(Received for publication, August 9, 1995)
From the
integrin (VLA-4) appears to
be unique among the leukocyte integrins in that it can initiate the
adhesion of circulating lymphocytes without cellular activation. It is
not known how lymphocytes or other cell types maintain constitutive
levels of
integrin activity. The
current report describes a monoclonal antibody, 15/7, that recognizes a
high affinity or ligand-occupied conformation of
integrin. Studies with 15/7 revealed that
integrin-dependent adhesion of
leukocytic cell lines is mediated by a population of low affinity
receptors that is conformationally responsive to ligand; the 15/7
epitope could be induced by nanomolar concentrations of soluble VCAM-1
or by micromolar concentrations of a peptide derived from the type III
connecting segment domain of fibronectin (as ligands for
integrin). The same receptors were
also responsive to adhesion activating reagents, such as
Mn
, activating anti-
integrin
antibodies, and phorbol myristate acetate, which induced the 15/7
epitope directly and/or decreased the concentration of ligand required
for epitope induction. In addition to the responsive receptor pool,
cells expressed a second population of
integrin that was conformationally
restrained, failing to respond to ligand or to any of the activating
reagents. The relative size of the responsive and inactive receptor
pools, as well as the affinity of the responsive receptors, represented
a stable phenotype of different cell types and played important roles
in defining the cells' adhesive capacity and ligand specificity.
Similar receptor populations were measured on lymphocyte subsets in
whole blood. These studies provide insight into how cells maintain
different constitutive levels of
integrin activity, and how the activity of
integrin can be modulated by activators of cell adhesion.
Integrins are heterodimeric adhesion molecules that contribute
to the specificity of cellular interactions through the recognition of
numerous matrix and cell-associated ligands(1) . Importantly,
the ligand binding activity of integrins can be modulated rapidly,
allowing cells to specify the timing and location of integrin-mediated
adhesive interactions. On circulating leukocytes, for example, the
integrins LFA-1 and Mac-1 are thought to be activated
by site-specific factors (cytokines or other adhesive interactions)
during transient cell interactions with the vascular wall; the
receptors then establish firm adhesive contacts and mediate leukocyte
extravasation(2, 3) . Likewise, platelets are
stimulated at sites of vascular injury allowing
II
integrin to bind to fibrinogen and
initiate thrombosis(4) .
The regulation of integrin activity on circulating immune cells appears to be
different from that of the other leukocyte integrins described above.
and
integrin can mediate leukocyte adhesion to their endothelial
ligands VCAM-1 (5) and MAdCAM-1(6) , respectively,
without cellular activation, and can do so even in the presence of the
shear forces encountered in normal blood
flow(7, 8, 9, 10) . These results
suggest that circulating leukocytes maintain a constitutive level of
integrin activity.
and
integrin also bind to the
alternatively spliced connecting segment III (CS1) (
)domain
of fibronectin (FN; 11-13), but freshly isolated blood
lymphocytes require activation in order to adhere to this
ligand(7) . Cell lines maintained in culture exhibit a range of
integrin activity; some cells bind
to both VCAM-1 and the CS1 domain of fibronectin, others bind VCAM-1
but require activation to bind FN CS1, while others cannot bind FN CS1
even in the presence of activating reagents(14) .
Similar
differences in activity and ligand specificity have also been described
for other integrins, such as II
integrin and its interaction with fibrinogen and
fibronectin(15) , as well as for
integrin and its interaction with collagen and
laminin(16) . Distinct subsets of integrin can also exist
simultaneously on the cell surface that exhibit different affinities
for ligand. Interestingly,
integrin-dependent cell adhesion appears to be mediated by a large
subset of low affinity receptors(17) , while Mac-1-dependent
adhesion is mediated by a small subset of high affinity
receptors(18) . Subsets of LFA-1 have also been described that
differ not only in their state of activation but also in their ability
to be activated(19, 20) .
