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(Received for publication, April 11, 1995; and in revised form, July 31, 1995) From the
Monoclonal antibodies to the The
transduction pathways depending on the Acrylamide gel electrophoresis of the protein bands
corresponding to the integrin
Type I collagen, a major component of the extra cellular matrix,
promotes the adhesion of a variety of cells in solid tissues,
influencing many processes such as proliferation, differentiation,
migration, and cell shape changes(1) . Several receptor
molecules have been demonstrated as promoting the adhesion to type I
collagen. Among these receptors, some belong to the family of
integrins, for instance Purified type I collagen is
able to bind to PMN in vitro. This binding is followed by the
stimulation of some main functions of PMN, such as emission of
pseudopods, secretion of lytic enzymes, and liberation of superoxide.
We demonstrated that the stimulation of PMN by collagen occurs through
two sequences of the By using monoclonal antibodies, we were able
to demonstrate that the The
aim of this paper is to demonstrate that the receptor of type I
collagen is the integrin
The synthetic peptides GRGD, DGGRYY, RFDS,
CGRGDSPC, and CGRGESPC were synthesized by Neosystem (Strasbourg,
France). Fura-2 was bought from Molecular Probes Inc (Eugene, OR).
Na
The evaluations of elastase
and of 92-kDa type IV collagenase were performed using N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide and
biotinylated type IV collagen as substrates, for incubation periods of
1 and 3 h, respectively, according to the methods of Nakajima et
al.(9) and of Wilkinson et al.(10) . The N-acetyl-
The bound material was eluted first with a 10 mM Tris-HCl
buffer, pH 7.4, containing 5 mM EDTA, 0.1% (w/v) n-octyl Fractions of 1 ml were collected and their
radioactivity counted with an automatic
In another series of
experiments, PMN were preincubated for 15 min at 37 °C with the
synthetic peptides RFDS, GRGD, CGRGDSPC, CGRGESPC, or DGGRYY at various
concentrations. At the end of this incubation, they were stimulated by
addition of 0.3 µM collagen I. The
O&cjs1138;
The experiment was performed after
several types of stimuli had been applied to the cells, either 0.1
µM fMet-Leu-Phe or 0.3 µM type I collagen.
The incubation was stopped at various times (0, 0.5, 1.0, 2.0, or 5.0
min) by addition of 0.15 ml of a 35% (w/v) sodium perchlorate solution.
The reaction mixture was centrifuged at 5,000
A 100-µl
aliquot of cell extract was immunoprecipitated by addition of anti-CD18
or anti-CD11a monoclonal antibodies (6 µg/ml), followed by
adsorption on protein A-Sepharose for 2 h at 4 °C. The
immunoprecipitated material was fractionated by SDS-PAGE in 10%
polyacrylamide gel under reducing conditions and blotted on a transfer
Immobilon membrane (Millipore, Bedford, MA). The membrane was saturated
by incubation for 1 h with a 10 mM Tris buffer, pH 7.4,
containing 150 mM NaCl and 5% (w/v) bovine serum albumin, then
incubated for 2 h in the presence of monoclonal antibodies to
phosphotyrosine (clone 6G9 or clone PY-20). Alkaline
phosphatase-conjugated anti-mouse IgG antibody (Organon Teknika,
Durham, NC) was used as a second antibody, for detection of the
positive protein bands visualized by the reaction with
5-bromo-4-chloro-3-indolyl phosphate in the presence of nitro blue
tetrazolium. For the evaluation of tyrosine phosphorylation of the
Figure 1:
Dose-dependent inhibiting effect of
monoclonal antibodies to the integrin subunits
Figure 2:
Elution profile of the affinity
chromatography of a PMN extract performed on type I collagen-Sepharose.
The left arrow represents the start of elution by 150 mM NaCl + 5 mM EDTA. The right arrow represents the start of elution by 1 M NaCl + 5
mM EDTA. Inset, characterization by SDS-PAGE of the
fractions eluted from the affinity column of collagen-Sepharose.
Electrophoresis carried out in 7.5% polyacrylamide gel at pH 8.3.
Detection by autoradiography. Lane 1, molecular size standards
revealed by Coomassie Blue; lane 2, total extract from
iodinated membrane; lane 3, peak 1; lane 4, peak
2.
