(Received for publication, June 13, 1995; and in revised form, October 31, 1995)
From the
The very late activation antigens (VLA) or 1 integrins
mediate cell attachment to different extracellular matrix proteins and
intercellular adhesions. The ligand binding activity of these adhesion
receptors is not constitutive and can be regulated by temperature,
presence of extracellular divalent cations, stimulatory monoclonal
antibodies (mAbs), and cellular activation. We have generated three
novel mAbs, HUTS-4, HUTS-7, and HUTS-21, recognizing specific epitopes
on the common
1 subunit (CD29) of VLA integrins whose expression
correlates with the ligand binding activity of these heterodimeric
glycoproteins. This correlation has been demonstrated for several
integrin heterodimers in different cell systems using a variety of
extracellular and intracellular stimuli for integrin activation. Thus,
the presence of micromolar concentrations of extracellular
Mn
, preincubation with the activating anti-
1 mAb
TS2/16, and cell treatment with phorbol esters or calcium ionophores,
induced the expression of the HUTS
1 epitopes on T lymphoblasts.
Using a panel of human-mouse
1 chimeric molecules, we have mapped
these epitopes to the 355-425 sequence of the
1 polypeptide.
This segment represents therefore a novel regulatory region of
1
that is exposed upon integrin activation. Interestingly, binding of
HUTS mAbs to partially activated VLA integrins results in maximal
activation of these adhesion receptors and enhancement of cell adhesion
to
1 integrin ligands collagen, laminin, and fibronectin.
The 1 or VLA (
)integrins are a subgroup within
the integrin family comprising at least 10 members, each with a
distinct
subunit non-covalently associated with the common
1
subunit (reviewed by Giancotti and Mainiero(1994), Hemler (1990),
Hynes(1992), and Sánchez-Madrid and Corbi(1992)).
The VLA integrins function as cellular receptors for extracellular
matrix proteins such as different types of collagen, laminin, and
fibronectin. However, VLA-4 not only acts as a cellular receptor for
fibronectin but also mediates the interaction with its cellular ligand
vascular cell adhesion molecule-1 expressed on cytokine-activated
endothelial cells (Elices et al., 1990), and has been
implicated in homotypic cell interactions as well (Campanero et
al., 1990).
The subunits of all integrins, including the
VLA subfamily, are transmembrane glycoproteins that contain in the
N-terminal region seven homologous domains of approximately 50
residues. Three or four of these domains are putative divalent cation
binding sites with homology to the EF-hand Ca
binding
motif (Sánchez-Madrid and Corbi, 1992; Tuckwell et al., 1992). The common
1 subunit of VLA integrins is
also a transmembrane glycoprotein with a large extracellular domain
containing a 4-fold repeat of a cysteine-rich segment and a highly
conserved N-terminal segment of 200 amino acids which contains an
EF-hand-like cation binding motif (Kühn and Eble,
1994). Divalent cations are essential for regulation of integrin ligand
specificity and binding affinity. General features of the control by
divalent cations of integrin functional activity are currently
emerging. Most data indicate that Mg
and
Mn
induce activation of integrins resulting in
effective interaction with corresponding ligands (Arroyo et
al., 1993; Dransfield et al., 1992a; Kirchhofer et
al., 1990; Masumoto and Hemler, 1993). Conversely, Ca
generally exerts inhibitory effects on integrin function
(Dransfield et al., 1992a; Grzesiak et al., 1992;
Hemler et al., 1990; Masumoto and Hemler, 1993; Staatz et
al., 1989; Weitzman et al., 1993). In addition, an intact
active cellular metabolism has been shown to represent a second type of
requirement for integrin function (Campanero et al., 1990;
Dransfield et al., 1990; Marlin and Springer, 1987; Rothlein
and Springer, 1986; van de Wiel-van Kemenade et al., 1992).
Functional activation of integrins can also be experimentally
induced from outside the cells with one of the several existing
stimulatory mAbs directed against a specific or the common
integrin subunits, which seem to act by imposing a conformation of
these molecules with high affinity for ligand binding (Keizer et
al., 1988; Melero et al., 1993; van Kooyk et
al., 1991). In particular, activation of VLA integrins has been
reported by treatment with different mAbs specific for the
1
subunit (Arroyo et al., 1992; Arroyo et al., 1993;
Faull et al., 1994; Kovach et al., 1992; Luque et
al., 1994; Masumoto and Hemler, 1993; van de Wiel-van Kemenade et al., 1992).