Changes in integrin
activity can be induced by several types of reagents in vitro,
and are associated with changes in receptor conformation. Antibodies
against integrin, such as TS2/16 and 8A2, bind to the
receptor regardless of its state of activation and induce a more active
form(21, 22, 23) . The divalent cation
Mn
also interacts directly with integrins to induce
an active conformation(24) . Although integrin-dependent
adhesion can be enhanced by cell stimulating agents such as PMA, the
effects of PMA are more complex than those of the direct integrin
activators; in some systems PMA stimulation enhances integrin
affinity(25, 26) , while in others, it causes cell
spreading without inducing receptor activation(27) . Changes in
receptor conformation have been characterized by anti-integrin
antibodies that recognize activation-dependent epitopes. These
antibodies can either inhibit adhesive function by engaging the active
binding site of the receptor(18, 28, 29) , or
promote adhesion by stabilizing an active integrin
conformation(30, 31, 32) . Another class of
antibodies recognize ligand-induced binding sites associated with
II
integrin(33) , and
document changes that occur in receptor conformation upon ligand
occupation.
Although much is known about integrin activity and
changes in receptor conformation, there is no clear understanding of
how cells modulate both integrin activity and ligand specificity, and
how cells maintain stable differences in integrin activation states. In
this report we describe a monoclonal antibody, 15/7, against an
activation/ligand-induced epitope on integrin.
Studies with 15/7 provided a model for the regulation of
integrin activity that differs from
the conventional view of integrin-mediated cell adhesion. In this
model, receptors that mediate cell adhesion under resting conditions
undergo a change in conformation in response to ligand, rather than a
change in conformation that triggers ligand binding. The
ligand-responsive receptors were also sensitive to Mn
and to activating antibodies against
integrin,
as well as to PMA. These receptors generally exhibited a low affinity
conformation (interacting reversibly with soluble ligand), but
supported cell adhesion through a multivalent interaction with
immobilized ligand. However, not all surface
integrin
could respond to ligand or to activating agents, and this subset
constituted an inactive receptor pool. The size of the inactive pool
was found to be a stable phenotype of different cells and played an
important role in determining the capacity of the cells to bind ligand,
as well as the specificity of ligands that the cells could bind.
Figure 2:
Antibody reactivity by FACS and Western
analysis. A, FACS analysis: Jurkat and THP-1 cells were
exposed to the indicated antibody for 30 min at room temperature,
washed, and exposed to PE-conjugated goat anti-mouse IgG Fc for FACS
analysis (``Materials and Methods''). In a separate
experiment it was found that integrin accounts for
the majority of
integrin expressed on both THP-1 and
Jurkat cells. The cells were exposed to
-chain-specific monoclonal
antibodies (see ``Materials and Methods'') and then to
FITC-conjugated anti-mouse Ig for FACS analysis. Fluorescence intensity
Jurkat/THP-1: IgG, 3/5; anti-
, 3/3;
anti-
, 5/6; anti-
, 9/25;
anti-
, 204/193; anti-
, 14/62;
anti-
, 25/49; anti-
integrin,
200/276. &cjs2108;, Jurkat cells; &cjs2110;, THP-1 cells. B,
Western analysis.
integrin was
isolated from a lysate of U937 cells with an anti-
integrin affinity column, subjected to SDS-PAGE (non-reduced),
and transferred to Immobilon. A separate sample of
integrin was isolated from a lysate
of U937 cells that had been surface biotinylated. Individual strips of
the blot were probed with control IgG
, 15/1, 15/7, and
TS2/16 (control anti-
integrin) at 10 µg/ml. Bound
antibody was detected with sheep anti-mouse horseradish peroxidase
(``Materials and Methods''). The strip derived from the
biotinylated cells was probed with streptavidin-horseradish peroxidase (SA) to visualize the separate
and
integrin bands. On U937 cells
integrin is expressed as the cleaved form of the molecule, as
described(48) , migrating as two bands at 70 and 80 kDa. A
small amount of uncleaved
integrin (150 kDa), as well
as a 180-kDa form described (49) is also present.
integrin runs as a 130-kDa protein.
