Mß2 (CD11b/CD18, Mac-1) integrin activation by a unique monoclonal antibody to M I domain that is divalent cation-sensitive
* Cell and Molecular Biology, Pharmacia Corporation, Kalamazoo, Michigan; and
Department of Pathology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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M subunit, which directly induces adhesion
of 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 M I domain. The M I domain is
essential for ligand binding, can directly bind divalent cations, and
participates in the regulation of Mß2
ligand-binding affinity. Thus, these studies have identified a novel
M I domain activation epitope of
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
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subunit [6
]. There are four ß2 integrins:
Lß2 [CD11a/CD18, lymphocyte
function-associated antigen-1 (LFA-1)],
Mß2 (CD11b/CD18, Mac-1, CR3),
Xß2 (CD11c/CD18, p150,95), and
dß2 (CD11d/CD18) [6
,
7
]. All four subunits contain a 200 amino
acid-inserted or "I" domain. The I domain is also present in five
other integrin subunits: 1 of
1ß1[8
],
2 of 2ß1
[9
], 10 of
10ß1[10
],
11 of 11ß1
[11
], and E of
Eß7 [12
]. The I domain
plays a significant role in ligand binding [13
]. In
addition, crystal structures of two of the ß2 integrins,
Lß2 and 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.
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
]. 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 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 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 M subunit-specific antibody, mAb 6C1, that
promotes Mß2 ligand-binding function. The
mAb 6C1 induces adhesion of Mß2 expressing
cells to multiple ligands, including fibrinogen, ICAM-1, and iC3b. The
binding of mAb 6C1 induces a conformational change in
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
M subunit, further substantiating the involvement of
this domain in integrin activation.
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M) [37
], mAb 8H1
(anti-ß2) [37
], mAb LM2/1
(anti-M) [38
], and mAb 8.4A6
(anti-ICAM-1) [37
]. Murine mAb 24
(anti-L, -M, -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 M
(Gly111 to Ala318), were described previously
[41
]. Recombinant 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 L
[42
] or 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 M or the mutant M subunit with
the wild-type ß2 subunit. Construction of the mutant
M constructs M(D140A),
M(D142A),
M(D140GS/A140GA),
M(S144A),
M(L250A), and
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 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
Mß2 was purified from
polymorphonuclear leukocytes by mAb LM2/1 immunoaffinity chromatography
as previously described [21
]. Mice (BALB/c) were
immunized with purified Mß2 (20 µg)
subcutaneously, which was followed by three intraperitonial injections
of purified 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
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
Mß2-transfected CHO cells. Positive
hybridomas were tested for their ability to enhance the adhesion of
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 M was immobilized on
Immunlon II plates (Dynatech Laboratories, Chantilly, VA) by the
addition of 50 µl/well of 10 µg/ml M I domain in 100
mM NaHCO3, pH 8.2, at 4°C overnight. Alternatively, CHO
cells expressing 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-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
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-M) and by using the
molecular weight of 150,000.
Surface iodination, immunoprecipitation, and SDS-PAGE
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.
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Mß2-transfected CHO cells to immobilized
Fg, ICAM-1, and iC3bMß2 were fused with myeloma cells to
produce hybridomas. Hybridoma supernatants were screened initially for
reactivity to Mß2-transfected CHO cells in
a whole-cell ELISA. Antibodies reacting in this assay were further
screened for their ability to enhance adhesion of
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,
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-M mAb
3H5 (unpublished results), consistent with mediation by
Mß2.
 View larger version (22K): [in a new window] |
Figure 1. mAb 6C1 induces adhesion of recombinant
Mß2 expressed on CHO cells to immobilized
ligand. Fluorescently labeled
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 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-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.
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Mß2-transfected CHO cells’ adhesion to
immunopurified ICAM-1 or to the complement C3 fragment iC3b, two other
known ligands for Mß2.
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-M mAb 3H5 (unpublished results). In
contrast to immobilized fibrinogen and ICAM-1,
Mß2-transfected CHO cells adhere to
immobilized iC3b under the coating conditions used. However, mAb 6C1
induced a further increase in binding of
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 M (unpublished results). Taken
together, these results demonstrate that mAb 6C1 promotes adhesion of
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
Mß2-transfected CHO-cell adhesion (Fig. 1C) .
mAb 6C1 recognizes an epitope on M
To identify the subunit specificity of mAb 6C1, immunoprecipitates
of detergent-lysed 125I-labeled CHO cells, expressing
recombinant Mß2or
Lß2and
125I-labeled human neutrophils, were analyzed by SDS-PAGE
(Fig. 2A
). The mAb 6C1 immunoprecipitated M and
ß2 in the stably transfected
Mß2 cells only, analogously to the control
anti-M mAb 3H5. In contrast, an antibody directed
against L, mAb TS1/22, precipitated L and
ß2 in the stably transfected
Lß2 cells only. The anti-ß2
mAb 8H1 precipitated bands concordant with
Lß2 and Mß2
complexes in the Lß2- and
Mß2-transfected cell lines, respectively.