SDS-PAGE was performed on the proteins contained in both peaks (Fig. 2, inset). The first peak of elution contained
two major bands with respective apparent molecular masses of 95 and 185
kDa as estimated by comparison to control globular proteins of known
molecular mass. In addition, this peak contained two minor bands, of 31
and 35 kDa, respectively. Peak two contained these 31- and 35-kDa
proteins as major bands. The identity of the proteins contained in
these peaks was verified by immunoprecipitation with the monoclonal
antibodies anti-CD18 and anti-CD11a. The precipitated molecules were
analyzed by SDS-PAGE. The antibody anti-CD18 precipitated the two
proteins of molecular masses 95 and 185 kDa from peak 1 (Fig. 3A). No material from peak 2 was precipitated by
this antibody. Similar results were obtained with the antibody
anti-CD11a (Fig. 3B). Monoclonal antibodies to CD11b
and CD11c did not precipitate any material.
Figure 3:
SDS-PAGE
of the immunoprecipitates. A, obtained with the monoclonal
antibody to CD18. Lane 1, peak 1 from the affinity
chromatography; lane 2, peak 2. B, obtained with the
monoclonal antibody to CD11a. Lane 1, peak 1 from the affinity
chromatography; lane 2, peak 2.
The synthetic peptides
CGRGDSPC and DGGRYY used either separately or together were not able to
elute significant amounts of proteins from the column by themselves
(data not shown).
In the same type of
experiments, the effect of pertussis toxin (at concentrations ranging
from 0 to 500 ng/ml) on the collagen-dependent formation of superoxide
was compared to the effect of this toxin on the fMet-Leu-Phe-dependent
stimulation. Pertussis toxin was found to be inactive on the
transduction of the message from type I collagen, whereas it exerted a
dose-dependent inhibiting effect on the stimulation by the peptide
fMet-Leu-Phe used as a control.
Figure 4:
Variations of the cytoplasm content of
inositol trisphosphate under various stimuli. PMN (5
Figure 5:
Increase of the cytoplasmic free
Ca
Figure 6:
Effect of the protein kinase C inhibitors
on superoxide production by PMN. A, inhibition by H-7; B, inhibition by staurosporine; C, inhibition by
calphostin C. An amount of 10
Figure 7:
Protein kinase C activity of the PMN. The
activity of protein kinase C was measured in the cytosolic (light
shaded bars) and in the particular (darker shaded bars)
fractions of PMNs stimulated with: 1, Dulbecco's
solution alone (control); 2, 0.3 µM collagen I; 3, 0.1 µM fMet-Leu-Phe; 4, 8 nM PMA. Protein kinase C was measured 2 min after stimulation. Data
represent mean of triplicate determinations. Bars represent 1
S.D. from the mean.
Figure 8:
Inhibitory effect of genistein on
superoxide production triggered by PMN. PMN (10
Figure 9:
Tyrosine phosphorylation of the
The The synthetic CGRGDSPC peptide, which
exposes the RGD sequence on a loop, inhibits the adhesion of PMN to
type I collagen, whereas linear GRGD and CGRGESPC do not. The
conformation of RGD sequences is critical for fulfilling its function
of fixation or message transmission(22) . The peptide DGGRYY
does not inhibit the binding of type I collagen to PMN, whereas it
inhibits superoxide and lytic enzyme secretions. The functions of
binding and stimulating are distributed between the two peptide
sequences involved in the ligand. Apparently, RGD, when correctly
folded, is in charge of binding, whereas DGGRYY is responsible for the
transmission of stimulation. Pepsinized Previously
we found that a Two monoclonal antibodies (clones MEM 25
and MHM 24) had been used to immunoprecipitate the The properties of the inhibiting antibody MEM 25
suggest that the I domain of Characterization of the receptor
protein by radioactive iodine labeling of whole cells, followed by
preparation of plasma membranes and affinity chromatography of proteins
on Sepharose-type I collagen columns, permitted isolation of two major
peaks. In SDS-PAGE, peak 1 was found to contain two protein bands of 95
and 185 kDa, respectively, apparent molecular masses corresponding to
those proposed in literature for As regards the transduction pathways linking the membrane receptor
of type I collagen to the effector systems of superoxide formation and
enzyme granule secretion, neither G Cytochalasin B, an
inhibitor of F actin assembly, and colchicine, a disrupting agent for
tubulin, exert severe inhibitory effects on type I collagen induced
superoxide formation. In contrast, the same cytochalasin B enhances the
stimulation by the peptide fMet-Leu-Phe. The latter effect points out
to the intervention of actin and tubulin in the reaction to type I
collagen. Inositol trisphosphate increases sharply after collagen
stimulation, and calcium ion also increases for a short period of about
30 s. From these results, it is inferred that the activation of the
type I collagen receptor is transmitted to a phospholipase C, which
liberates inositol trisphosphate and diacylglycerol. Few studies have