The
inhibitory antibody against human integrin,
AN100226m, has been described(34) , and F(ab`)
fragments of the antibody were prepared by TSD BioServices
(Newark, DE). The activating antibody against
integrin, TS2/16(21, 22) , was generously
provided by Dr. F. Sanchez-Madrid (University of Madrid, Spain), as a
hybridoma supernatant (used at a dilution of 1:5), or as an ascites
(used at 1:2000). 8A2, also an activating antibody against
integrin(23) , and its F(ab`)
fragments were
kindly provided by Dr. N. Kovach (University of Washington, Seattle,
WA) as purified reagents and were used at 1 and 10 µg/ml,
respectively. The inhibitory anti-
integrin antibody,
AIIB2(35) , was kindly provided by Dr. C. Damsky (University of
California, San Francisco). Antibody TS2/7 (against
integrin) was obtained from T Cell Sciences (Cambridge, MA). Antibodies
Gi9 (against
integrin), GoH3 (against
6
integrin), K20 (against
integrin), and
FITC-conjugated K20 were purchased from Immunotech, Inc. (Westbrook,
ME). Antibodies P1B5 (against
integrin) and P1D6
(against
integrin) were obtained from Life
Technologies, Inc. (Grand Island, NY). The hybridoma cell line
secreting TS1/18, against
integrin, was obtained from
the ATCC (#HB203). 15/1, 15/7, AN100226m, and TS1/18 were grown as
ascites, and purified by ammonium sulfate precipitation and fast
protein liquid chromatography. The purified antibodies contained less
than 1 unit of endotoxin/mg of protein. 15/10 was used as a hybridoma
supernatant diluted 1:4. For some experiments, 15/7 was conjugated to
fluorescein isothiocyanate (FITC; Sigma) according to the
manufacturer's instructions and used within 30 days.
FITC-conjugated goat anti-mouse IgG, FITC-conjugated goat anti-rat IgG,
and FITC-conjugated mouse IgG (MOPC-21; negative control) were obtained
from Sigma. FITC-conjugated goat anti-human IgG was obtained from
Vector Laboratories, Inc. (Burlingame, CA), and PE-conjugated goat
F(ab`)
anti-mouse IgG Fc was obtained from Immunotech
(Westbrook, ME). FITC-conjugated CD45Ro was obtained from DAKO
(Denmark).
Recombinant
soluble VCAM-1 was expressed as a fusion protein with the heavy chain
of human IgG. The construct carried the seven
immunoglobulin domains of VCAM-1 on the N terminus (to phenylalanine
697) and the Fc domain of human IgG
at the C terminus
(including the hinge, CH2, and CH3 regions; the hinge region cysteine,
normally disulfide-bonded to the light chains, was mutated to
arginine). The cDNA for the soluble construct was ligated into pVL.MCS,
a derivative of pVL1393 (40) , and the plasmid was designated
pVL.sVCAM.Fc. Recombinant baculovirus was generated by co-transfecting
Sf9 cells with 3 µg of pVL.sVCAM.Fc plasmid DNA and 1 µg of
BaculoGold
viral DNA (Pharmingen) and plaque-purified by
visual screening. High titer virus (10
plaque-forming
units/ml) was used to infect Trichoplusia ni High Five
cells (Invitrogen, San Diego, CA). The supernatant 72 h
post-infection was collected and sVCAM-1-IgG was affinity-purified by
Protein A-Sepharose chromatography. Its purity was established by
SDS-PAGE and silver staining.
Whole human fibronectin, rat laminin, and the peptides GRGDSP and GRGESP were purchased from Life Technologies, Inc. An eight amino acid peptide (EILDVPST), derived from the CS1 region of fibronectin, was prepared (430 ABI peptide synthesizer) and purified by high performance liquid chromatography. For adhesion studies, the same peptide was synthesized with a linker sequence on the N terminus (CGGG) and was conjugated to rabbit serum albumin as described by Wayner and Kovach (41) . Recombinant soluble human ICAM-1 was a generous gift from Dr. Mary Perez (Wyeth-Ayerst, Princeton, NJ).
