binding by ---40% to 50% but markedly suppressed
Xa generation and only partially inhibited monocyte adherence
to unstimulated endothelium. Concordance was observed between binding of factor X or fibrinogen and competence for
leukocyte adhesion to unstimulated endothelium. Antibody
competition assays segregated the anti-neo antibodies into the
same two distinct sets, consistent with recognition of separate
neoepitopes that are linked to aMP, function. These data
support the conclusion that the activated conformer of aM/
that binds fibrinogen and factor X also mediates monocyteendothelial interactions as well as the alternative cellular coagulation pathway. (Circ Res. 1994;75:165-171.)
Key Words * integrins * leukocytes * adhesion .
neoepitopes * antibodies
T he leukocyte integrins play key roles in a diverse
set of functions central to leukocyte biology.
Most widely noted is the determinant role in
leukocyte traffic responsible for targeting inflammatory
responses to appropriate tissues. Three members of the
leukocyte /2 integrin family are known: aLP2 (CDlla/
CD18, LFA-11), aMp2 (CDllb/CD18, Mac-1), and ax f2
(CDllc/CD18, p150,95). These transmembrane glycoproteins are organized as a pair of noncovalently linked
cal3 heterodimers and serve as major cell-surface receptors involved in cell-cell and cell-protein recognition
functions.1 -4
aMP2 is expressed predominantly in leukocytes of
myeloid and monocytic lineage but also in natural killer
lymphocytes. aMP2 iS the cognate receptor for the complement fragment iC3b and also participates in cell
adhesion to vascular activated endothelium.' 8 aCm,2 has
been also implicated in a novel mechanism for initiation
of the coagulation serine protease cascade, resulting in
thrombin and fibrin formation on cell surfaces.9-12 aMP,2
interacts specifically with the zymogen form of the
serine protease coagulation factor X via three peptidyl
substructures and with fibrinogen via a single established peptidyl site.912-14 The specific receptor-ligand
interaction requires transition of aMP2 from a latent
relaxed state to a high-affinity activated state. This
transition is presumed to require a qualitative remodeling of the receptor through conformational changes
associated with exposure or reorganization of new protein surface structures, ie, neoepitopes.15'16
The functional transition of this receptor to the
activated state has been previously described in response to physiological agonists such as ADP, ATP,
GTP, or the bacterial chemotactic peptide N-formylMet-Leu-Phe-benzylamide (fMLP).941t2 Recently, we
found that cytochalasin B, the actin filament-capping
agent, can induce a more stable transition of aMP2 to the
activated conformer.14 After incubation with cytochalasin B, isolated human monocytes and cells of the
monocytic cell-line THP-1 bind factor X and fibrinogen
in a dose-dependent, saturable, high-affinity, and Ca2dependent manner.14 Cytochalasin B-treated monocytic cells express aMI32 in the activated state for extended lengths of time compared with that provoked by
other agonists, thus facilitating immunization to such
neoepitopes. Using an intrasplenic hyperimmunization
protocol in mice, we first described the generation of
monoclonal antibodies (mAbs) specific for neoepitopes
of the activated conformational state of aM!32.17 In the
present study, we demonstrate that these anti-neo mAbs
inhibit (1) ligand binding by aMI32, (2) activation of the
alternative coagulation cascade by the aM,82-factor X
complex, and (3) cell adhesion function (however, to a
different extent). Taken together with anti-neo mAb
competition studies, these results indicate the generation of at least two distinct neoepitopes on aM!32 on
Received November 18, 1993; accepted March 22, 1994.
From the Vascular Cell Molecular Biology Program, Department of Immunology, The Scripps Research Institute, La Jolla,
Calif.
Correspondence to Thomas S. Edgington, MD, Department of
Immunology/IMM-17, The Scripps Research Institute, 10666
North Torrey Pines Rd, La Jolla, CA 92037.
© 1994 American Heart Association, Inc.
166
Circulation Research Vol 75, No 1 July 1994
activation as well as linkage of these neoepitopes to
function. These mAbs also provide potential analytical
probes for aMI32 activation in vivo.
Materials and Methods
Reagents
All chemicals and reagents, except otherwise stated, were
ing 1% Triton X, 50 mmol/L Tris-HCl, 150 mmol/L NaCl, and
protease inhibitors.19 Lysates were precleared, and immunoprecipitations were performed indirectly as previously described'2'14'16'9 with 50 ,ug/mL purified mAbs. Samples containing 5 x 10' cpm were electrophoresed on 8.5 % polyacrylamide/
0.1% sodium dodecyl sulfate (SDS) slab gels under reducing
conditions and exposed for autoradiography with intensifying
screens.16
purchased from Sigma Chemical Co.