In addition, the mAb 6C1 immunoprecipitated M and
ß2in human neutrophils similarly to the
control anti-M mAb 3H5 and anti-ß2mAb 8H1 antibodies. Analysis by immunoblots of purified
Mß2from neutrophils revealed
that mAb 6C1 did not recognize the M subunit under
reduced or nonreduced conditions (unpublished results).
 View larger version (56K): [in a new window] |
Figure 2. mAb 6C1 recognizes an epitope on the M subunit. (A)
Detergent lysates from surface-iodinated
Lß2- or
Mß2-transfected CHO cells and human
neutrophils were immunoprecipitated with mAbs 8H1
(anti-ß2, TS2/4 (anti-L), 6C1, or LM2/1
(anti-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
Mß2- or
Lß2-transfected CHO cells was examined by
flow cytometry. Mß2- (top panels) or
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-M (mAb 3H5), anti-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.
|
Mß2or
Lß2(Fig. 2B) . The mAb 6C1
bound to the Mß2-transfected CHO cells
only, similar to the control anti-M mAb 3H5; the
anti-L mAb TS1/22 bound to the
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 M subunit alone or
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
M subunit.
mAb 6C1 induces a conformational change in
Mß2
To determine whether mAb 6C1 induces a conformational change in
the 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 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 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
Mß2 integrin conformation recognized by
mAb 24.
 View larger version (16K): [in a new window] |
Figure 3. mAb 6C1 induces expression of the activation-dependent neoepitope
recognized by mAb 24 on Mß2-transfected
CHO cells. The binding of mAb 24 to
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.
|
MMß2function, the expression
of the mAb 6C1 epitope on 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
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.
 View larger version (18K): [in a new window] |
Figure 4. Expression of mAb 6C1 epitope is divalent cation-dependent.
Mß2-transfected CHO cells were incubated
with fluorescein-conjugated antibodies mAb 6C1 or anti-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.
|
|
View this table: [in a new window] |
Table 1. 125I-6C1 binding affinities to recombinant
Mß2-expressing CHO cells
|
Mß2. These divalent
cation-binding site(s) are important regulators of
Mß2 ligand-binding function. The I domain
of 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
M I domain, modulates a divalent cation conformation
recognized by mAb 24 [47
]. Consequently, the effects of
the M I domain mutations, which disrupt the interaction
between bound divalent cation and 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 Mß2 stained brightly
with mAb 6C1 when compared with the anti-M control
antibody 2LPM19c. In contrast, mAb 6C1 binding in the presence of 0.5
mM Mn2+ to cells expressing
M(D140A)ß2 or
M(S142A)ß2 was reduced
considerably when compared with the anti-M control
antibody 2LPM19c. However, cells expressing
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
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 Mß2.
 View larger version (15K): [in a new window] |
Figure 5. Expression of mAb 6C1 epitope on recombinant wild-type or mutant
Mß2 receptors. The binding of mAb 6C1 to
COS-7 cells transfected with wild-type or mutant
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.
|
MMß2
within the I domain of M, have been identified.
Furthermore, a novel Lß2 activation
epitope recognized by mAb 83, which is located within the I domain of
L, has also been identified [32
]. To
determine whether the mAb 6C1 activation epitope was located within the
I domain of M, mAb 6C1 was tested for its ability to
recognize a purified I domain in an ELISA assay. The M I
domain was isolated and purified from a glutathione
S-transferase fusion protein as previously described
[41
]. The recombinant M I domain was
recognized specifically by anti-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-M antibody mAb 24 did not recognize the
M I domain. None of the antibodies tested reacted with
BSA. No differences in the binding of mAb 6C1 to the M I
domain were detected using different cation conditions in a way that
mirrors the effects of cation binding of mAb 6C1 to recombinant
Mß2 expressed on CHO cells (unpublished
results). It is possible that once the M I domain is
immobilized, it is unable to undergo further cation-dependent
conformational changes.