Figure 5:
The effect of 15/7 on THP-1 cell adhesion
to different substrates. Cells were treated with the indicated
-chain-specific antibodies for 30 min on ice, then added to wells
coated with purified laminin, fibronectin, ICAM-1 (each at 500
ng/well), or with membrane VCAM-1 (3 ng/well). The binding assay was
carried out in the presence of no additions, 1 mM Mn
, or 5 µg/ml 15/7 (which were added at the
time of the adhesion incubation) for 30 min at room
temperature.
Figure 1:
THP-1 and Jurkat adhesion to VCAM-1. A, THP-1 and Jurkat cells were added to wells that had been
coated with recombinant VCAM-1, and allowed to adhere for 30 min at
room temperature. , Jurkat cells;
, THP-1. B,
THP-1 cells were pretreated with 8A2, Mn
(1.5
mM), or with no additions for 30 min on ice and then added to
recombinant VCAM-1-coated wells. Another set of cells was added to
wells containing PMA (50 nM). The cells were allowed to adhere
for 30 min at 37 °C. It was found in separate experiments that
adhesion of both cell types was completely inhibited by
anti-
integrin at all densities of VCAM-1 examined.
, no stimulation;
, PMA;
, Mn
;
, 8A2.
Figure 10:
Induction of the 15/7 epitope on whole
blood lymphocytes by LDV peptide. Samples of freshly isolated whole
human blood (containing heparin as an anticoagulant) were incubated for
5 min at 37 °C with or without PMA (50 nM). Samples of the
blood were then exposed to 15/7 (10 µg/ml) in the presence of LDV
peptide (at the indicated concentration) for 30 min at room temperature
and processed for double color FACS analysis with anti-CD45Ro
(``Materials and Methods''). --,
CD45Ro
; -
-, CD45Ro
;
, PMA,
CD45Ro
;
,
PMA, CD45Ro
.
THP-1 and
Jurkat cells were used as the basis of a screen to identify potential
activation epitopes associated with integrin; it was expected that such an epitope would be expressed
at higher levels on Jurkat cells in correspondence to their higher
level of VCAM-1 binding activity (both cell types expressed similar
levels of
integrin). Antibodies were
raised against purified
integrin
(see ``Materials and Methods'') and their ability to
recognize THP-1 and Jurkat cells was examined by FACS analysis; the
reactivity of three of these antibodies is shown in Fig. 2(15/1, 15/7, and 15/10) compared to that of control
reagents against
and
integrin (all
antibodies were mouse IgG
). Jurkat and THP-1 cells
expressed similar levels of
integrin, while THP-1
cells expressed higher levels of the
integrin
subunit. Of the 70 reactive antibodies raised against purified
integrin, only 15/7 produced a
staining profile that was markedly different than that of the control
reagents. 15/7 reacted at low levels with Jurkat cells, and even lower
levels with THP-1 (fluorescence intensity of 24 and 2 units above
control IgG background, respectively). Interestingly, addition of the
integrin activating reagent Mn
(1.5 mM)
caused a 10-fold increase in the expression of the 15/7 epitope on both
cell types (fluorescence intensity of 253 and 22 for Jurkat and THP-1
cells, respectively), but did not affect the reactivity of the other
antibodies (not shown). Western analysis indicated that 15/7 recognized
the
integrin subunit (Fig. 2B). Even
though THP-1 cells expressed higher levels of
integrin than Jurkat, they expressed much lower levels of the
15/7 epitope (in the presence or absence of Mn
); this
difference could not be explained by expression of different
-chain subunits, since both cell types expressed similar levels of
integrin, and expression of
integrin could account for the majority of
integrin on both cell types (see Fig. 2, legend). Neither
THP-1 nor Jurkat cells expressed
integrin (not
shown).