Proteins
Cells
Human factor X was isolated and purified as previously
described.24 It was radiolabeled with "25I-sodium by the iodogen method23 to a specific activity of 0.5 ,UCi/Lg protein.
Purified mAbs 7A10 and SF10 were radiolabeled by the same
method to a specific activity of 0.7 gCi/,ug.
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Peripheral blood mononuclear cells were isolated from the
blood of disease-free volunteers by centrifugation over FicollHypaque at 400g for 30 minutes at 22°C. Monocytes were
separated by adherence to plastic Petri dishes precoated with
autologous serum for 1 hour at 37°C.9 Cells were resuspended
in endotoxin-free and phosphate-free RPMI 1640 medium
containing 10% heat-inactivated endotoxin-free fetal calf serum (Irvine) 2 mmol/L L-glutamine (Irvine), and 20 mmol/L
HEPES (Calbiochem). The monocytic leukemia cell line
THP-1 (American Type Culture Collection)'1 was maintained
in continuous culture in RPMI 1640 medium supplemented as
described above with 10 gmol/L 2-mercaptoethanol (Eastman
Kodak).
Agonists
The chemoattractant fMLP was used at 1 ,mol/L and ADP
at 10 ,mol/L final concentration in phosphate-buffered saline.
mAb Production, Purification,
and Characterization
The immunization protocol has been published in detail
elsewhere.19 Briefly, 6-week-old female BALB/c mice were
immunized with cytochalasin B-treated THP-1 monocytic
cells bearing aMP2 in the activated state.'4 One intraperitoneal
injection of 1 x 107 cells in complete Freund's adjuvant was
followed by two intrasplenic injections of stimulated cells
(2x107 cells) at 6-week intervals. Mice received an intrasplenic boost of unstimulated cells (5 x 106 cells) 10 days before
fusion and an intrasplenic boost of stimulated cells (5 x 106
cells) 4 days before harvest of spleen cells for fusion to P3X63
Ag 8653.1 myeloma cells. Four hundred hybridomas were
screened for antibody to stimulated versus unstimulated
THP-1 cells by indirect immunofluorescence microscopy.
Seven selected hybridomas were recloned by limiting dilution
and grown as ascites tumors in mice. Immunoglobulins were
purified by chromatography on protein A-Sepharose columns
(Biorad). Isotypes were determined using a commercial kit
(Immunoselect, GIBCO).
Other mAbs
The anti-aM mAb OKM-10 (IgG2b) and anti-/32 mAb IB-4
(IgG2a) were used as published previously.5,9'1020-22 The mAb
to the p53 nonviral T antigen (TIB 116, IgG2b) and mAb to the
simian virus 40 large T antigen (TIB 115, IgG,) served as
irrelevant controls.
Fluorescence-Activated Cell Sorter Analysis
THP-1 monocytic cells (1x 106 cells per milliliter) were
stimulated (fMLP, 1 ,umol/L; ADP, 10 ,umol/L) or not and
incubated with mAbs at room temperature for 20 minutes,
washed in phosphate-free RPMI 1640 medium containing 2%
bovine serum albumin (BSA), incubated with fluorescein
isothiocyanate-conjugated goat F(ab')2 anti-mouse IgG for 20
minutes, washed again, and analyzed in a Beckton Dickinson
IV/40 fluorescence-activated cell sorter (FACS).
Binding Assays
Antibody Competition Assay
Competitive inhibition of mAb binding was used to assess
epitope topographical identity. These assays were performed
according to the protocol described in detail previously.9 Stimulated THP-1 cells (2x 107 cells per milliliter) were incubated
with a reference mAb, eg, `1I-7A10 (1.2x10` molIL), in the
presence of a 100-fold molar excess of unlabeled competing
mAb, eg, SF7, SF10, 4E1, 4D5, 2C11, or 8B2. Specific binding of
the radiolabeled reference mAb to cells was quantified.
Antibody-Ligand Competition
THP-1 cells were stimulated with ADP (10 ,mol/L) for 2
minutes at 22°C and incubated with saturating concentrations
of different mAbs (7A10, SF7, SF10, 4E1, 4D5, 2C11, and
8B2). Increasing concentrations of '25I-factor X were added
and incubated for 15 minutes at room temperature. Specific
binding was calculated by subtracting from the total the
amount of '25I-factor X in the presence of a 100-fold excess of
unlabeled factor X (nonspecific binding).9 Nonspecific binding
varied from 5% to 10%.