Intracellular signals generated as a result
of cell activation also regulate the affinity and conformation of
integrins in many cellular systems. For instance, monocyte and
neutrophil activation by inflammatory mediators, such as tumor necrosis
factor-, C5a, or fMet-Leu-Phe, is required for
1 and
2
integrin-mediated adhesion to the endothelium and subsequent
transmigration across the endothelial lining (Zimmerman et
al., 1992). Thrombin-induced activation of platelets, which
involves G-proteins and protein kinase C, is accompanied by an increase
in the affinity of the
II
3 integrin for soluble fibrinogen
(Phillips et al., 1991). Similarly, the
2 integrin
leukocyte function-associated antigen-1 only mediates adhesion of T
lymphocytes to intercellular adhesion molecule-1-expressing target
cells after intracellular activation signals are induced through the
TcR/CD3 or CD2 molecules (Dustin and Springer, 1989; van Kooyk et
al., 1989). Phorbol esters such as phorbol 12-myristate 13-acetate
or phorbol 12,13-dibutyrate, which are potent and sustained activators
of protein kinase C, have been widely used in a number of cellular
systems for induction of integrin activation. At least two different
mechanisms have been implicated in the up-regulation of
integrin-mediated adhesion induced by treatment with phorbol esters.
The first one consists in the induction of transitions to a high
affinity state in a small fraction of integrin receptors, probably as a
consequence of conformational changes of these molecules. This seems to
be the case for the leukocyte integrin leukocyte function-associated
antigen-1 on T lymphocytes (Lollo et al., 1993). The second
type of mechanism by which phorbol esters stimulate integrin-mediated
cell adhesion is by altering events that occur after integrin occupancy
such as the induction of cytoskeleton-driven cell spreading (Danilov
and Juliano, 1989; Faull et al., 1994). Cell spreading as well
as the induction of other post-receptor morphological changes can
potentially favor integrin-mediated adhesion in some instances without
affecting receptor affinity. The relative contribution of these two
types of phorbol ester effects on integrin-mediated adhesion may well
depend on the cell system under consideration. In addition to protein
kinase C-mediated signaling, an increase in cytosolic free calcium has
been recently demonstrated to play a major role in integrin activation
through the use of calcium ionophores such as A23187 and ionomycin (van
Kooyk et al., 1993; Hartfield et al., 1993).
Several ``reporter'' mAbs with the ability to discriminate
between different states of integrin activation have revealed their
usefulness in many studies on the regulation of activation of integrins
belonging to the 2 and
3 subfamilies
(Cabañas and Hogg, 1993; Dransfield et
al., 1992a, 1992b; Du et al., 1991; Frelinger et
al., 1991; O'Toole et al., 1990; van Kooyk et
al., 1991, 1994). For the
1 subgroup of integrins, however,
availability of similar ``activation-reporter'' mAbs with the
capacity to distinguish between different states of activation has been
very limited.
In this report we describe a group of three mAbs,
HUTS-4, HUTS-7, and HUTS-21, recognizing epitopes in the 355-425
region of the common 1 subunit of VLA integrins, whose expression
parallels the ligand binding activity of these adhesion receptors
induced by various extracellular and intracellular stimuli.
