(Received for publication, November 14, 1994; and in revised form, January 13, 1995)
From the
The vascular cell adhesion molecule-1 (VCAM-1) plays an
important role in diverse physiological and pathological processes. The
homologous first and fourth immunoglobulin-like domains of the seven
domain form of VCAM-1 present binding motifs for
integrin. Using a panel of VCAM-1
domain deletion mutants we show that
integrin interacts with both domains 1 and 4. In contrast to
their identical domain usage,
and
integrins differ in the activation
states required for binding to domains 1 and 4 of VCAM-1. We show that
integrin
required significantly
higher concentrations of Mn
than integrin
to support half-maximal adhesion to
domain 4. Moreover, a clear difference in the capacity of integrins
and
to interact with domain 4 was detected in the presence of
Ca
and Mg
cations. Adhesion to
domain 1 of VCAM-1, however, was not affected by integrin heterodimer
composition. Instead, the activity level of integrin
for domain 1 binding was regulated
by CD24 expression. Binding to seven domain VCAM-1 was not altered
significantly by
and
subunits or
CD24. These data indicate that integrin heterodimer composition and
CD24 expression differentially modulate integrin binding to domains 1
and 4 of VCAM-1. Mechanisms that alter integrin binding specificity or
monovalent versus divalent interactions may affect the
strength of adhesion as well as signal transmission in adherent cells
and may therefore be critical to controlling the cellular response to
integrin occupancy.
The inducible vascular cell adhesion molecule-1 (VCAM-1) ()is a member of the immunoglobulin superfamily and may be
involved in numerous physiological and pathological processes,
including embryonic development of skeletal muscle, hematopoiesis,
inflammatory reactions, and the development of autoimmune
disorders(1, 2, 3) . In addition to the seven
domain form of VCAM-1 (VCAM-7D), a six-domain form (VCAM-6D) that is
generated by alternative splicing of domain 4, a glycolipid-anchored
form containing the first three domains, and an eight-domain form of
VCAM-1 have been described(1, 2) . The homologous
first and fourth immunoglobulin-like domains of VCAM-1 (VCAM-7D)
present binding motifs for
integrin(4, 5, 6, 7, 8, 9, 10) .
Based on temperature sensitivity and phorbol 12-myristate 13-acetate
activation, it has been suggested that
integrin requires different states of activation for binding to
VCAM-1 domains 1 or 4 (11) . Recently, it has been demonstrated
that integrin
functions as an
alternative leukocyte receptor for
VCAM-1(12, 13, 14, 15) . Integrins
and
also share binding specificity for the the CS-1 peptide derived
from
fibronectin(12, 13, 14, 15, 16) .
Adhesion to the mucosal addressin MAdCAM-1, however, is predominantly
mediated by integrin
(15, 17) .
The
heat-stable antigen (CD24) has been described as a maturation marker of
murine lymphocytes (18, 19, 20) and has
co-stimulatory activity for the clonal expansion of CD4 T
cells(21) . On B lymphocytes, cross-linking of CD24 results in
a rapid rise in cytoplasmic calcium levels suggesting a role for CD24
in signal transduction(22) . Structurally, CD24 consists of a
small glycolipid-anchored peptide core with a large number of potential N-linked and O-linked glycosylation
sites(23, 24) . The carbohydrate moiety may be
involved in homophilic CD24 interactions (25) and in presenting
binding sites for P-selectin(26) . In addition, CD24 alters
integrin adhesion to fibronectin and
TNF-stimulated endothelioma cells(27) .
In the present study
we show that integrin independently
binds to domains 1 and 4 of VCAM-1. When compared with
, adhesion of
integrin to domain 4 required
significantly lower levels of activation, suggesting that domain 4
binding is regulated by the
subunits of
integrins. By contrast, efficient adhesion of
integrin to VCAM-1 domain 1 was
dependent on CD24. Differential regulation of
integrin binding to individual domains of VCAM-1 may affect the
strength of adhesion as well as signal transmission in adherent
leukocytes.