Cellular Coagulant Response
Agonist-stimulated THP-1 cells were incubated with factor
X (10 ,g/mL) in the presence of Ca21 (2.5 mmol/L CaCl2 in
medium) and in the presence or absence of mAbs (20 /Lg/mL
purified IgG). After a 10-minute incubation at room temperature, a 0.1-mL aliquot of cell suspension was transferred to
37°C and mixed with 0.1 mL bovine plasma deficient in both
factors X and VII. The assay was initiated with 0.1 mL of 20
mmol/L Ca 2+, and the clotting time was read. This rate was
converted to nanograms of factor Xa per milliliter by using a
standard curve constructed with serial dilutions of Xa, which
was produced by activation of factor X to Xa by the activating
enzyme from Russell's viper venom.9,24
Cell Adhesion Assay
The monocyte adhesion assay to unstimulated human umbilical vein endothelial cells (HUVECs) has been described in
detail previously.'9 Briefly, monocytes or THP-1 monocytic
cells were surface-labeled with 1251-sodium, washed, stimulated, and incubated in the absence or presence of factor X (45
nmol/L) or fibrinogen (0.5 gmol/L) at room temperature for
10 minutes. Cells (lx 106) were allowed to settle on endothelial monolayers at 22°C for 30 minutes. Nonadherent cells were
removed by several washes of RPMI 1640 medium containing
1% BSA. Adherent cells were quantified by calculating the
radioactivity after subtraction of background binding determined with cells incubated with 1% BSA or with irrelevant
mAbs TIB 116 or TIB 115.
Immunoprecipitation
Results
mAbs to Activated aMI32
Monocytes were isolated and labeled (1.5 x 107 cells) with 1251
by the iodogen method23 and solubilized in lysis buffer contain-
mAbs were generated by intrasplenic immunization
of mice with monocytic cells stimulated with cytochala-
Elemer and Edgington CrMI2 Neoepitopes
167
FIG 1. Graphs showing reactivity of monoclonal
antibodies (mAbs) with stimulated and nonstimulated THP-1 cells. Flow cytometric analysis of
reactivity of mAbs with ADP (10 gmol/L)-stimulated THP-1 monocytic cells (solid line) is compared with that of nonstimulated cells (broken
line). Anti-aM mAb OKM-10 and anti-fl2 mAb IB-4
served as controls.
G)
in
E
z
C.)
10o
1o0
102
103
104
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Fluorescence Intensity
sin B to stably express aMuI2 in the activated state.'4
After antigenic diversion with cells expressing nonactivated aMI32 and boost with activated aMI32 cells, splenic
lymphocytes were harvested and fused with myeloma
cells. Hybridoma culture fluid supernatants were
screened for reactivity with stimulated monocytes, granulocytes. and THP-1 monocytic cells. Seven hybridomas
producing desired antibodies were selected by the
strong signal obtained on immunohistochemical reaction of the supernatant with cells stimulated by two
different agonists and little if any reaction with unstimulated cells. The mAb 7A10 was IgG2h, the mAbs 5F7
and 2C11 were IgGa, and the mAbs 4E1, 4D5, 5F10,
and 8B2 were IgG,. All mAbs had kappa light chains.
Flow cytometry indicated a marked increase in fluorescence intensity of THP-1 cells when stimulated with
ADP compared with the reactivity with nonstimulated
cells of each of the selected mAbs (Fig 1). By contrast,
cells reacted with OKM-10 anti-aM mAb (Fig 1) and
with IB-4 anti-P,2 mAb showed equivalent reactivity
regardless of whether the cells were stimulated or not.
The cell activation-dependent mAbs immunoprecipitated a protein pair from detergent lysates of surface
1251-labeled and stimulated monocytes (Fig 2). All had
comparable migration on reduced SDS-acrylamide electrophoretic gels and similar position on the autoradiographs. They were identified by comparison with valid
bands observed after immunoprecipitation of monocyte
lysates by the reference anti-am mAb OKM-l0 (Fig 2).
Preclearing the cell lysate with OKM-10 removed aM/32
and eliminated precipitation by the activation-dependent
mAb 7A10 (Fig 2). Neither the irrelevant mAbs TIB 116
nor TIB 115 generated bands (Fig 2).
Anti-Neo mAbs Block the aM,82-Dependent
Alternative Coagulation Pathway
Myelomonocytic cells expressing aMI8 have the potential to initiate the coagulation cascade by surface binding
and activation of the zymogen factor X.9-Y1 To determine
the role of activation-dependent neoepitopes, we first
examined the effect of these anti-neo mAbs on activation-specific binding function by inhibition of factor X
binding. THP-1 cells or monocytes stimulated with fMLP
(1 gzmol/L) or ADP (10 gmol/L) were incubated at 22°C
for 15 minutes with each of the different anti-a MI3 neo
mAbs or with the control TIB 115 or TIB 116 mAbs.