1 Integrins were purified from Triton
X-100 lysates of surgery samples of human lung, liver, and skeletal
muscle tissues (obtained from the Department of Pathology, Hospital de
la Princesa, Madrid). These tissues were diced, sieved, and lysed in
300 ml of lysis buffer (20 mM Tris-HCl, 150 mM NaCl,
2 mM MgCl
, 200 µM MnCl
,
1% Triton X-100, 0.02% NaN
, 1 mM PMSF, 0.2
units/ml aprotinin, and 5 mM iodoacetamide, pH 8.0) for 2 h at
4 °C. The cell lysate was centrifuged at 3,000
g for 30 min at 4 °C and then ultracentrifuged at 100,000
g for 1 h at 4 °C. The lysate was precleared by
passing through a 2-ml pre-column of glycine-Sepharose CL-4B
(pre-equilibrated in lysis buffer) and then loaded onto the 3-ml column
of TS2/16-Sepharose CL-4B (pre-equilibrated in lysis buffer) at a flow
rate of 0.5 ml/min. The column was then washed sequentially with 15 ml
of lysis buffer and 15 ml of washing buffer (50 mM ethanolamine, 0.2% Triton X-100, 0.5 M NaCl, 2 mM MgCl
, 200 µM MnCl
, 1 mM PMSF, pH 10.0) and bound
1 integrins were finally eluted with
50 mM ethanolamine, 0.5 M NaCl, 2 mM MgCl
, 200 µM MnCl
, 1% octyl
glucoside, 1 mM PMSF, pH 12.0, at a flow rate of 0.5 ml/min.
Fractions of 0.5 ml were collected and neutralized with 0.1 volume of 1
mM Tris, pH 6.7. Fractions containing
1 integrins were
identified by SDS-7% PAGE followed by silver staining. The yield of
total
1 integrins was 1.5 mg in 10 ml of neutralized elution
buffer as estimated using the bicinchoninic-Protein Assay Reagent
(Pierce Chemical Co).
To demonstrate the effects
of different divalent cation conditions, T lymphoblasts were lysed in
20 mM Hepes, 150 mM NaCl, 1% Triton X-100, 1% BSA, 1
mM PMSF, pH 7.4. After centrifugation and pre-clearing,
appropriate amounts of divalent cations were added to different
aliquots of the cell lysates to yield final concentrations of
Ca (1 mM) and Mg
(1
mM) or Mn
(1 mM) prior to the 2-h
incubation with the corresponding purified mAb (5 µg). Precipitates
were subsequently removed with protein A-Sepharose as described above
and the concentrations of divalent cations were preserved throughout
the washing and processing procedures. Analysis of samples was carried
out in SDS-7% PAGE and autoradiography. Immunoprecipitations of
dissociated VLA integrin subunits after high pH treatment of cell
lysates were done essentially as described (Bednarczyk et al. 1994). Basically,
I-surface-labeled T lymphoblasts
were lysed at 3
10
cells/ml in 10 mM Tris-HCl, pH 8.0, containing 1% Triton X-100, 150 mM NaCl, 1 mM Mn
, 2% BSA, 1 mM PMSF. Aliquots of the cell lysates were mixed with a 10-fold (v/v)
excess of 10 mM Tris-HCl, 500 mM NaCl, 0.2% Triton
X-100, 1 mM Mn
, pH 8.0; or 20 mM triethylamine, 500 mM NaCl, 0.2% Triton X-100, 1 mM Mn
, pH 11.0. These mixtures were incubated at 37
°C for 30 min and then rapidly neutralized by the addition of 1/10
volume of 1.0 M Tris-HCl, pH 6.8. Immunoprecipitation of
integrin polypeptides with the respective mAbs and processing and
analysis of samples were carried out as described above.
In
cross-competitive mAb binding assays, 5 10
T
lymphoblasts were preincubated in round-bottomed wells with an excess
of unconjugated mAbs (15-20 µg/ml) for 10 min at 37 °C in
Hepes/NaCl buffer containing 0.5 mM Mn
.
Then, 2 µg of biotinylated mAbs were added to the wells and
incubation proceeded for an additional period of 15 min at 37 °C.
Unbound mAbs were then removed by washing the cells three times with
200 µl of RPMI, and 75 µl of a 1:300 dilution of fluorescein
isothiocyanate-conjugated avidin (Sigma) in Hepes/NaCl buffer were
added to the wells and incubated for 30 min at 4 °C. Finally, cells
were washed three times in PBS, fixed in 200 µl of 5% formaldehyde
in PBS, and their fluorescence determined by flow cytometry.