The C3H/He
B cell lymphoma 38C13 was demonstrated to express membrane-bound
subunits, but lacks detectable cell surface protein
and RNA transcripts for integrin
and
subunits(28) . Integrin
or
chains were expressed de novo in 38C13 lymphoma cells by
retrovirus-mediated gene transfer generating cell lines 38-
and 38-
(15) . TK1.cl14 is a spontaneous
AKR/cum T cell lymphoma expressing integrin subunits
and
but not
, whereas RAW112
is an Abelson leukemia virus-induced BALB/c pre-B cell lymphoma that
expresses
and
but not
subunits(15) . CHO transfectants expressing the human
VCAM-7D receptor and CHO/CDM8 control cells have been
described(15) . The pre-B cell line N232.18 was generated by
infection of bone marrow-derived cells from a CD24-deficient chimeric
mouse with a replication defective A-MuLV(27) . 18H18 cells
were obtained by transfection of N232.18 cells with the CD24 expression
plasmid pHSEX62.8(27) .
38- cells
were allowed to adhere to CHO transfectants in the presence of 1.0
mM Mn
to determine plateau levels of cell
binding. To examine
integrin-dependent adhesion, the fraction of cells bound in the
presence of mAb PS/2 (anti-
) was subtracted from the
percent of adherent cells treated with mAb M17/4.2 (anti-LFA-1). As is
shown in Fig. 1A, plateau levels of 38-
cell binding to VCAM-1 were not altered significantly by
1.2.3 or
4.5.6 deletions. Plateau binding of 38-
cells was also not affected by deletion of either domain 1 or 4
(data not shown). By contrast, deletion of both domains 1 and 4 almost
completely abrogated adhesion of 38-
cells (Fig. 1A), indicating that binding of integrin
to VCAM-1 requires the presence of
either domain 1 or 4. Structural integrity of the
1.4 mutant was
confirmed by reactivity with several mAbs specific for epitopes present
in domains 2, 3, or 6 of VCAM-1 (data not shown). Consistent with
previous reports on the domain requirements of
binding to VCAM-1 (11) ,
adhesion of Mn
-treated 38-
cells was
also abrogated by the
1.4 deletion, but was not affected by the
1.2.3 or
4.5.6 mutations (Fig. 1B). The
results therefore indicate that domains 1 and 4 of VCAM-1 independently
support binding of the
integrin.
Figure 1:
Domain requirements for binding of
integrin to VCAM-1. Confluent monolayers of CHO cells transfected with VCAM-7D or
1.2.3,
4.5.6, and
1.4 domain deletion mutants were
incubated with fluorescence-labeled 38-
(A)
or 38-
(B) cells in the presence of 1 mM Mn
and adherence was measured by fluorimetry. To
determine
or
integrin-specific adhesion the
percent of cells bound in the presence of mAb PS/2 was subtracted from
the percent of adherent cells treated with mAb M17/4.2. The results are
expressed as percent of specifically adherent cells and represent the
mean ± S.D. from four to six independent experiments.
Statistical analysis was performed by the Student's t test.
The
Mn titration profiles for adhesion of
and
integrins to distinct domains of VCAM-1 were further analyzed. As
is depicted in Fig. 2A, the average ED
values for binding of 38-
cells to full-length
VCAM-7D or the
1.2.3 and
4.5.6 mutants did not differ
significantly. Half-maximal adhesion was observed at 19-23
µM for each VCAM-1 construct (Table 1). By contrast,
deletion of domains 1-3 or 4-6 differentially affected
binding of 38-
cells. When compared with full-length
VCAM-7D, the average ED
values for 38-
binding to the
1.2.3 but not to the
4.5.6 mutant were
significantly increased (Fig. 2B). Direct comparison of
and
integrins revealed a significant difference (p <
0.01) between the ED
values for binding to domain 4 of
VCAM-1 (Table 1). The average ED
value for
integrin was 23.0 ± 2.2
µM, whereas the
receptor required 63.4 ± 27.9 µM Mn
for half-maximal adhesion. Similarly, on
18H18 cells the ED
values for adhesion of
integrin to domain 4 were 94.2
± 10.0 µM Mn
and 24.4 ±
5.7 µM Mn
for domain 1 binding (Table 1). These results indicate that
requires a significantly higher
level of activation than integrin
to
bind domain 4 of VCAM-1.