 View larger version (15K): [in a new window] |
Figure 6. mAb 6C1 recognizes an epitope within the M I domain. (A)
Recombinant 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 L
I domain expressed on BHK/VP16 cells was examined by flow
cytometry. The L I domain-transfected cells were
incubated with anti-ß2 mAb 8H1,
anti-LmAb TS1/22,
anti-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.
|
M I domain,
the mAb 6C1 was tested for its ability to recognize BHK cells
expressing the GPI-anchored form of the L I domain
[48
], as determined by flow cytometry (Fig. 6B)
. The
L I domain was recognized specifically by the
anti-L mAb TS1/22. In contrast, mAb 6C1 did not
recognize the L I domain. Similarly, the
anti-ß2 mAb TS1/18 and anti-L mAb 24 did
not react with L I domain-expressing cells. Thus, the
mAb 6C1 activation epitope is located with the I domain of
Mß2. |
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
M-specific antibody,
mAb 6C1, which induces 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
M subunit. Thus, these studies have identified a novel
M activation epitope within the I domain of
Mß2 and further suggest the involvement of
this domain in regulating ß2 integrin-ligand
interactions.
With the exception of iC3b,
Mß2-expressing cells are competent to bind
ligands only following cellular activation [53
].
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
Mß2-adhesive interactions. mAb 6C1 was
identified by its ability to enhance
Mß2-adhesive interactions with fibrinogen
and ICAM-1. Although Mß2-transfected CHO
cells adhere to iC3b, mAb 6C1 enhanced this interaction further as
well. Binding of Mß2-transfected CHO cells
induced by mAb 6C1 was dose-dependent and inhibited by blocking mAbs to
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 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-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 Mß2 within the
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 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 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
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
Mß2 molecules (10–30%) on activated
neutrophils [57
, 58
]. However, this
subpopulation of Mß2 molecules mediates at
least 90% of the adhesion activity of
Mß2. The mAb CBRM1/5, like mAb 6C1,
recognizes the 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 M I domain. Unfortunately, we were unable to map
precisely within the 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 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
Mß2 ligand binding by the localization of
blocking antibody epitopes and by direct ligand binding to the isolated
I domain [13
, 41
]. The M I
domain binds cations also [14
, 15
].
Moreover, localization of the mAb 6C1 activation epitope to the
M I domain indicates that this domain modulates the
ligand-binding conformation state of Mß2.
This concept is supported by the crystal structures of the
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 Mß2 within the
M I domain, has been identified [59
].
In summary, we have described a mAb directed against the
M I domain that promotes the adhesion of
Mß2 to multiple ligands, including iC3b,
fibrinogen, and ICAM-1. The anti-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.
Received May 20, 1999; revised May 18, 2000; accepted May 23, 2000.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
dß2, binds preferentially to ICAM-3 Immunity 3,683-690[Medline]1 subunit J. Biol. Chem. 268,2989-29962 subunit (platelet GPIa): homology to other integrins and the presence of a possible collagen-binding domain J. Cell Biol. 109,397-40710, a ß1-associated collagen binding integrin expressed on chondrocytes J. Biol. Chem. 273,20383-20389E subunit. Unusual structure and restricted RNA distribution J. Biol. Chem. 269,6016-6025 subunit of integrin CR3 (CD11b/CD18) Cell 81,631-638[Medline]Lß2) integrin Proc. Natl. Acad. Sci. USA 92,10277-10281-chain of human leukocyte function-associated antigen 1 induces homotypic cell-cell interactions J. Immunol. 140,1393-1400[Abstract]Lß2 and Mß2 ligand interactions J. Biol. Chem. 271,23729-23736Lß2, CD11a/CD18) and Mac-1 (Mß2, CD11b/CD18) J. Biol. Chem. 270,94-98subunits EMBO J 8,3759-3765[Medline] subunit of a leukocyte adhesion glycoprotein, p150,95 EMBO J 6,4023-4028[Medline]Mß2 that differentially affect a divalent cation-dependent conformation J. Biol. Chem. 270,25866-25871IIbß3 (platelet glycoprotein IIb-IIIa) J. Biol. Chem. 267,22211-22216Mß2 ligand recongition J. Leukoc. Biol. 64,767-773[Abstract]IIbß3 integrin (platelet GPIIb-IIIa) is an intrinsic property of the receptor Cell Regul 1,883-893[Medline]This article has been cited by other articles:
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