(Received for publication, November 27, 1995; and in revised form, February 12, 1996)
From the
Several studies indicate that the I domain located in the
chain (CD11a) of leukocyte function-associated antigen-1 (LFA-1;
CD11a/CD18) plays an essential role in ligand recognition. We recently
identified three distinct epitopes (IdeA, IdeB, and IdeC) within the
CD11a I domain, recognized by antibodies that block binding of LFA-1 to
intercellular adhesion molecules (ICAM) 1, 2, and 3. In the present
study, we used a series of human/murine CD11a I domain chimeras, to
localize a fourth I domain epitope (IdeD), recognized by three
independently derived anti-CD11a antibodies that selectively block the
binding of LFA-1 to ICAM-3, but not to ICAM-1. The IdeD epitope
depended on human CD11a residues Asp
and Ser
and was not present in CD11b or CD11c. Although mutation of
Asp
and Ser
failed to abolish ICAM-3
adhesion of LFA-1 transfectants, alignment of these residues with the
crystal structure of the CD11a I domain suggested that the IdeD epitope
is located in close proximity to residues (Ile
and
Asn
) recently implicated in the ICAM-3 binding
site(1) . Interestingly, the IdeB and IdeC epitopes appeared to
be in close proximity of a divalent cation binding pocket within the
CD11a I domain that regulates both ICAM-1 and ICAM-3 adhesion. Taken
together, these data indicate that distinct regions of the CD11a I
domain contain epitopes for antibodies that either selectively inhibit
binding of LFA-1 to ICAM-3, or interfere with both ICAM-1 and ICAM-3
binding of LFA-1.
The leukocyte integrin LFA-1 ()(CD11a/CD18) is a cell
surface receptor that mediates adhesive interactions and signal
transduction in the immune
system(2, 3, 4, 5) . LFA-1 is
expressed by leukocytes and belongs to the
family of
integrins, in which a common
subunit (CD18) is associated with
any of three distinct, but structurally homologous,
subunits;
(CD11a, LFA-1),
(CD11b, Mac-1), and
(CD11c, p150, 95)(2) . The extracellular
domain of the LFA-1
subunit contains two domains thought to be of
functional significance. These include a putative divalent cation
binding region, consisting of three tandem repeats of an EF-hand motif,
also found in other integrins (6, 7) and a 200-amino
acid inserted or ``I'' domain (8) , which is also
present in
,
,
,
, and
subunits(2, 9) . The I domain contains sequences
homologous to the type A domains of von Willebrand factor, cartilage
matrix-binding protein, and complement factor B(8) .
LFA-1 is known to recognize three ligands: ICAM-1(10) , ICAM-2(11) , and ICAM-3(12, 13, 14) , all of which are members of the Ig superfamily, and have five, two, and five Ig-like domains, respectively. ICAM-1 is expressed on many cells, including lymphocytes and endothelial cells, and its expression is cytokine inducible(15) . ICAM-2 is expressed on lymphocytes, endothelial cells, and platelets(16, 17) , whereas ICAM-3 is only expressed on leukocytes(18) . In contrast to ICAM-1, expression of ICAM-2 and -3 is not induced by cytokines(18, 19) .
LFA-1-mediated adhesion
requires activation of the LFA-1 molecule (20, 21, 22) . Activation can be induced by
intracellular signals generated upon cross-linking of cell surface
receptors (T cell receptor/CD3)(21, 22) , upon binding
of activating anti-LFA-1
antibodies(23, 24, 25, 26, 27) ,
or divalent cations, such as Mn(28) .
Activation of LFA-1 and subsequent ligand binding is thought to result
from conformational changes in the
/
heterodimer and requires
binding of divalent cations, such as Mg
and
Ca
, an intact cytoskeleton, and a physiological
temperature(29) . While Ca
binding supports
clustering of LFA-1 on the cell surface, presumably resulting in
enhanced ligand binding avidity, Mg
binding to LFA-1
has been suggested to alter the affinity of LFA-1 for its
ligands(28, 30) .
Recent findings indicate that the
I domains of CD11a, CD11b, and CD11c, as well as I domain sequences of
the and
chains of
class of integrins, are involved in ligand binding. Evidence
comes from the finding that purified
(CD11a),
(CD11b), and
I domains directly
bind their respective ligands, ICAM-1, fibrinogen and iC3b, or
collagen(31, 32, 33) . Second, mutation of
aspartic acid or threonine residues within the I domains of
,
,
, and
affects cation binding and impairs
adhesion(33, 34, 35, 36, 37) .
Furthermore, we identified residues Ile
and Asn
within the CD11a I domain to be critical for adhesion to ICAM-3,
but not for ICAM-1 binding, indicating that the CD11a I domain contains
distinct binding sites for different ligands (1, 27) .