Increasing concentrations of 125I-factor X were added,
and after incubation at 22°C for 10 minutes, specific
binding of radiolabeled factor X was determined. Monocytes incubated in the presence of irrelevant mAbs TIB
116 or TIB 115 bound 12I-factor X in a concentrationdependent manner, reaching saturation at 45 nmol/L
125I-factor X (Fig 3). Incubation with mAb 5F7, 7A10, or
8B2 (set 1) inhibited 125-factor X binding by >90% at six
different concentrations of the ligand, whereas incubation with mAb 2C11, 4D5, 4E1, or 5F10 (set 2) diminished 1251-factor X binding by z50% (Fig 3).
When factor X is bound to crMf2 on the surface of
stimulated monocytic cells, it can be activated to the
serine protease Xa.'0 This mediates thrombin formation
on cell surfaces.
To determine whether expression of
aMI32 activation-dependent neoepitopes may be linked
1
Mr
200k
--
_
..
97k -w
.
;.
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
i
s
66k1
2
3
4
5
6
7
8
9
FIG 2. Immunoprecipitation of CaMI2 from detergent lysates of
1251-labeled monocytes. Monocytes were isolated as described in
"Materials and Methods," surface-iodinated, stimulated with
N-formyl-Met-Leu-Phe-benzylamide (1 gmol/L) for 1 minute, and
lysed in 1% Triton X. Immunoprecipitates with the following
monoclonal antibodies (mAbs) were subjected to reducing 8.5%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis and
autoradiography: lane 1, OKM--10 anti-aM mAb; lane 2, 7A10
mAb; lane 3, 7A10 after preclearing with OKM-10; lane 4, 4E1
mAb; lane 5, 5F1 0 mAb; lane 6, SF7 mAb; lane 7, 8B2 mAb; lane
8, TIB 116 control mAb; and lane 9, TIB 115 control mAb.
168
Circulation Research Vol 75, No
1
July 1994
TABLE 1. Effect of Anti-Neo a:MJ32 Monoclonal
Antibodies on the Coagulant Response of THP-1 Cells
mAb
No stimulation
None
120f
00
0
m
40
15
30
45
60
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1251-X (nM)
FIG 3. Graph showing the effect of anti-am4 monoclonal antibodies (mAbs) on 1251-factor X binding. Monocytes were isolated,
stimulated with ADP (10 gmol/L), and incubated with mAbs at
220C for 15 minutes. Increasing concentrations of 1251-factor X
(1251_)) were added, and cell-associated total binding was determined by centrifugation of cell suspension through a mixture of
silicone oil.9 Specific binding was calculated by subtracting from
the total the nonspecffic binding that was determined in the
presence of 100-fold molar excess of unlabeled factor X. *
indicates binding with control mAb TIB 116 or TIB 115; A, binding
with mAb 2C11, 4D5, 4E1, or 5F10; and o, representative of
binding with mAb 5F7, 7A1 0, or 8B2.
directly or indirectly to factor X activation, we investigated whether various anti-neo mAbs inhibited this
novel alternative cellular coagulant response. Monocytes or THP-1 cells stimulated with ADP (10 ,umol/L)
or fMLP (1 ,umol/L) accelerated the coagulation rate of
plasma compared with unstimulated cells (Table 1).
Addition of anti-neo mAbs immediately after stimulation diminished this acceleration. The inhibition was,
however, independent of differences in relative inhibition of factor X binding to aMI32 by these mAbs (Table
1, Fig 3). All anti-neo mAbs suppressed the formation
of Xa by stimulated cells (Fig 4). Thus, the set 2
anti-neo mAbs inhibited the coagulant activity in excess
of their ability to inhibit binding.