Figure 1:
Expression of the epitopes
recognized by mAbs HUTS-4, HUTS-7, and HUTS-21 on T lymphoblasts is
induced by Mn and correlates with
1
integrin-mediated cell adhesion. A, flow cytometry analysis of
the differential expression of epitopes HUTS-4, HUTS-7, and HUTS-21 on
T lymphoblasts depending on whether incubation is done in the absence
of extracellular divalent cations by removal with 3 mM EDTA (left panel) or in normal RPMI medium to which 1 mM Mn
has been added (right panel). B, T lymphoblast adhesion to
1 integrin ligands type I
collagen (20 µg/ml), laminin (10 µg/ml), and fibronectin (10
µg/ml) is stimulated by the addition of Mn
(1
mM) to the extracellular medium and by the presence of
anti-
1 mAb TS2/16 (5 µg/ml) and completely blocked in the
absence of extracellular divalent cations by removal with 3 mM EDTA. Percentages of adherent cells represent means of triplicates
± standard deviation and one representative experiment out of
six independent ones is shown.
The nature of the molecules
recognized by the three HUTS mAbs was investigated by
immunoprecipitation and SDS-PAGE analysis from I
surface-labeled lysates of human T lymphoblasts. Fig. 2shows
that the precipitates obtained with these three mAbs from cells
solubilized in a standard lysis buffer (1% Triton X-100, 1% BSA in PBS,
pH 7.4, without Ca
or Mg
) consisted
of one major band with an electrophoretic mobility identical to the
1 integrin subunit, both under reducing (130 kDa) and nonreducing
conditions (110 kDa). In order to confirm the
1 specificity of the
epitopes recognized by the HUTS mAbs and also the dependence of their
expression upon activation of solubilized integrins, we compared the
precipitates obtained from T lymphoblasts lysed under different
divalent cation conditions. Fig. 3shows that when the lysis and
the subsequent immunoprecipitation protocol were performed under
conditions where VLA integrins are inactive, i.e. in the total
absence of divalent cations obtained by removal with 3 mM EDTA, only one major band corresponding to the
1 subunit
could be detected in the precipitates obtained with the three HUTS
mAbs. Interestingly, when lysis and immunoprecipitation were carried
out in the presence of both Ca
(1 mM) and
Mg
(1 mM) (which in terms of divalent
cations represents a situation equivalent to the physiological
extracellular conditions), again only the same single band
corresponding to the
1 subunit could be observed in the HUTS
precipitates. In contrast, the presence of Mn
(2
mM) in the lysis buffer and throughout the immunoprecipitation
protocol induced important qualitative and quantitative changes in the
immunoprecipitates of these mAbs. Thus, Mn
induced
the appearance in the precipitates of additional bands corresponding to
1,
2, and the 80 kDa fragment of
4, associated with the
common
1 subunit. Moreover, the presence of Mn
also induced an increase in the intensity of the band
corresponding to the
1 subunit. Definitive demonstration of the
1 specificity of the epitopes detected by the HUTS mAbs was
obtained from lysates of T lymphoblasts containing activated VLA
integrin heterodimers (in the presence of 2 mM Mn
) that had been previously dissociated in
their
and
constituent subunits by a short high pH
pretreatment (pH 11.0, 30 min at 37 °C) followed by rapid
reneutralization to pH 8. After this high pH pretreatment of the cell
lysates only the
1 subunit was observed in the HUTS precipitates
(not shown).
Figure 2:
MAbs HUTS-4, HUTS-7, and HUTS-21
immunoprecipitate a single polypeptide with identical electrophoretic
mobility to the 1 integrin subunit.
I-Labeled T
lymphoblasts were lysed in PBS, 1% Triton X-100 buffer without
Ca
and Mg
and immunoprecipitation
carried out as described under ``Materials and Methods'' with
the following mAbs: TS2/16 (
1), lanes A and K;
TS2/7 (VLA-1), lanes B and L; Tea 1/41 (VLA-2), lanes C and M; P1B5 (VLA-3), lanes D and N, HP2/1 (VLA-4), lanes E and O; Lia 1/2 (
1), lanes F and P; GoH3 (VLA-6), lanes G and Q; HUTS-4, lanes H and R; HUTS-7, lanes
I and S; and HUTS-21, lanes J and T.
Figure 3:
The addition of Mn to
lysates of T lymphoblasts induces the appearance of the
1-associated integrin
subunits in the immunoprecipitates
with mAbs HUTS-4, HUTS-7, and HUTS-21. Cell surface labeled T
lymphoblasts were lysed in 20 mM Hepes, 1% Triton X-100
without divalent cations (-cation) and then
Mn
or Ca
+ Mg
were added to aliquots of lysates to give a 1 mM final
concentration of corresponding divalent cation. Immunoprecipitation was
performed as described under ``Materials and Methods.''