Finally, most anti-CD11a, CD11b, and CD11c antibodies that block ligand
interactions recognize the I
domain(38, 39, 40) . Previous investigations
have revealed that anti-human CD11a antibodies do not cross-react with
murine LFA-1, implying that sequences in the I domain important for mAb
binding can be located by replacing human CD11a sequences with the
murine homologues(40) . Using human/mouse I domain mutants in
which sequences from the human CD11a I domain were substituted into
murine I domain residues, we recently demonstrated that anti-CD11a I
domain antibodies that inhibit the interaction of LFA-1 with ICAM-1,
-2, and -3 (
)(e.g. TS1/22, 25.3, and
MHM.24)(39, 40) , recognized three distinct epitopes
within the CD11a I domain (IdeA, residues 126-129; IdeB, residues
143-148; IdeC, residues 198-204)(40) .
In the
present studies, we identified a fourth epitope within the CD11a I
domain (IdeD), that is recognized by three anti-CD11a antibodies that
selectively inhibit the binding of LFA-1 to ICAM-3. Alignment of the
IdeD epitope with the recently solved crystal structure of the CD11a I
domain (41) suggested that it is located in close proximity to
I domain residues Ile and Asn
, critical for
ICAM-3, but not ICAM-1 binding of LFA-1(1) .
Figure 1: The CD11a I domain-specific mAbs MEM-83, YTH81.5 and 122.2A5 selectively inhibit the LFA-1/ICAM-3 interaction. After activation of LFA-1 with the CD18-activating antibody MEM-48 (5-20 µg/ml), the capacity of HSB T cells or 293 cells transfected with CD11a/CD18 constructs, to bind purified ICAM-1Fc or ICAM-3Fc proteins (300 ng/ml; 50 µl/well) was determined, in the absence of presence of mAbs MEM-83, YTH81.5, 122.2A5, or NKI-L15 (CD11a) at 10-50 µg/ml. Results are expressed as the mean percentage of adherent cells from triplicate wells. One representative experiment out of three is shown.
Figure 2:
MEM-83, YTH81.5, and 122.2A5 inhibit
ICAM-3-dependent costimulation of T cells. Resting T cells were plated
on ICAM-1Fc or ICAM-3Fc, together with suboptimal concentrations of
coated anti-CD3 antibodies (30 ng/ml; 50 µl/well), in the presence
or absence of mAbs MEM-83, YTH81.5, 122.2A5, NKI-L15 (CD11a), REK-1
(ICAM-1), or AZN-IC3.1 (ICAM-3), at 10 µg/ml. After 3 days of
culture, H incorporation was determined. Results are
expressed as the mean counts/min of triplicate wells. Data are
representative of three experiments.
Figure 3: Amino acid sequences of murine and human CD11a I domains and human/murine substitution mutants. A, schematic representation of CD11a with respective locations of the I domain and the putative metal binding (EF-hand) domains. B, in the human/murine chimeras (H/M48-54), one to five human residues were substituted for murine residues. In D137A and D239A mutants, aspartic acid residues were substituted for alanine.
To identify residues within the CD11a I domain critical for binding
of ICAM-3 blocking CD11a antibodies, we determined the ability of
MEM-83, 122.2A5 and YTH81.5 to immunoprecipitate chimeric CD11a
proteins from transiently transfected 293 cells. The MEM-83, 122.2A5,
and YTH81.5 antibodies all readily immunoprecipitated H/M53, H/M52, and
H/M54 proteins (Fig. 4), showing that these antibodies did not
bind to the previously identified IdeA, IdeB, or IdeC epitopes within
the CD11a I domain(40) . In contrast, the ICAM-3 blocking
antibodies were unable to immunoprecipitate the H/M48 protein,
suggesting that antibody binding depended on Asp and
Ser
residues, replaced by Thr and Leu, respectively, in
the H/M48 chimera. The H/M48 mutation did not simply inhibit antibody
binding by disrupting the overall conformation of CD11a, since
monoclonal antibodies recognizing the IdeA, IdeB, or IdeC epitopes were
still able to bind the H/M48 protein(40) . Thus, H/M48 appeared
to define a new epitope recognized by several independently isolated
monoclonal antibodies.
Figure 4:
Immunoprecipitation of human CD11a
variants with ICAM-3 blocking anti-CD11a monoclonal antibodies MEM-83,
YTH81.5, and 122.2A5. The human kidney adenocarcinoma cell line 293 was
transfected with plasmids directing the expression of full-length human
CD11a (CD11a), or human CD11a I domain variants in which human CD11a I
domain residues were replaced by corresponding murine residues
(H/M48-H/M54). A diagram listing the mutations introduced in each
variant is provided in Fig. 3. ICAM-3 blocking anti-CD11a mAbs
(MEM-83, YTH81.5, 122.2A5) were added to detergent lysates of
transfected cells metabolically labeled with
[S]methionine and subjected to
immunoprecipitation. MEM-83 is shown in panel A, YTH81.5 in panel B, and 122.2A5 in panel
C.