mAbs Inhibit Monocyte Adherence to
Unstimulated Endothelium
Cellular activation that induces amI32 activation results in an enhanced but transient adherence of monocytes to unstimulated HUVEC monolayers.19 Furthermore, stimulated monocytes and monocytic cells with
bound factor X19 or fibrinogen1719 on their surfaces
exhibit a sustained adhesion to unstimulated endothelial monolayers. Quantification of adhesion with surface-labeled monocytes or THP-1 monocytic cells in the
absence or presence of either factor X or fibrinogen or
both confirmed the increased cell adhesion to unstimulated HUVEC monolayers when cells were stimulated
and bound factor X or fibrinogen. To determine the
potential role of aMf32 activation conformers, we evaluated the ability of anti-neo mAbs to block monocytic
cell adhesion in the presence of factor X and fibrinogen
(Fig 5). The three mAbs of set 1, namely 7A10, 5F7, and
8B2, were potent inhibitors of monocytic cell adhesion
in the presence of both factor X and fibrinogen. The
four mAbs of set 2, namely 4D5, 4E1, 5F10, and 2C11,
diminished but did not abolish monocytic cell adherence
to the endothelial monolayers. Although the correlation
between the blocking action of these mAbs on factor X
Clotting Time, s
44.5+1
7A1 0
435+3
Stimulation
ADP
None
18.5±1
2C1 1
32.0+6
4E1
33.6+5
4D5
37.0±4
5F10
35.0±0.5
5F7
41 .0+3
8B2
40.6±4
7A1 0
40.8±3
fMLP
None
15.5±0.5
2C1 1
29.0±3
4E1
32.6±3
4D5
32.0±3
5F10
34.0±4
5F7
43.0±3
8B2
40.0+5
7A1 0
41.5±6
ADP (10 ,lmol/L)- or N-formyl-Met-Leu-Phe-benzylamide
(fMLP, 1 ,mol/L)-stimulated THP-1 cells complemented with 2.5
mmol/L Ca2+ and factor X (10 jig) were incubated in the
presence or absence of saturated amounts of monoclonal antibodies (mAbs) at room temperature. After 10 minutes, cell
suspensions were mixed with factor VIl- and factor X-deficient
plasma at 370C and recalcified with 25 mmol/L Ca2+, and
clotting time was observed. Values are mean±SD of three
experiments.
and fibrinogen binding and blocking of cell adhesion
was apparent, there was not a direct correlation with the
degree of inhibition of cellular coagulant activity.
mAbs Define Different Neoepitopes
To determine whether these anti-neo mAbs may
define topographically identical, overlapping, or spatially separate neoepitopes, we analyzed whether mAbs
defining the two sets competed with one another. Reference anti-neo mAb 7A10 was 125-Ilabeled. THP-1
monocytic cells were stimulated with fMLP (1 gmol/L)
and incubated with 1251-7A10 (1 x 10-7 mol/L) alone or in
the presence of a 100-fold molar excess of an unlabeled
competing mAb (Table 2). Ninety-five percent inhibition was observed in the presence of the homologous
inhibitor (7A10), providing evidence for specificity of
1251-7A10 binding. The heterologous competitor mAbs
separated into two sets. The SF7 and 8B2 mAbs (from
set 1) cross-competed with 7A10, suggesting that they
bind to an identical epitope or to allosterically linked or
spatially overlapping epitopes. The other mAbs 4E1,
4D5, 5F10, and 2C11 (set 2) inhibited '251-7A10 binding
Elemer and Edgington aMP2 Neoepitopes
169
TABLE 2. Competition of Binding of Anti-Neo aMI32
Monoclonal Antibodies to THP-1 Cells
Competing mAb
Bound
Antibody,
molecules/
cell
Inhibition, %
151 7A1 0 studies
Xa
(ng/mi)
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FIG 4. Bar graph showing the effect of anti-aM,82 monoclonal
antibodies on the cellular procoagulant response. The experiments were performed in a manner similar to that described in
Table 1. The mean clotting time was converted to nanograms of
factor Xa per milliliter by using a standard curve as described in
"Materials and Methods." Values are mean+±SD of three experiments done in duplicate. fMLP indicates N-formyl-Met-Leu-Phebenzylamide.
by 50% to 60%, indicating that these mAbs do not
recognize the same epitope as 7A10 but rather bind to
partially overlapping or distinct sites. To address this
question, mAb SF10 (set 2) was radiolabeled, and mAb
binding in the absence or in the presence of competitors
was monitored. Whereas the homologous competitor
blocked "25I-5F10 binding by 92%, the heterologous
competitors of set 1 diminished the binding by only 25%
to 30%. The noncompetitive binding of these mAbs
supports the conclusion that the two sets of anti-neo
mAbs identify two or more distinct neoepitope sites on
aM132.
Discussion
To explore the structural biology of aM/2 in the
multiplicity of recognitive functions of this integrin in
x
FG
STIM
7A10
5F7
8B2
4D5
4E1
5F10
2C11
h
9
bJ4
STIM
7A10
5F7
8B2
-1
H
---Eh
4D5
4E1
5F10
2C11
.
.
0
60
-1-4
.
100
150
200
300
cpm/well-3
FIG 5. Bar graph showing the effect of anti-aM,82 monoclonal
antibodies (mAbs) on THP-1 cell adhesion to human umbilical
vein endothelial cells. Surface-iodinated THP-1 monocytic cells
were stimulated with N-formyl-Met-Leu-Phe-benzylamide (1 ,mol/
L for 2 minutes at 220C) and incubated with soluble factor X (X,
45 nmol/L) or fibrinogen (Fg, 0.5 ,mol/L) in the absence (STIM,
stippled bar) or presence of anti-neo aM132 mAbs (clear bar).