4-F corresponds to the 80-kDa proteolytic fragment of the
4
integrin subunit. The stimulatory anti-
1 mAb TS2/16 was used as an
invariant control in immunoprecipitations. Identical results were
obtained when the blocking anti-
1 mAb Lia 1/2 was used as an
invariant control.
Figure 4:
Expression of the epitope recognized by
mAb HUTS-21 is enhanced by cell preincubation with stimulatory
anti-1 mAb TS2/16, phorbol 12,13-dibutyrate, and calcium ionophore
A23187. T lymphoblasts were preincubated with 5 µg/ml TS2/16, 100
nM phorbol 12,13-dibutyrate (PDBU), or 2 µM calcium ionophore A23187 in Hepes/NaCl buffer containing 1 mM Mg
and 1 mM Ca
for 30
min at 37 °C. Biotin-conjugated mAb HUTS-21 was then added and
binding subsequently detected with avidin-fluorescein isothiocyanate.
Flow cytometric analysis was performed as described under
``Materials and Methods.'' Numbers represent
percentage of positive cells.
To further characterize the parallelism observed between the process
of functional activation of VLA integrins and augmented levels of
expression of these 1 epitopes, the effects of temperature and
divalent cation conditions on the T lymphoblast surface expression of
HUTS epitopes were analyzed. Fig. 5A shows that
Mg
alone (in the presence of the Ca
chelator EGTA) was able to induce increased expression of the
epitope recognized by mAb HUTS-21 on T lymphoblasts (compared to
background levels observed in the absence of divalent cations) only
when the incubation was carried out under physiological temperature
conditions (37 °C). Ca
, however, did not induce
detectable expression of this epitope either under 37 °C nor under
4 °C temperature conditions. Interestingly, when both extracellular
Mg
and Ca
were present, the level
of expression of the HUTS-21 epitope was only marginally above
background. Mn
induced an important increase in
epitope expression at 37 °C but, in contrast, this induction was
minimal at 4 °C. Similar results were obtained for epitopes HUTS-4
and HUTS-7 under all conditions (data not shown). We also studied in
parallel the regulation exerted by these different divalent cation
conditions on functional activation of
1 integrins expressed on T
lymphoblasts, under physiological temperature conditions, by measuring
the percentage of these cells that effectively adhere to collagen,
laminin, and fibronectin (Fig. 5B). A close correlation
between functional activity of
1 integrins and expression of HUTS
epitopes was observed under all the divalent cation conditions tested.
Figure 5:
Regulation of HUTS-21 epitope expression
and 1 integrin-mediated T cell adhesion by extracellular divalent
cations. A, expression of the
1 epitope HUTS-21 is
induced at 37 °C by Mg
or Mn
and is inhibited by the presence of Ca
or low
temperature. The anti-
1 mAb TS2/16 is included as an invariant
control. B, T lymphoblast adhesion to
1 integrin-specific
ligands type I collagen, laminin, and fibronectin at 37 °C under
different divalent cation conditions. Divalent cations and
Ca
chelator EGTA were used at the following
concentrations: 1 mM Ca
, 1 mM Mg
, 0.5 mM Mn
, 2
mM EGTA.
To rule out the possibility that the observed increase in the
expression of the HUTS epitopes during the process of 1 integrin
activation on T lymphoblasts was a phenomenon restricted to this cell
system or a consequence of their in vitro culturing and
stimulation with phytohemagglutinin/interleukin-2, we analyzed the
expression of HUTS epitopes on freshly isolated peripheral blood
lymphocytes. We also extended our studies to other
1-positive cell
types including the B lymphoblastoid cell lines RAMOS (which only
expresses VLA-4 among
1 integrins), the erythroleukemic cell line
K562 (which only expresses the VLA-5 member), and the colocarcinoma
cell line COLO-320 (which expresses VLA-1, VLA-2, VLA-3, VLA-5, and
VLA-6). Table 1shows that, similarly to the results already
described with T lymphoblasts, Mn
induced an
important increase in the expression of the HUTS-21 epitope (above the
level observed in medium containing Ca
+
Mg
) on peripheral blood lymphocytes and on the RAMOS,
K562, and COLO-320 cell lines only when physiological temperature
conditions were used. In contrast, Mn
did not induce
the expression of this epitope in the JY B lymphoblastoid cells, which
express hardly detectable amounts of the
1 integrin subunit on
their surface. Similar results were obtained with epitopes HUTS-4 and
HUTS-7 (data not shown).
