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(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
The inducible vascular cell adhesion molecule-1 (VCAM-1) ( 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
In the present study
we show that
The C3H/He
B cell lymphoma 38C13 was demonstrated to express membrane-bound
38-
Figure 1:
Domain requirements for binding of
integrin
The
Mn
Figure 2:
Integrins
We next asked whether different binding
efficiencies of
Figure 3:
Binding of
In control
experiments, the Mn
Figure 4:
Expression of CD24 on lymphoma lines and
transfectants. Cell lines 38-
The Mn
Figure 5:
Integrin
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
Incubation of cells with Mn The activity state of integrins can be
influenced by numerous factors. Integrin phosphorylation (33, 34, 35) and glycosylation(36) ,
structural elements of Triggering of lymphoid cells or monocytes through
Previous studies have shown
that the loss of CD24 expression on pre-B cells is associated with a
strongly reduced binding activity of
Volume 270,
Number 11,
Issue of March 17, 1995 pp. 5979-5984
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Integrin-dependent Binding to Domains 1
and 4 of Vascular Cell Adhesion Molecule-1 (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
)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) . integrin adhesion to fibronectin and
TNF-stimulated endothelioma cells(27) . 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.
Antibodies and Cell Lines
Antibodies used
included rat-anti-murine integrin , PS/2 (ATCC,
Rockville, MD), rat-anti-murine LFA-1, M17/4.2 (ATCC),
mouse-anti-human VCAM-1 domain 1, 4B2(11) , mouse-anti-human
VCAM-1 domain 6, 15D10(11) , rat-anti-murine CD24, J11d.2
(ATCC), and rat-anti-murine T cell receptor V14 (Dianova, Hamburg,
Federal Republic of Germany). All antibodies were used at saturating
concentrations as determined by flow cytometry analysis. 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) .Stable Expression of VCAM-1 Deletion Mutants
CHO
cells were co-transfected with pCDM8 plasmids containing deletion
mutants of the human VCAM-7D receptor lacking domains 1-3
(
1.2.3), domains 4-6 (4.5.6), domains 1 and 4
(1.4), domain 1 (1), or domain 4 (4) and with the
plasmid pLXSN carrying the neomycin resistance gene. Clones expressing
mutant VCAM-1 receptors were identified by flow cytometry analysis
subsequent to selection in G418. Construction of VCAM-1 domain deletion
mutants has been described(11) .Flow Cytometry Analysis
Cells were incubated with
saturating amounts of mAbs, washed, and stained with FITC-conjugated
rabbit F(ab)
fragments reacting with mouse or rat Ig
(Dianova, Hamburg, Federal Republic of Germany). Incubations were
performed for 20 min at 4 °C in the presence of 0.05%
NaN
. Cells were fixed with 1% paraformaldehyde and analyzed
on an EPICS Elite cytometer (Coulter Corp., Hialeah, FL).Adhesion Assays
CHO transfectants (2-3
10/well) were added to 96-well plates and allowed
to grow for 24 h prior to the assay. Confluent cell monolayers were
washed with PBS and fixed for 10 min at room temperature with 0.05%
glutaraldehyde. Recombinant soluble VCAM-1 was coated at 0.3
µg/well in PBS for 16 h at 4 °C. Plates were washed extensively
with PBS containing 1 mM EDTA and subsequently with divalent
cation-free cell adhesion buffer (24 mM Tris, pH 7.4,
containing 137 mM NaCl, 2.7 mM KCl, 2 mM glucose, and 1% bovine serum albumin). 38- and
38- lymphoma cells were labeled for 30 min at 37
°C with 12 µg/ml H33342 dye (Calbiochem) in RPMI 1640
containing 1% bovine serum albumin and washed twice with PBS 1 mM EDTA. Cells (8 10
/ml) were resuspended in cell
adhesion buffer supplemented with various concentrations of
Mn and saturating amounts of mAb PS/2 or M17/4.2. For
some experiments, cells were resuspended in adhesion buffer containing
1.0 mM Ca and 1.0 mM Mg instead of Mn. After incubation for 10 min at
room temperature cells (100 µl/well) were added to the plates and
centrifuged for 10 min at 10 g. Cells were allowed to
adhere for 30 min at 37 °C, and nonadherent cells were removed by
inverse centrifugation for 10 min at 50 g. Adhesion
assays were quantified by fluorimetry using a Cytofluor 2300
(Millipore, Bedford, MA). Specific adhesion of 38- or
38- cells was determined by subtracting the percent of
cells adherent in the presence of mAb PS/2 from the percent of adherent
cells treated with mAb M17/4.2. mAb PS/2 has been shown previously to
completely inhibit ligand binding functions of murine
and integrins(15) .