Adherent cells were quantified as described in "Materials and
Methods." Data are representative of two experiments, and each
value is the mean+SD of eight samples.
None
93 380±9120
95
7A10
7300±996
90
5F7
8652±1330
8B2
93
6378±946
4E1
65
32 770+2930
64
4D5
34 340±5530
50
5F10
46680+1980
50
2C1 1
47 190±3450
1251 5F10 studies
None
83 810±1190
92
5F10
6390+512
7A10
35
62 910±1920
5F7
26
59 930+2180
THP-1 monocytic cells stimulated with N-formyl-Met-Leu-Phebenzylamide (1 gmol/L for 2 minutes at 220C) were incubated
with 1251-7A10 monoclonal antibody (mAb) alone (None) or in the
presence of a 100-fold molar excess of unlabeled competing
mAbs (7A10, 5F7, 8B2, 4E1, 4D5, 5F10, and 2C11) or with
1251-5F10 alone (None) or in the presence of a 100-fold molar
excess of unlabeled competing mAbs (5F10, 7A10, and 5F7),
and specific binding of 1251-7A10 and 1251-5F10 was determined.
Values are mean±SD.
vascular biology, we generated mAb probes preferentially recognizing newly conformed sites on activated
aMI62. These anti-neo mAbs inhibited the three classes
of functions under analysis, namely the factor X and
fibrinogen recognitive function of aM132, the alternative
coagulation pathway, and the heterotypic cell-cell adhesive interaction with unstimulated endothelium. The
results are consistent with the notion that one or more
new epitopes are created by conformational transitions
of aM/32 to the activated state in response of an appropriate cellular agonist.15'16 Based on the effects of these
anti-neo mAbs, adhesion of monocytes to unstimulated
endothelium as well as the binding of factor X and of
fibrinogen, independent ligands, can be blocked, suggesting that there is structural proximity or linkage of
the aM/82 structure responsible for each of these different functions and the associated cellular events.
Several mAbs against activation-dependent neoepitopes on /2 integrins have been reported recently.1925-28
In accordance with our description of neoepitopes on
aM/32, Diamond and Springer26 reported that neutrophil
activation prompts a subset of aMP2 molecules to acquire structurally competent sites to bind fibrinogen and
to engage counterreceptors on the endothelium.17,26
The present studies with a panel of such types of mAbs
suggest that distinct and presumably spatially distant
epitopes may become exposed or constituted in response to cell stimulation, consistent with the acquisition of multiple functions. These anti-neo mAbs may
recognize a functional protein surface interactive site in
170
Circulation Research Vol 75, No 1 July 1994
proximity to those responsible for factor X binding,
fibrinogen binding, or cellular adhesion. Alternatively,
the binding of a mAb to a neoepitope may induce
allosteric effects, such as the relation of the aM and P2
chain to one another, and result in modification of the
conformation and function of these various binding
sites. This has been observed for an activation inducing
mAbto aIb/33.29
The major recognition site for several ligands for aMf2
has been mapped to the 200-amino acid "I" domain on
the aM subunit.30 However, differences in pattern of
inhibition by different anti-aM mAbs suggested the
existence of several functional subdomains to which the
I domain contributes structure directly or indirectly.30
The existence of more than one recognition site on aM/2
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is indicated. Current data support the idea that this
integrin is an oligoreceptor with multiple docking sites
rather than a receptor with a promiscuous single binding site for a selected set of ligands. In respect to
discrete recognitive substructures in aMP2 ligands, three
spatially distant surface loops in the latent catalytic
domain of factor X were identified as discrete ligand
structures that mediate the interaction of this protein
with aM132.31 Further, the binding of fibrinogen is mediated by a novel peptidyl structure in the 30-kD carboxyl
terminal domain of fibrinogen that bears no similarity to
those mediating factor X binding.32 None of these are
RGDX peptidyl ligands. Neither the 30-kD ligand
fragment or peptidyl ligand from fibrinogen compete for
or inhibit binding of factor X (unpublished data, D.
Altieri). These data lead to the conclusion that ligand
binding depends on the recognition of ligands by a
limited number of independent sites on aM02.31 Binding
of the zymogen factor X to activated aMI2 has been
established as an initial step in a novel alternative
pathway triggering the coagulation cascade on monocyte surfaces.9-11 The bound factor X is converted to the
protease factor Xa by limited proteolytic activation,
leading to the local generation of thrombin.10,1 The
expression of aM/2 activation neoepitopes is correlated
with the binding of factor X and the subsequent conversion to Xa. However, there was a notable discordance between inhibition by some anti-neo mAbs of
factor X binding and of Xa generation. Although such
anti-neo mAbs only partially inhibited factor X binding,
they strongly inhibited proteolytic activation to Xa. This
suggests an indirect mechanism of inhibition of this
proteolytic activation event.