Figure 6:
mAb HUTS-21 () stimulates the
adhesion of T lymphoblasts to
1-specific ligands fibronectin, type
I collagen, and laminin. Adhesion assays were performed in RPMI medium
as described under ``Materials and Methods.'' mAbs were used
at a final concentration of 5 µg/ml. Adhesion stimulatory
anti-
1 mAb TS2/16 (
), and the irrelevant anti-CD11c mAb
HC1/1 (
) were used as controls. The total dependence of this
cell adhesion system on VLA integrin function was demonstrated by the
complete blockade with the inhibitory anti-
1 mAb Lia 1/2 (not
shown). Similar results were obtained with mAbs HUTS-4 and
HUTS-7.
Figure 7:
A,
mAbs HUTS-4, HUT-7, and HUTS-21 cross-compete for binding to the 1
polypeptide. In each sample, T lymphoblasts were first incubated with
the mAb shown at the left side of the arrow and then
with the biotinylated mAb shown at the right side of the arrow, as described under ``Materials and Methods.''
Anti-VLA-1 (CD49a) mAb 5E8D9 was used as a control. B, the
epitope recognized by mAb HUTS-21 maps within the region 355-425
of the human
1 polypeptide. Wild type and human/mouse chimeric
1 molecules h587/m, h425/m, and h354/m were immunopurified as
described under ``Materials and Methods.'' Purified materials
were separated by SDS-PAGE under nonreducing conditions, transferred
onto Immobilon-P membrane, and blotted with mAbs HUTS-21 or TS2/16.
Identical results were obtained with mAbs HUTS-4 and
HUTS-7.
Unlike other cell surface receptor molecules which are constitutively activated, integrins can only bind ligand and mediate cellular adhesion after their activation is induced by specific stimuli. Reversible functional transitions between inactive and activated states of integrins represent therefore a key mechanism by which cells control their adhesive properties.
In this report, we
describe a group of three mAbs, named HUTS-4, HUTS-7, and HUTS-21, that
recognize specific epitopes on a novel regulatory region of the common
1 subunit of VLA integrins whose expression parallels functional
activation and ligand binding by these adhesion receptors. These mAbs
were obtained after immunization of mice with a purified preparation of
activated human
1 integrins. The protocol for purification of VLA
integrins was designed to yield these molecules in active conformation
with high ligand binding affinity. The initial selection of the HUTS
hybridomas was based on the marked increase in the binding of these
mAbs induced by the presence of Mn
, suggestive of
their specificity for epitopes on VLA integrins whose expression
accompanies functional activation of these molecules.
Interestingly,
immunoprecipitation analysis revealed that, when divalent cations were
not present in the lysis buffer, the HUTS mAbs only precipitated the
1 subunit of VLA integrins. However, the presence of
Mn
(but not of Ca
and
Mg
) in the lysis buffer resulted in the appearance in
the precipitates of the associated
subunits. A possible
explanation for these results is that upon T cell solubilization a
certain proportion of VLA integrin heterodimers are dissociated in
their
1 and
constituent subunits. In the absence of divalent
cations or presence of both Ca
and Mg
in the lysis buffer, the epitopes recognized by these mAbs are
only expressed on the population of dissociated
1 polypeptides.
The implication is that, under these two divalent cation conditions, i.e. complete absence of divalent cation and presence of both
Ca
and Mg
, these
1 epitopes
are masked on the inactive integrin
:
heterodimers. On the
contrary, in the presence of Mn
these epitopes are
expressed not only by the fraction of dissociated
1 chains but
also by the
1 polypeptides (in a specific
Mn
-induced conformation) which are forming part of
activated intact integrin
:
1 heterodimers. These activated
conformations of
1 integrins can result from direct binding of
Mn
to the cation binding site present in the
N-terminal region of the
1 chain. Alternatively, the allosteric
changes in
1 polypeptide detected by the HUTS mAbs can be
transmitted by the
subunits upon Mn
binding to
one or more of their cation binding sites.