The sensitivity of the 15/7 epitope to Mn,
and the higher expression levels of the epitope on Jurkat than on THP-1
cells suggested that 15/7 recognized an activation epitope associated
with
integrin. In order to examine this possibility
further, the antibody was conjugated to FITC and incubated with Jurkat
and THP-1 cells in the presence of different activators of
integrin (Fig. 3). Consistent with the results above,
15/7-FITC reacted with resting Jurkat cells better than with THP-1,
although reactivity with both cell types was low. Treatment of the
cells with PMA produced a small increase in expression of the 15/7
epitope, while higher expression levels were induced by Mn
(1.5 mM), and by two activating antibodies against
integrin, TS2/16 and 8A2 (tested at saturating
concentrations). 8A2 induced the highest expression levels of the 15/7
epitope, representing a 10-fold increase above resting levels for both
cell types. The inductive effects of Mn
and TS2/16
were additive, resulting in an expression level of the 15/7 epitope
equivalent to that produced by 8A2 alone. Mn
did not
affect the already high expression levels of the epitope induced by
8A2. In contrast to the activating antibodies, the inhibitory antibody
against
integrin, AIIB2, completely eliminated the
15/7 epitope. A control antibody against
integrin
(K20), that has no effect on
integrin adhesive
function, did not significantly affect expression of the 15/7 epitope.
15/7-FITC reactivity with the cells was specific since it was
effectively competed by excess unlabeled 15/7. Mn
,
PMA, and the activating antibodies against
integrin
influence receptor activity through distinct mechanisms; therefore,
these results suggest that 15/7 recognizes an epitope associated with
the activation state of
integrin, rather than a
conformation particular to a given activating agent.
Figure 3:
Modulation of the 15/7 epitope by reagents
that affect integrin-dependent cell adhesion. THP-1
and Jurkat cells were exposed to 15/7-FITC or IgG-FITC (each at 10
µg/ml) in the presence of saturating concentrations of the
indicated antibodies for 30 min at room temperature; Mn
was tested at 1.5 mM. One set of samples was pretreated
with PMA (50 nM) for 5 min at 37 °C, while another was
preincubated with excess unlabeled 15/7 (100 µg/ml) in the presence
of Mn
for 30 min at room temperature (XS 15/7). After
incubation with the FITC reagents, the cells were washed and examined
by FACS analysis. Inset, THP-1 and Jurkat cells were treated
with 15/7 in the presence of the indicated concentrations of
Mn
for 30 min at room temperature. The cells were
washed and exposed to FITC-conjugated goat anti-mouse IgG
for FACS analysis.
Regardless of
how the cells were stimulated, THP-1 cells exhibited 3-10-fold
lower levels of the 15/7 epitope than Jurkat cells, even though THP-1
cells expressed higher levels of integrin (Fig. 2). This result was not due to limiting quantities of the
activating reagents since the stimulatory antibodies were tested at
concentrations above saturation. Furthermore, the responsive receptors
on the two cell types demonstrated nearly identical sensitivity to
Mn
(excitatory concentration for half maximal
expression, or EC
= 0.7 mM); higher
concentrations of Mn
could not compensate for low
expression of the 15/7 epitope on THP-1 cells (Fig. 3, inset). These results indicate that THP-1 cells exhibited a
lower number of activation-responsive receptors than Jurkat cells.
Figure 4: THP-1 and Jurkat cell adhesion to FN CS-1 peptide. THP-1 and Jurkat cells were pre-exposed to the indicated reagent for 5 min at 37 °C and then added to wells coated with a saturating level of FN CS1 peptide-albumin conjugate (3 µg/ml). Adhesion was allowed to occur for 30 min at room temperature. &cjs2108;, Jurkat; &cjs2109;, THP-1 cells.
Figure 6:
Induction of the 15/7 epitope by soluble
VCAM-1. Jurkat and THP-1 cells were treated with F(ab`) fragments of anti-
integrin (AN100226m; 10
µg/ml), or with no additions for 30 min on ice. The cells were then
exposed to 15/7 (10 µg/ml) in the presence of recombinant soluble
VCAM-1 (at the indicated concentration) for 30 min at room temperature,
washed, and exposed to PE-conjugated goat anti-mouse Fc (in the
presence of human serum) to detect 15/7 by FACS analysis.
-
-, Jurkat;
, Jurkat +
anti-
integrin; -
-, THP-1;
, THP-1 +
anti-
integrin.