Figure 2:
Integrins and
require distinct levels of
activity for binding to domain 4 of VCAM-1. Adhesion of
fluorescence-labeled 38-
(A) or
38-
(B) cells to CHO cells transfected with
VCAM-7D or deletion mutants
1.2.3 and
4.5.6 was analyzed over
a wide range of Mn
concentrations. The percent of
cells bound in the presence of mAb PS/2 was subtracted from the percent
of adherent cells treated with mAb M17/4.2 for each Mn
concentration and adherence was measured by fluorimetry. The
ED
values for
integrin-dependent binding
were calculated and average ED
values were obtained from
four to six independent experiments. The results are presented as mean
± S.D. Statistical analysis was performed by the Student's t test. NS = not
significant.
We next asked whether different binding
efficiencies of and
integrins for VCAM-1 domain 4 are
also detected under more physiological divalent cation conditions. When
adhesion assays were performed in the presence of Ca
and Mg
at 1.0 mM each, deletion of
VCAM-1 domains 1-3 strongly reduced binding of integrin
lymphoma lines
38-
and RAW112, but only minimally affected adhesion
of
38-
and TK1.cl14 cells (Fig. 3A). By contrast, both
and
lymphoma lines
bound to the
4.5.6 mutant with the same efficiency as to the
full-length VCAM-7D receptor (Fig. 3B). Thus, the
Mn
titration experiments revealed significant
differences in the capacity of integrins
and
to interact with domain 4
of VCAM-1 that are also relevant for adhesion in the presence of
Ca
and Mg
cations.
Figure 3:
Binding of and
integrins to VCAM-1
deletion mutants in the presence of Ca
and
Mg
cations. Adhesion of fluorescence-labeled
lymphoma lines
38-
and RAW112 (solid bars) or
cell lines
38-
and TK1.cl14 (hatched bars) to CHO cells
transfected with VCAM-1 deletion mutants
1.2.3 (A) or
4.5.6 (B) was analyzed in the presence of Ca
and Mg
at 1.0 mM each. The percent of
cells bound to CHO/CDM8 control cells was subtracted from the percent
of cells adherent to CHO/VCAM-7D, CHO/
1.2.3, or CHO/
4.5.6
transfectants. Adhesion to VCAM-1 deletion mutants is given as percent
of VCAM-7D binding for each lymphoma line. The results were obtained
from four to six independent experiments and are presented as mean
± S.D.
In control
experiments, the Mn requirement for binding of
integrin to fibronectin was
analyzed. When compared with recombinant soluble VCAM-1, plateau levels
of fibronectin binding were similar (61.7 ± 5.8 versus 59.7 ± 5.5% specific adhesion; p > 0.05),
whereas the average ED
values were significantly increased
(18.5 ± 7.7 versus 70.3 ± 27.2 µM Mn
; p < 0.01). Therefore these data
demonstrate that distinct activity levels of
integrin can be distinguished by
Mn
titration experiments.
Figure 4:
Expression of CD24 on lymphoma lines and
transfectants. Cell lines 38-, 38-
,
N232.18, 18H18, RAW112, and TK1.cl14 were incubated with mAb J11d.2
specific for CD24 (solid lines) or an isotype-matched control
mAb directed against the T cell receptor V
14 segment (dotted
lines) followed by FITC-conjugated rabbit-anti-rat Ig
serum.