Our data show that
expression of the HUTS epitopes correlates with functional activation
of VLA integrins. We have demonstrated that this parallelism not only
applies when activation of 1 integrins is induced from outside the
cells with the stimulatory CD29 mAb TS2/16 or Mn
, but
also when integrin activation is induced by intracellular signals
(inside-out signaling) generated upon cell treatment with phorbol
esters or calcium ionophores.
Several recent reports have shown that
Ca generally exerts inhibitory effects on the
functional activity of integrins belonging to the
1,
2, and
3 subfamilies (Dransfield et al., 1992a; Grzesiak et
al., 1992; Hemler et al., 1990; Masumoto and Hemler,
1993; Staatz et al., 1989; Weitzman et al., 1993). In
contrast, in the absence of extracellular Ca
(i.e. in the presence of the Ca
chelator EGTA), Mg
supports effective
integrin-mediated cell adhesion in a dose-dependent manner. We have
characterized in detail the divalent cation regulation of
1
integrin functional activity on T lymphoblasts by measuring their
adhesion to three different ligands specific for VLA integrins: type I
collagen, laminin, and fibronectin. As previously reported in other
cellular systems (Masumoto and Hemler, 1993; Luque et al.,
1994; Weitzman et al., 1993), we find that Mg
and Mn
induce activation of
1 integrins
and support cell adhesion. In contrast, Ca
exerts
inhibitory effects on
1 integrin function as no adhesion to
ligands collagen and laminin (and only weak adhesion to fibronectin)
occurred when Ca
was the only divalent cation present
in the extracellular medium. Furthermore, the presence of extracellular
Ca
partially inhibited the level of cell adhesion
supported by Mg
. The level of expression of the
epitopes recognized by the HUTS mAbs correlated with functional
activity of
1 integrins under all the divalent cation conditions.
Physiological temperature has been characterized as a requirement
for integrin activation and subsequent adhesion in a number of cell
systems, reflecting the need for an intact cell metabolism (Campanero et al., 1990; Dransfield et al., 1990; Marlin and
Springer, 1987; Rothlein and Springer, 1986; van de Wiel-van Kemenade et al., 1992). The use of the HUTS activation-reporter mAbs
has also allowed us to confirm that an intact cell metabolism is an
absolute requirement for 1 integrin activation, as only minimal
levels of expression of these epitopes on T lymphoblasts could be
detected at 4 °C, regardless of the stimulus used.
Our findings
on the temperature and divalent cation regulation of 1 integrin
function and correlated expression of the HUTS epitopes have been
confirmed for different VLA heterodimers using other cellular systems,
including the B lymphoblastoid cell line RAMOS (VLA-4), the
erythroleukemic cell line K562 (VLA-5), and the colocarcinoma cell line
COLO 320 (which expresses several members of the VLA integrin
subfamily). The fact that a similar regulation by temperature and
divalent cations was also observed in freshly isolated lymphocytes
rules out possible in vitro culturing effects and indicates
that the HUTS epitopes indeed represent physiologic epitopes.
Competitive cell binding studies indicate that mAbs HUTS-4, HUTS-7,
and HUTS-21 recognize common or overlapping epitopes on the 1
polypeptide. A small region of the human
1 subunit (residues
207-218) with important regulatory properties on the function of
VLA integrins has been recently identified using a panel of
1-specific mAbs (Takada and Puzon, 1993). All these mAbs recognize
overlapping epitopes in the 207-218 region but they have
completely different effects on VLA integrin function: while some of
these antibodies (including TS2/16) activate
1 integrins and
induce cell adhesion, others exert a blocking effect on
1
integrin-mediated adhesion. The epitopes recognized by the three HUTS
mAbs have been mapped to a distant segment (residues 355-425)
which is in the vicinity to the cysteine-rich domains in the primary
sequence of the
1 polypeptide (Argraves et al., 1987).