Since we observed some reduced
immunoprecipitation of the H/M50 chimera by 122.2A5, we investigated
the ability of human CD18 to form a heterodimeric complex with chimeric
CD11a, to verify the conformational integrity of the H/M chimeras. In
these experiments full-length chimeric CD11a variants were
co-transfected with wild type human CD18 into 293 cells and
transfectants were assayed for antibody binding by
fluorescence-activated cell sorter analysis. Co-expression of CD18 with
all CD11a variants was detected on the cell surface (Table 1),
suggesting that the CD11a I domain mutations preserved the structural
elements required for heterodimer formation and export to the cell
surface. Another indication for the conformational integrity of the H/M
variants was provided by binding of a murine antibody to human CD18
(MHM.23), that recognizes an epitope critically dependent on
/
association of CD11a and CD18(44, 49) .
The observation that MHM.23 bound all of the I domain variants examined (Table 1) provided further data that the I domain variants used
for epitope mapping studies did not interfere with structural features
required for heterodimer formation. Similarly, the binding of the
NKI-L16 mAb (CD11a) to the conformation-sensitive L16 epitope (23) located outside the CD11a I
domain(37, 50) , provided additional evidence that
mutations in the CD11a I domain do not change the overall conformation
of CD11a. In contrast to the immunoprecipitation studies, mAb 122.2A5
readily bound the H/M50 chimera when complexed with CD18 (Table 1), suggesting that residues mutated in H/M50 do not
directly contribute to the 122.2A5 epitope, but rather affect antibody
binding by inducing subtle conformational changes in CD11a, which are
not apparent in the CD11a/CD18 heterodimer. Together, these studies
demonstrate that antibodies that selectively inhibit the LFA-1/ICAM-3
interaction, recognize a novel epitope within the CD11a I domain,
termed IdeD, dependent on residues
Asp
-Ser
.
Figure 6:
Ribbon representation of CD11a I domain
based on coordinates provided by Leahy et al.(41) .
Epitopes recognized by antibodies to CD11a (IdeA, IdeB, IdeC, and IdeD)
have been aligned with the CD11a sequence and are shown as black
ribbon or tube. Mn ion (black
sphere), and N and C termini have also been labeled. Ribbon
diagram was drawn using the MIDAS program (University of California,
San Francisco). Positions corresponding to the IdeB epitope (residues
143-148), the IdeC epitope (residues 198-204), and the IdeD
epitope (Asp
and Ser
) are indicated. Of the
IdeA epitope (Ile
and Asn
) only the fourth
residue (Asn
) is indicated, since the first three
residues were not included in the CD11a I domain
crystal.
Figure 5:
Adhesion of CD11a I domain mutants to
ICAM-1 and ICAM-3. The capacity of 293 cells transfected with chimeric
CD11a and wild type CD18 to bind ICAM-1Fc or ICAM-3Fc was determined,
in the presence of the activating CD18 mAb KIM185 (5-10
µg/ml). A, 293-cells were transfected with CD18 alone
(293-mock), with CD18 and CD11a (CD11a), or with human/mouse chimeric
CD11a constructs and CD18 (H/M48 and H/M53). B, the capacity
of 293 cells cotransfected with CD18 and CD11a point mutants D137A or
D239A, to bind to ICAM-1Fc or ICAM-3Fc was determined in the same
experiment. Results are expressed as the mean OD from
triplicate wells. One representative experiment out of three is
shown.
We have demonstrated that the CD11a I domain-specific
antibodies MEM-83, YTH81.5 and 122.2A5 that selectively inhibit the
interaction of LFA-1 with ICAM-3(27, 39) , bind to a
novel epitope (IdeD) in the I domain of CD11a. This is distinct from
the previously identified IdeA, IdeB and IdeC epitopes, recognized by
antibodies that block LFA-1 binding to ICAM-1, -2 and
-3(39, 40) . Site-directed mutagenesis
demonstrated that the IdeD epitope comprises amino acids Asp
and Ser
, although mutation of these residues failed
to inhibit the binding of LFA-1 to ICAM-3. Thus antibody binding to the
IdeD epitope appears to interfere with ICAM-3 binding by steric
hindrance rather than by competitive binding to the ligand binding
site. Placement of the IdeD epitope on the crystal structure of the
CD11a I domain (41) suggested that the IdeD epitope was located
in close proximity to residues recently identified as being critical
for ICAM-3 binding to LFA-1(1) .
Interestingly, residues
critical for ICAM-3 binding (Ile and Asn
)
and the IdeD epitope are both unique to human CD11a and are not found
in CD11b or CD11c, suggesting that these represent a structural feature (i.e. all or part of a ligand binding domain) unique to LFA-1.