The Mn titration profiles for adhesion of N232.18 and 18H18 cells to
VCAM-1 domains 1 and 4 were further analyzed. Similar to the results
obtained with 38-
cells, the average ED
values for binding of 18H18 transfectants to full length VCAM-7D
or the
4 mutant did not differ, whereas half-maximal adhesion to
the
1 mutant required significantly higher Mn
concentrations (Fig. 5A). By contrast, binding of
CD24-deficient N232.18 cells to VCAM-1 was in addition sensitive to
deletion of domain 4. As is shown in Fig. 5B, the
average ED
values for binding to the
1 as well as the
4 mutants were significantly increased. When N232.18 and 18H18
cells were compared directly, a clear difference between the ED
values required for binding to the
4 (p <
0.0001) but not to the
1 mutant (p > 0.05) was
observed. The average ED
value for binding of 18H18
transfectants to CHO/
4 cells was 24.4 ± 5.7
µM, whereas N232.18 cells required 118.7 ± 20.1
µM Mn
for half-maximal adhesion (Table 1). It therefore appears that the activation state of
for adhesion to domain 1 of VCAM-1
is controlled by CD24.
Figure 5:
Integrin binding to VCAM-1 domain 1 is regulated by CD24. Adhesion of
fluorescence-labeled CD24
18H18 transfectants (A) or CD24-deficient N232.18 cells (B) cells to CHO
cells transfected with VCAM-7D or deletion mutants
1 and
4
was analyzed over a wide range of Mn
concentrations.
The percent of cells bound in the presence of mAb PS/2 was subtracted
from the percent of adherent cells treated with mAb M17/4.2 for each
Mn
concentration and adherence was measured by
fluorimetry. The ED
values for
integrin-dependent binding were
calculated and average ED
values were obtained from three
to five independent experiments. The results are presented as mean
± S.D. Statistical analysis was performed by the Student's t test. NS = not
significant.
Previous studies have shown that structurally related
integrins may interact with non-homologous binding sites present in
matrix proteins or cellular adhesion receptors. For example, LFA-1 and
Mac-1 recognize distinct domains of ICAM-1(30) , and
fibronectin contains separate binding sites for VLA-4 and
VLA-5(31) . For binding of integrin
to its alternate counter-receptor
VCAM-1, however, epitopes present in the homologous domains 1 and 4 are
of critical
importance(4, 5, 6, 7, 8, 9, 10) .
In the present study, domain deletion mutants of VCAM-1 were used to
localize the binding sites for integrin
. The results indicate that integrins
and
share the domain requirements for adhesion to VCAM-1, as binding
of both integrins was almost completely abolished by simultaneous
deletion of domains 1 and 4. Structural integrity of the
1.4
mutant was confirmed by reactivity with several mAbs specific for
VCAM-1 domains 2, 3, or 6. Amino acid residues in domains 1 and 4 have
been identified that are critical to integrin
binding(6, 8, 9) . It will be
interesting to determine whether the same residues are also involved in
interactions of
with VCAM-1.
Incubation of cells with Mn induces the high
avidity conformation of
integrins even in the absence
of other activating signals(29, 32) . Studies on
mutant integrin
subunits indicated that distinct
levels of integrin binding activity can be distinguished by the
concentrations of Mn
required for half-maximal
adhesion(29) . These studies also showed that ED
values are independent of integrin expression levels and the
overall adhesive state of the cell(29) . The results presented
here extend these findings, as differences in the Mn
titration profiles revealed a critical role of integrin
heterodimer composition and CD24 expression for differentially
regulating binding of
and
integrins to domains 1 and 4 of
VCAM-1. Importantly, a clear difference in the capacity of
and
integrins to bind domain 4 of VCAM-1 was also detected in the
presence of Ca
and Mg
cations.
Taken together, these data suggest that Mn
titration
experiments may be applied to determine integrin activity states
necessary for ligand binding.