Figure 7:
Induction of the 15/7 epitope by LDV
peptide. THP-1 () and Jurkat (
) cells were treated with
15/7 (10 µg/ml) and LDV peptide (at the indicated concentration)
for 30 min at room temperature in the presence of F(ab`)
fragments of 8A2 (-
-
-
, 5 µg/ml),
Mn
(- - -, 1 mM), PMA (
,
50 nM), or no additions(-). The cells with PMA were
preincubated for 5 min at 37 °C. The cells were washed and exposed
to PE-conjugated goat anti-mouse Ig Fc for FACS
analysis.
Also
shown in Fig. 7, 15/7 could be used to measure the relative
number and affinity of receptors that were available for ligand
occupancy in the presence of three different activators of cell
adhesion. While Mn induced the 15/7 epitope by itself
(as shown in Fig. 3), it further potentiated the response of
receptors to LDV peptide (EC
Jurkat with Mn
= 2 µM; THP-1 = 5 µM).
Interestingly, Mn
did not change the overall number
of receptors responsive to LDV peptide, suggesting that Mn
and the peptide interact with the same receptor population. In
contrast, 8A2 increased both the sensitivity of receptors to LDV
peptide, as well as the number of receptors that expressed the 15/7
epitope at peptide saturation; in the presence of 8A2, receptors on
both Jurkat and THP-1 cells exhibited an EC
of 2
µM for the peptide, while fluorescence at saturation
increased 30 and 100% on the two cell types, respectively. PMA
increased the apparent affinity of receptors on THP-1 cells by 5-fold,
but did not affect receptor affinity on Jurkat cells. Thus, in the
presence of PMA, both cell types exhibited the same apparent affinity
for the peptide ligand (EC
= 20 µM).
PMA did not significantly increase the number of responsive receptors
on either cell type; Jurkat cells continued to express 4-fold higher
levels of the 15/7 epitope than THP-1 cells in the presence of PMA. Table 1summarizes the EC
for induction of the 15/7
epitope on Jurkat and THP-1 cells in the presence of the activating
reagents.
Figure 8:
15/7 stabilizes an active conformation of
integrin induced by Mn
or by LDV peptide. A, FACS analysis. THP-1 cells were
treated with Mn
(1.5 mM), LDV peptide (500
µM), or no additions for 30 min on ice. Half of the cells
in each sample were washed (&cjs2108;) free of the reagents, while the
remaining cells were left unwashed (&cjs2110;). Both sets of cells were
exposed to 15/7 (10 µg/ml) for 30 min on ice, and then to
PE-conjugate goat anti-mouse Ig Fc for FACS analysis. B, cell
adhesion. THP-1 cells were treated with the following reagents either
individually or in combination (as indicated in the figure) for 30 min
on ice: 15/7 (10 µg/ml), Mn
(1.5 mM),
LDV peptide (500 µM), anti-
integrin
(AN100226m; 5 µg/ml), or TS2/16 (5 µg/ml). The cells were
washed quickly or left unwashed, as in A, and immediately
tested for their ability to bind to purified membrane VCAM-1 (3
ng/well) as described under ``Materials and
Methods.''
Figure 9:
Mn activates receptors
that normally mediate cell adhesion. THP-1 cells were treated with a
combination of 15/7 (10 µg/ml) and Mn
(1.5
mM), or with no additions for 15 min at room temperature. The
cells were washed rapidly, resuspended in buffer with or without
recombinant soluble VCAM-1 (30 nM), and tested immediately for
their ability to bind to purified membrane VCAM-1 (plated at the
indicated concentration). The adhesion incubation was allowed to occur
for 15 min at room temperature.
, no treatment;
, rsVCAM-1;
, 15/7 + Mn
;
, 15/7 +
Mn
with rsVCAM-1.
Several conclusions can be drawn from the studies with 15/7,
and are illustrated in the model shown in Fig. 11. 1) 15/7
recognized an activation epitope on integrin that was
strongly associated with the adhesive capacity of cells. The antibody
promoted
integrin-dependent cell adhesion by
recognizing and directly stabilizing an active conformation of the
receptor. 2) 15/7 identified both high affinity and ligand-occupied
integrin, and provided evidence that these receptor
populations share a similar conformation. 3) Studies with 15/7
indicated that there are at least three subsets of
integrin on the surface of resting cells: constitutively active
or high affinity, transiently active or low affinity, and receptors
that are inactive or resistant to activation. 4) The low affinity
receptors were available for interaction with ligand, were responsive
to Mn
and activating antibodies, and were the
receptors that normally mediated cell adhesion when ligand was present
at sufficient density. 5) The size of the low affinity receptor pool,
and its relative affinity for ligand, was a distinct phenotype of THP-1
and Jurkat cells and represents a mechanism by which
integrin-mediated cell adhesion is
likely to be regulated by cells in the circulation.