VCAM-1 Binding Sites for Integrin
Both
and integrins function as lymphocyte receptors for
VCAM-1(12, 13, 14, 15, 16) .
For integrin to bind VCAM-1,
epitopes present in domains 1 and 4 are
required(4, 5, 6, 7, 8, 9, 10) .
To identify VCAM-1 domains interacting with the
integrin, CHO cells were stably
transfected with pCDM8 expression plasmids encoding VCAM-1 deletion
mutants lacking domains 1-3 (1.2.3), domains 4-6
(4.5.6), domains 1 and 4 (1.4), domain 1 (1), or domain
4 (4). Immunofluorescence staining and flow cytometry analysis
using mAbs specific for domains 1 or 6 of VCAM-1 showed that the
VCAM-7D receptor and deletion mutants were expressed at similar levels
on CHO transfectants (data not shown). 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.
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 Binding of
To analyze Integrins to Domain 4 of
VCAM-1 Is Modulated by and Subunits and
integrin activation states required
for binding to individual domains of VCAM-1, adhesion of 38- and 38- cells was analyzed over a wide range of
Mn concentrations, and the concentrations resulting
in half-maximal adhesion (ED
values) were determined. It
has been shown previously that distinct activation states of
integrin can be distinguished by
Mn titration experiments and that ED values are independent of integrin expression levels (29) . 38- and 38- cells
differ exclusively in the expression of integrin or
subunits(15) , thus excluding cell
type-specific differences in the basal activation states of
and integrins. The Mn titration experiments reveled
that the average ED values for 38- and
38- adhesion to CHO/VCAM-7D cells did not differ
significantly (19.2 ± 6.5 µMversus 22.8
± 3.9 µM Mn, p >
0.05). In addition, similar ED values were obtained for
binding of (18.5 ± 7.7
µM Mn) and integrins (14.4 ± 8.2 µM Mn) to recombinant-soluble VCAM-7D. These
results therefore indicate that integrins and require the same level of
activation for binding to the full-length VCAM-7D receptor. 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.
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.
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.
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.
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.The Binding of
Using
N232.18 and 18H18 pre-B lymphoma cells that differ exclusively in the
expression of CD24, it has been shown previously that CD24 alters
binding of integrin Integrin to Domain 1 of VCAM-1 Is Regulated by CD24 to endothelial
cells and fibronectin (27) . Immunofluorescence staining and
flow cytometry analysis confirmed the differential expression of CD24
by N232.18 and 18H18 cells (Fig. 4) and revealed that both cell
lines do not express integrin subunits (data not
shown). Fig. 4also shows that lymphoma lines
38-, 38-, TK1.cl14, and RAW112
express high levels of CD24, with the expression on TK1.cl14 cells
being more heterogeneous than on other lymphoma lines. To investigate
the role of CD24 in regulating the VCAM-1 binding activity of
integrin, the Mn titration profiles were determined. Interestingly, the average
ED values for adhesion of CD24 18H18
transfectants to recombinant soluble VCAM-7D or CHO/VCAM-7D cells (12.4
± 5.9 and 26.5 ± 4.4 µM Mn; p < 0.01) were almost identical
to those determined for 38- cells. In addition, it was
shown that the ED values for binding of CD24-deficient
N232.18 cells to both purified and cellular VCAM-7D (31.3 ± 5.7
and 36.3 ± 6.0 µM Mn) were
increased as compared with 18H18 cells. However, the average ED values for adhesion to CHO/VCAM-7D cells did not differ
significantly and differences in binding purified VCAM-7D were of low
statistical significance (p = 0.03). Thus, the
expression of CD24 has only minor effects on the
integrin activation state required
for binding to full length VCAM-7D.
, 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 V14 segment (dotted
lines) followed by FITC-conjugated rabbit-anti-rat Ig
serum.
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.
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.
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. 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. 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. 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. 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.
)
We thank Drs. C. Evans and K. Pfeffer for critically
reading the manuscript and A. Lifka for expert technical assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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