The ability of both sets of anti-neo mAbs to inhibit
factor X binding was correlated with inhibition of
monocyte adhesion to the endothelial monolayers in the
presence of factor X or fibrinogen. This suggests that
the aMP2-mediated binding of factor X and fibrinogen
may be directly involved in a mechanism for stabilizing
monocyte adherence to the vascular endothelium without a necessary activation of the endothelial cells. This
aMP2 adhesion phenomenon may be important for trafficking mononuclear cells through the vascular wall. The
different inhibitory effect of these mAbs on factor X
binding and adhesion to endothelium, together with
results on antibody competition, suggests the existence
of two distinct or several overlapping sites regulating
aM132 adhesive functions. The reagent mAbs provide
potential analytical probes for activation of aMI3 in vivo
in health and disease.
Acknowledgments
This study was supported by National Institutes of Health
(NIH) grant P01 HL-16411 and by NIH General Clinical
Research Center grant M01 BR-00833. Dr Elemer was supported by a fellowship from the American Heart Association,
California Affiliate, Inc. The technical assistance of J. Royce
and the preparation of the manuscript by B. Parker are
appreciated.
References
1. Albelda SM, Buck CA. Integrins and other cell adhesion molecules. FASEB J. 1990;4:2868-2880.
2. Arnaout MA. Structure and function of the leukocyte adhesion
molecules CDli/CD18. Blood. 1990;75:1037-1050.
3. Hynes RO. Integrins: versatility, modulation, and signaling in cell
adhesion. Cell. 1992;69:11-25.
4. Larson RS, Springer TA. Structure and function of leukocyte
integrins. Immunol Rev. 1990;114:181-217.
5. Arnaout MA, Lanier LL, Faller DV. Relative contribution of the
leukocyte molecules Mo-1, LFA-1, p150,95 (LeuM5) in adhesion
of granulocytes and monocytes to vascular endothelium is tissueand stimulus-specific. J Cell Physiol. 1988;137:305-309.
6. Lo SK, Detmers PA, Levin SM, Wright SD. Transient adhesion of
neutrophils to endothelium. J Exp Med. 1989;169:1779-1793.
7. Lo SK, van Seventer GA, Levin SM, Wright SD. Two leukocyte
receptors (CD1la/CD18) and CD11b/CD18) mediate transient
adhesion to endothelium by binding to different ligands. J Immunol.
1989;143:3325-3329.
8. Smith CW, Marlin SD, Rothlein R, Toman C, Anderson DC.
Cooperative interactions of LEA-1 and Mac-1 with intercellular
adhesion molecule-1 in facilitating adherence and transendothelial
migration of human neutrophils in vitro. J Clin Invest. 1989;83:
2008-2017.
9. Altieri DC, Edgington TS. The saturable high affinity association
of factor X to ADP-stimulated monocytes defines a novel function
of the Mac-1 receptor. J Biol Chem. 1988;263:7007-7015.
10. Altieri DC, Edgington TS. Sequential receptor cascade for coagulation proteins on monocytes: constitutive biosynthesis and functional prothrombinase activity of a membrane form of factor V/Va.
J Biol Chem. 1989;264:2969-2972.
11. Altieri DC, Morrissey JH, Edgington TS. Adhesive receptor Mac-1
coordinates the activation of factor X on stimulated cells of
monocytic and myeloid differentiation: an alternative initiation of
the coagulation protease cascade. Proc Natl Acad Sci U SA. 1988;
85:7462-7466.
12. Altieri DC, Bader R, Mannucci PM, Edgington TS. Oligospecificity of the cellular adhesion receptor Mac-1 encompasses an
inducible recognition specificity for fibrinogen. J Cell Biol. 1988;
107:1893-1900.
13. Wright SD, Weitz JI, Huang AJ, Levin SM, Silverstein SC, Loike
JD. Complement receptor type three (CDlib/CD18) of human
polymorphonuclear leukocytes recognizes fibrinogen. Proc Natl
Acad Sci USA. 1988;85:7734-7738.
14. Elemer GS, Edgington TS. Microfilament reorganization is associated with functional activation of aMf3S on monocytic cells. J Biol
Chem. 1994;269:3159-3166.
15. Plow EF, Edgington TS. Molecular events responsible for modulation of neoantigenic expression: the cleavage-associated neo-
antigen of fibrinogen. Proc NatlAcad Sci USA. i972;69:208-212.