Figure 11:
Subsets of integrin: a model. Three subsets of
integrin were identified in studies
with 15/7. One receptor population (shown in the middle) interacted
transiently with ligand to express the 15/7 epitope, and mediated cell
adhesion to immobilized ligand when it was present at sufficient
density. Receptors within this pool also responded to Mn
and to activating antibodies against
integrin
to exhibit the 15/7 epitope and a high affinity conformation.
Stimulation of cells with PMA increased the sensitivity of these
receptors to ligand, but did not induce the highest affinity form. It
is possible that receptors within this pool normally exhibit a rapid
equilibrium between active and inactive conformations, as suggested for
integrin(32) . In the
presence of Mn
or LDV peptide, 15/7 stabilized the
conformation of the responsive receptors in a high affinity form (shown
on the right). Receptors that constitutively expressed the 15/7 epitope
(in the absence of ligand or activating reagents) were present at only
low levels on Jurkat cells, and were almost absent on THP-1 cells and
on whole blood lymphocytes. Inactive receptors (shown on the left)
resisted expression of the 15/7 epitope in the presence of saturating
concentrations of peptide ligand, soluble VCAM-1, Mn
,
activating antibodies against
integrin, or PMA. The
large percentage of inactive receptors on THP-1 cells appeared to limit
their adhesive capacity, even when the affinity of receptors in the
responsive pool was increased by Mn
or
PMA.
The ligand occupied forms of other integrins may also
exhibit an active conformation, since it has been shown that peptide
ligand can induce the activity of purified or fixed
II
integrin(33) . In
addition, 15/7 has characteristics similar to antibody 24, which
recognizes an activation epitope associated with the
integrins(30) . Following transient cell stimulation with
PMA, antibody 24 engages LFA-1 in the presence of ligand (ICAM-1 on
adjacent lymphocytes) and prevents the normal decline in receptor
activity. However, since LFA-1 on lymphocytes cannot interact with
ICAM-1 in the absence of cell stimulation(43) , its activity is
likely to be regulated in a manner different than that of
integrin.
The second population of
receptors was detected by 15/7 in the presence of saturating
concentrations of peptide ligand or Mn and is likely
to correspond to the population of ``low affinity'' receptors
that have been described in other reports(17, 27) .
These receptors were required for cell adhesion to VCAM-1, as shown in Fig. 9. Resting THP-1 cells bound well to VCAM-1 when the ligand
was plated at a sufficient density, and binding was not affected
significantly by soluble VCAM-1. These results suggest that the
affinity of the receptors that normally mediate THP-1 cell adhesion is
too low to form a stable association with soluble VCAM-1. Presumably,
such low affinity receptors can mediate cell adhesion to immobilized
ligand through multivalent interactions. When the
Mn
-responsive receptors were held active by 15/7,
soluble VCAM-1 was able to completely inhibit even the resting levels
of cell adhesion. These results indicate that Mn
affects the population of receptors that are normally available
for ligand binding. These receptors do not constitutively express the
15/7 epitope, nor do they exhibit a stable high affinity conformation
for interaction with ligand, and, therefore, are not normally occupied
by soluble VCAM-1; however, these receptors can initiate or mediate
cell adhesion when ligand is present at sufficient density. Recently,
it has been shown that in vivo administration of recombinant
soluble VCAM-1, at initial circulating levels of 100-200
nM, will inhibit the onset of experimental diabetes, even
though interaction of the construct with lymphocytes in the circulation
could not be measured(44) . Based on the results shown in Fig. 6, it is likely that the majority of low affinity
integrin receptors were occupied by
the VCAM-1 construct at this concentration, but that the interaction of
the construct with the lymphocytes could not be measured by indirect
FACS analysis (which involves multiple cell washings).