The HUTS mAbs, therefore, define this sequence as a novel regulatory
region in the
1 subunit that is exposed upon activation of
integrin heterodimers. Interestingly, a mAb (D3GP3) which recognizes a
conformational epitope of the
3 integrin subunit and induces
fibrinogen binding to a limited population of integrin
IIb/
3
molecules has also been mapped to a similar region close to or within
the cysteine-rich core of the
3 subunit (422-692) (Kouns et al., 1990). Therefore, common structural and functional
features which can be evidenced by the expression of activation
epitopes seem to be conserved among the
subunits of the integrin
family.
Binding of the HUTS mAbs to specific epitopes on the 1
integrin chain results in enhanced cell adhesion to VLA integrin
ligands fibronectin, laminin, and type I collagen. This stimulation by
HUTS mAbs of
1 integrin-mediated adhesion is similar to that
induced with mAb TS2/16. Although the overall effect of these mAbs on
cell adhesion is similar, the mechanism by which the HUTS and TS2/16
mAbs induce enhanced adhesion may be somehow different. In this regard,
the epitope recognized by mAb TS2/16 in the 207-218 region of the
1 subunit is constitutively expressed, regardless of the state of
integrin activation. This is evidenced by the high level of expression
of this epitope even in the presence of EDTA. It has been recently
shown that upon binding of mAb TS2/16 to the
1 polypeptide an
important increase in the ligand binding affinity of VLA integrins
occurs, presumably mediated by a change in conformation which favors
their interaction with ligands (Arroyo et al., 1992, 1993; van
de Wiel-van de Kemenade et al., 1992). In contrast, the
expression of the epitope(s) recognized by the HUTS mAbs is(are) not
constitutive but depends on the state of integrin activation.
Therefore, it is tempting to speculate that upon binding of HUTS mAbs
to the
1 subunit of partially preactivated VLA heterodimers
already showing weak interaction with ligand, the conformation of these
integrin molecules is ``locked'' in a state where ligand is
bound with high affinity and their reversion to an inactive state is
thus prevented. In fact, some preliminary data indicate that under some
conditions, the HUTS epitopes behave as ligand-induced binding sites
since their expression on
1 integrins also depends on the
interaction with ligands. A similar mechanism has been proposed to
explain the functional effects exerted by other activation-reporter
mAbs specific for the
2 or
3 integrins. For instance, binding
of activation-reporter mAb 24 to the
subunits of leukocyte
(
2) integrins inhibits T cell proliferative responses,
lymphokine-activated killer activity of T lymphoblasts, and
f-Met-Leu-Phe-induced neutrophil chemotaxis. All these functional
effects of mAb 24 could be explained by assuming that it prevents
``de-adhesion'' of integrin/ligand pairs, possibly by
freezing integrins in an active conformation with ligand firmly bound
(Dransfield et al., 1992b). Furthermore, the epitope
recognized by mAb 24 has been shown to be of the ligand-induced binding
site type since its expression also depends on the interaction of
integrin LFA-1 with ligand intercellular adhesion molecule-1
(Cabañas and Hogg, 1993).
Two recent reports
describe the use of a mAb (15/7), which detects an activation dependent
conformational epitope on the 1 molecules, in studies of the T
cell responses in secondary lymphoid tissue (Picker et al.,
1993) and of the expression of activated forms of
1 integrins in
chronic inflammatory diseases (Arroyo et al., 1995). However,
full characterization of the specificity and properties of this mAb has
not been published. Two additional mAbs specific for activation
dependent epitopes of the
1 molecule have been recently
characterized. One of these mAbs, named 9EG7, detects an epitope on the
mouse
1 subunit which is specifically expressed in the presence of
Mn
(Lenter et al., 1993). In contrast to the
adhesion-enhancing effect of the HUTS mAbs, mAb 9EG7 inhibits
integrin-ligand interactions. A second mAb, named SG/7, detects an
epitope on the human
1 molecule whose expression depends on the
presence of either Mn
or Ca
and
cannot be induced on the cell surface after phorbol ester treatment
(Miyake et al., 1994). The control of the expression of the
epitope detected by mAb SG/7 by Ca
is therefore
clearly different from that of HUTS epitopes. Since none of the
mentioned studies have characterized the regions of
1 integrin
subunit containing these activation epitopes it would be interesting to
carry out comparative studies between these and the HUTS mAbs.