16. Altieri DC, Edgington TS. A monoclonal antibody reacting with
distinct adhesion molecules defines a transition in the functional
state of the receptor CDiib/CD18 (Mac-1). J Immunol. 1988;141:
2656-2660.
17. Languino LR, Plescia J, Duperray A, Brian AA, Plow EF,
Geltosky JE, Altieri DC. Fibrinogen mediates leukocyte adhesion
to vascular endothelium through an ICAM-1-dependent pathway.
Cell. 1993;73:1-20.
18. Tsuchiya S, Yamabe M, Yamaguchi Y, Kobayashi Y, Konno T,
Tada K. Establishment and characterization of a human acute
monocytic leukemia cell line (THP-1). It J Cancer. 1980;26:
171-176.
19. Elemer GS, Edgington TS. Monoclonal antibody to an activation
neoepitope of aMi%2 inhibits multiple aut32 functions. J Immunol. In
press.
20. Ho MK, Springer TA. Biosynthesis and assembly of the a and ,B
subunits of Mac-1, a macrophage glycoprotein associated with
complement receptor function. J Biol Chem. 1983;258:2766-2769.
Elemer and Edgington aMf2 Neoepitopes
21. Keizer GD, Borst J, Fidgor CG, Spits H, Miedema F, Terhorst C,
de Vries JE. Biochemical and functional characteristics of the
human leukocyte membrane antigen family LFA-1, Mo-1 and
p150,95. Eur J Immunol 1985;15:1142-1147.
22. Sanchez-Madrid F, Nagy JA, Robbins E, Simon P, Springer TA. A
human leukocyte differentiation antigen family with distinct
a-subunits and a common ,3-subunit: the lymphocyte functionassociated antigen (LFA-1), the C3bi complement receptor
(OKM1/Mac-1) and the p150,95 molecule. J Exp Med. 1983;158:
1785-1803.
23. Fraker PJ, Speck JC Jr. Protein and cell membrane iodinations
with a sparingly soluble chloroamine, 1,3,4,6-tetrachloro-3a,6a
diphenylglycoluril. Biochem Biophys Res Commun. 1978;80:
849-857.
24. Fair DS, Plow EF, Edgington TS. Combined functional and immunochemical analysis of normal and abnormal human factor X.
J Clin Invest. 1979;64:884-894.
25. Elemer GS, Edgington TS. Integrin "activation" associated with
cytochalasin B depolymerization of cellular actin. J Cell Biol. 1991;
115:291a. Abstract.
26. Diamond MS, Springer TA. A subpopulation of Mac-1 (CDiib/
CD18) molecules mediates neutrophil adhesion to ICAM-1 and
fibrinogen. J Cell Bio. 1993;120:545-556.
171
27. van Kooyk Y, Weder P, Hovervorst F, Verhoeven AJ, van Seventer
G, te Velde AA, Borst J, Keizer GD, Figdor CG. Activation of
LFA-1 through a Ca2+-dependent epitope stimulates lymphocyte
adhesion. J Cell Biol 1991;112:345-354.
28. Landis RC, Bennett RI, Hogg N. A novel LFA-1 activation epitope
maps to the I domain. J Cell Biol. 1993;120:1519-1527.
29. Du X, Gu M, Weisel JW, Nagaswami C, Bennett JS, Bowditch R,
Ginsberg MH. Long range propagation of conformational changes
in integrin ajb/l33. J Biol Chem. 1993;268:23087-23092.
30. Diamond MS, Garcia-Aguilar J, Bickford JK, Corbi AL, Springer
TA. The I domain is a major recognition site on the leukocyte
integrin Mac-1 (CD11b/CD18) for four distinct adhesion ligands.
J Cell BioL 1993;120:1031-1043.
31. Altieri DC, Etingin OR, Fair DS, Brunck TK, Geltosky JE, Hajjar
DP, Edgington TS. Structurally homologous ligand binding of
integrin Mac-1 and viral glycoprotein C receptor. Science. 1991;
254:1200-1202.
32. Altieri DC, Plescia J, Plow EF. The structural motif glycine 190valine 202 of the fibrinogen gamma chain interacts with CD1lb/
CD18 integrin (aMl82, Mac-1) and promotes leukocyte adhesion.
J Biol Chem. 1993;268:1847-1853.
Downloaded from http://circres.ahajournals.org/ by guest on July 28, 2017
Two independent sets of monoclonal antibodies define neoepitopes linked to soluble ligand
binding and leukocyte adhesion functions of activated alpha M beta 2.
G S Elemer and T S Edgington
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Circ Res. 1994;75:165-171
doi: 10.1161/01.RES.75.1.165
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