The third
population of receptors on the cell surface appeared to be
conformationally restrained and unavailable for ligand interactions
regardless of ligand concentration or density. This population
represented the majority of integrin on the surface
of THP cells. In the presence of saturating concentrations of
Mn
, sVCAM-1, or peptide ligand, THP-1 cells expressed
3-10-fold lower levels of the 15/7 epitope than Jurkat cells (Fig. 3, Fig. 6, and Fig. 7), even though THP-1
cells expressed higher levels of
integrin than
Jurkat, and equivalent levels of
integrin (Fig. 2). This population of receptors resisted
expression of the 15/7 epitope even when directly engaged by the
activating antibodies against
integrin ( Fig. 3and Fig. 7). However, in lysates of THP-1 and
Jurkat cells,
integrin exhibited
equivalent levels of activity for binding immobilized VCAM-1 (not
shown). Furthermore, by Western analysis, 15/7 and the control antibody
against
integrin, 15/1, reacted equally well with the
isolated
integrin subunit (Fig. 2), regardless
of the cell type from which the receptor was obtained (not shown).
These results suggest that factors associated with the integrin
heterodimer on the cell surface, or within the cytoplasm, prevent
exposure of the 15/7 epitope and regulate receptor activity. Such
negative regulatory factors are likely to play a pivotal role in
controlling patterns of cell migration and localization by defining the
capacity of cells to respond to ligand at a given density.
The
regulation of II
integrin appears to
be different than that of
integrin,
in that subsets of
II
integrin were
not apparent on the platelet cell surface; essentially all of the
platelet receptors were responsive to RGD peptide or to PMA (45) . The existence of
integrin subsets is
likely to reflect the specialized function of the many different cell
types that express this receptor.
The activating
antibody against integrin, 8A2, affected the same
receptor population that was responsive to Mn
since
their inductive effects were not additive (Fig. 3); however, 8A2
also recruited additional receptors from the otherwise inactive pool.
In the presence of 8A2, maximal expression of the 15/7 epitope was
achieved with the same low concentrations of peptide ligand as with
Mn
(IC
= 2 µM) but
the level of response to peptide was significantly higher (100%
increase on THP-1 cells). Thus, in contrast to Mn
,
8A2 increased both the affinity and the number of receptors available
for ligand binding. These results are consistent with the finding that
8A2 induced higher levels of cell adhesion than Mn
( Fig. 1and Fig. 4).
PMA produced a small increase in
expression of the 15/7 epitope, indicating a conformational response,
or activation, of a small number of receptors (Fig. 3A). Of more significance, PMA increased the
apparent affinity of the ligand responsive receptors by up to 10-fold,
as measured by the concentration of LDV peptide required to induce the
15/7 epitope (Table 1). Regardless of the cell type and the
resting level of receptor activity, the apparent affinity of the
responsive receptors in the presence of PMA was the same; THP-1 cells,
memory, and naive lymphocytes all demonstrated an EC of
20 µM LDV peptide. It is interesting that the
receptors on nonstimulated Jurkat cells already exhibited this level of
apparent affinity and showed minimal response to PMA. Mn
or the activating antibodies against
integrin
increased the apparent affinity of
integrin on both THP-1 and Jurkat cells to a level that was
10-fold higher than that induced by PMA (EC
= 2
µM peptide). These results indicate that although PMA
induced low levels of the 15/7 epitope and increased receptor affinity
to a certain level, the receptors still exhibited a low affinity
conformation; separate experiments have demonstrated that these
receptors will not form stable association with soluble VCAM-1 (not
shown). Mn
and the activating antibodies directly
induced high levels of the 15/7 epitope as well as a high affinity
receptor conformation, and were found to promote stable association
with soluble VCAM-1 ( Fig. 9and not shown). It is possible that
the differing effects of PMA that have been reported for different
integrin systems (see Introduction) may reflect its ability to increase
receptor affinity without inducing the highest affinity receptor form.