These data are consistent with earlier observations that MEM-83,
YTH81.5, and 122.2A5 failed to cross-react with CD11b or
CD11c(39) . Although MEM-83, YTH81.5, and 122.2A5 all bind the
IdeD epitope, they show some functional differences, since MEM-83 can
activate LFA-1-mediated adhesion to ICAM-1(24, 27) ,
whereas 122.2A5 and YTH81.5 cannot(39) . This suggests that
MEM-83 can induce or stabilize an active ICAM-1 binding conformation of
LFA-1(24) . Surprisingly, we observed that MEM-83, YTH81.5 and
122.2A5, as well as blocking antibodies directed against ICAM-3,
inhibited ICAM-1-induced T cell proliferation. These data suggest that
although T cell costimulation in this system is dependent on engagement
of LFA-1 by coated ICAM-1, optimal proliferation may require
LFA-1/ICAM-3 interactions between proliferating cells, which are
prevented by IdeD-specific anti-CD11a antibodies. We previously
reported that MEM-83 (27) , as well as YTH81.5 and 122.2A5
(data not shown), are potent inhibitors of the LFA-1/ICAM-2
interaction, suggesting that residues critical for ICAM-2 and ICAM-3
binding may be located in close proximity. It will therefore be
important to determine whether the sequence that has been shown to be
essential for ICAM-3 binding, is also involved in LFA-1 binding to
ICAM-2.
In addition to ligand-specific sequences, the LFA-1 I domain
contains conserved sequences required for adhesion of LFA-1 to all
ligands. Interestingly, when aligned with the CD11a I domain
structure(41) , two of the recently identified CD11a I domain
epitopes (IdeB and IdeC) recognized by antibodies that block ICAM-1,
ICAM-2, and ICAM-3 binding(39, 40) were
located in close proximity to the divalent cation binding pocket, or
MIDAS motif(36) . Residues within this motif
(Asp
, Asp
, and Thr
) have been
implicated in cation binding (34) and/or ligand binding of the
I domain containing integrins Mac-1,
,
,
and LFA-1 (Refs. 1, 33-37, and 51; Fig. 5). In the CD11a I
domain crystal structure, a Mn
ion is coordinated by
five cation coordinating residues. A critical acidic glutamate residue
(E) within the integrin binding motif I/L-E-T-P/S-L in the first
Ig-like domains of ICAM-1, -2, or -3 (47, 52) may
provide the sixth cation coordinating residue in vivo,
implying a role for metal ions in the stabilization of LFA-1/ligand
interactions(36, 53) . In addition, residues in
proximity of the divalent cation binding pocket (Met
,
Glu
, Thr
, and Ser
) were shown
to be critical for binding of LFA-1 to ICAM-1(50) , underlining
the importance of this I domain region in LFA-1/ligand interactions. It
is tempting to speculate that antibodies recognizing the IdeB and IdeC
epitopes interfere with LFA-1 function by inhibiting actual ligand
binding residues in this area, or by altering the conformation of such
residues. Alternatively, these antibodies may affect adhesion by
altering the conformation of residues involved in cation coordination,
resulting in destabilization of the cation binding site.
Our data identify two distinct regions within the CD11a I domain that contain residues critical for ICAM-3 binding: the region involved in cation binding and the region defined by the IdeA epitope at the other side of the I domain. These regions may both contain actual contact sites for ICAM-3, implying that ICAM-3 contacts a relatively large binding face on LFA-1. However, it is also possible that mutations introduced in either one of these regions induce subtle conformational changes in the CD11a I domain, which reduce binding to ICAM-3. As yet we cannot distinguish between these possibilities.
In conclusion, our data
indicate that distinct regions of the CD11a I domain contain epitopes
recognized by antibodies that either selectively inhibit binding of
LFA-1 to ICAM-3, or inhibit both ICAM-1 and ICAM-3 adhesion of LFA-1.
These antibodies may inhibit LFA-1 function by either interfering with
ligand-specific sequences, or conserved domains within the CD11a
domain, that are required for binding of LFA-1 to both ICAM-1 and
ICAM-3. The challenge of future research will be to understand how
during integrin activation subtle conformational changes within the
and
subunits lead to exposure of these functionally
important domains and subsequent ligand binding. The ability of
antibodies to selectively inhibit LFA-1-ligand binding might find
utility in the development of immune response and inflammatory response
modulators. Moreover, these results suggest that the capacity of
anti-CD11a antbodies to interfere with leukocyte function (e.g. antigen presentation, cytotoxic killing, and B cell activation),
historically attributed to disruption of LFA-1/ICAM-1 interactions,
should be re-examined to evaluate the possible role of ICAM-2 and
ICAM-3.