The activity state of integrins can be
influenced by numerous factors. Integrin phosphorylation (33, 34, 35) and glycosylation(36) ,
structural elements of and
subunit cytoplasmic
tails(37, 38, 39) , divalent cation sites of
subunits(29) , expression of CD24(27) ,
composition of membrane phospholipids(40) , lipid
factors(41) , putative signal transduction
molecules(42) , and unknown cell type-specific factors (29) , all have been suggested to regulate the level of
integrin activity. Whereas most of these mechanisms appear to alter
ligand binding activity of integrins in general, the results reported
here identify mechanisms that differentially modulate the binding
activity of
integrins for homologous binding sites in
VCAM-1. It was demonstrated that integrin
binds much more efficiently to domain 4 of VCAM-1 than
, whereas both integrins showed the
same binding activity for domain 1. Previous results showing that
phorbol 12-myristate 13-acetate induces binding of
integrin to domain 4, but not to
domain 1, on human U937 cells support these findings(11) . In
contrast to the effects of integrin heterodimer composition, the
activation state of
integrin
required for binding to domain 1 but not domain 4 was modulated by CD24
expression. Interestingly, flow cytometry analysis revealed that low
levels of CD24 expressed by 18H18 transfectants are sufficient to
enhance domain 1 adhesion of
integrin. Thus, the regulatory effects of CD24 appear to be
largely independent of its cell surface expression levels. Since in
these experiments defined sets of B lymphoma transfectants were used
that differ exclusively in the expression of
and
subunits (38-
and 38-
cells) or CD24 (N232.18 and 18H18 cells), it seems highly
unlikely that cell-specific differences can account for these results.
In summary, we conclude from these data that
and
subunits are critical for modulating
integrin binding to domain 4 of VCAM-1, whereas adhesion of
to domain 1 is regulated by CD24.
Triggering of lymphoid cells or monocytes through
may regulate gene expression and
tyrosine phosphorylation of a 105-kDa protein, induce or inhibit cell
death, deliver co-stimulatory signals for T cell proliferation, and
promote cell migration(3) . As the full-length VCAM-1 receptor
expresses two potential binding sites for both
(4, 5) and
integrin (Fig. 1),
interaction of a single versus two integrin molecules with
VCAM-1 may affect the strength of adhesion as well as signal
transmission to adherent leukocytes. Therefore, mechanisms such as
those described here that modulate binding to domains 1 and 4
differentially may be critical to controlling the intensity and/or
chemical nature of signals and consequently the cellular response to
integrin occupancy.
Previous studies have shown
that the loss of CD24 expression on pre-B cells is associated with a
strongly reduced binding activity of integrin to fibronectin(27) . However, the same cells
still bound to TNF-stimulated endothelioma cells via
integrin. Whereas adhesion of
CD24
transfectants was blocked by anti-VCAM-1 mAb
MK/2.7, binding of CD24-deficient cells was not sensitive to inhibition
by mAb MK/2.7(27) . These data suggested that cells lacking
CD24 may interact with a ligand distinct from VCAM-1 on TNF-stimulated
endothelium. Alternatively,
on
CD24-deficient cells may interact with alternate domains in VCAM-1 not
inhibited by the domain 1 + 2-specific mAb MK/2.7. However, our
results demonstrate that VCAM-1 binding of CD24-deficient N232.18 cells
was completely dependent on the presence of domains 1 and 4 (not
shown), and binding activity for domain 4 was decreased as compared
with CD24
transfectants. Hence, it is highly unlikely
that
-mediated binding of N232.18
cells to TNF-stimulated endothelium results from VCAM-1 interactions
independent of domain 1. We therefore suggest that alternate
endothelial ligands for
integrin may
exist. Consistent with this interpretation, it has been reported that
the adhesion of certain human B and T lymphoma cells to TNF-stimulated
umbilical vein endothelial cells is completely inhibited by
anti-
integrin mAb but only partially by mAbs directed
against VCAM-1(5) . The existence of alternate endothelial
ligands may have important implications for current efforts to
understand and manipulate
integrin
functions in vivo.