pointed to cascades of stimulations involving A role for
diacylglycerol in the process of PMN stimulation with type I collagen
by activating a protein kinase C was confirmed by the inhibiting effect
exerted by staurosporine and calphostin C. Nevertheless, another
inhibitor of protein kinase C, H-7, did not inhibit the process,
whereas it inhibited the stimulation exerted by the peptide
fMet-Leu-Phe. Collagen stimulates the translocation of protein kinase C
to the plasma membrane, as demonstrated by measurements of changes in
enzyme activity. The Polymorphonuclear neutrophils
undergo multiple stimulations in order to cope with all the situations
of defense of the organism against the many invader cells or toxic
substances. Specific membrane receptors exist for every stimuli. At the
present time, only a few steps of the transduction pathways for these
messages are known and most of them concern the effect of the peptide
fMet-Leu-Phe. In this paper, we describe a somewhat different system of
signaling, depending on type I collagen or, more probably, on the large
fragments of collagen that are liberated during the initial degradative
steps of tissue inflammation or wound healing. The transduction system
is characterized by several distinct features; the receptor is an
integrin (namely
Volume 270,
Number 46,
Issue of November 17, 1995 pp. 27495-27503
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ROLE OF CALCIUM SIGNALING AND TYROSINE PHOSPHORYLATION OF LFA 1 (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
integrin inhibit the binding of type I collagen to PMN
(polymorphonuclear neutrophil leukocytes) as well as the subsequent
stimulation of superoxide production and enzyme secretion elicited by
this collagen. Pepsinized collagen still binds PMN but no longer
stimulates them. The I domain of the chain of the integrin is
involved in the binding. Two sequences of the 
(I)
polypeptide chain of collagen participate in the process. Experiments
of competitive inhibition by synthetic peptides showed that the
sequence RGD (915-917) is used for binding to the cells and
DGGRYY (1034-1039) serves to stimulate PMN. Experiments of
radioactive labeling of the cells and affinity chromatography on
Sepharose-collagen confirmed the presence in PMN extracts of two
proteins, 95 and 185 kDa, respectively, corresponding to the molecular
weights of the and chains of the
integrin and recognized by their specific monoclonal antibodies. integrin do not involve a G protein (ruled out by the use of
cholera and pertussis toxins), whereas the cytoskeleton was found to
participate in the process, as evidenced by inhibition by cytochalasin
B. After collagen stimulation, cytoplasmic inositol trisphosphate and
calcium ion increased sharply for less than 2 min. The use of the
inhibitors staurosporine and calphostin C demonstrated that protein
kinase C was involved. Evaluation of the activity of this enzyme showed
that, upon stimulation of PMN with collagen I, it was translocated to
plasma membrane.,
followed by immunoblotting using monoclonal antibodies to
phosphotyrosine, permitted us to demonstrate that, prior to stimulation
by type I collagen, there was no phosphorylation, whereas after
stimulation, both and chains were
stained by anti-phosphotyrosine antibodies. The adhesion of PMN to
pepsinized type I collagen triggered tyrosine phosphorylation of the
chain of the integrin, without stimulating
O&cjs1138; production by these cells, whereas
their stimulation by complete type I collagen induced the tyrosine
phosphorylation of both and subunits. The tyrosine phosphorylation of both integrin subunits
during transduction of stimuli is a heretofore undescribed phenomenon
that may correspond to a new system of transmembrane communication.
,
, and
(2) . On the other hand, some
types of mobile cells also interact with collagens, for instance
polymorphonuclear neutrophils (PMN), a variety of
leukocytes circulating in blood, capable of crossing the vascular wall
in order to invade the inflamed tissues and to participate in defenses
against bacteria or foreign molecules through their property of
phagocytosis. In several previous papers, we demonstrated that PMN and
type I collagen do interact and began to describe their interactions (3, 4, 5) .(I) chain, both located in the
C-terminal region of the molecules, an RGD sequence corresponding to
residues 915-917, and a DGGRYY sequence corresponding to residues
1034-1039, located at the C-terminal extremity of the chain. The
type I collagen molecule, either fibrillar or denatured, is active,
whereas pepsinized collagen, lacking the C-terminal telopeptide, is
not. The cyanogen-bromide cleaved peptide 1(I)-CB6, which contains
the C-terminal residues (823-1039) of the chain, is also active. In contrast, the addition of the peptides
RGD and DGGRYY either separately or together induces an inhibition of
PMN. Both sequences must be contained in the same peptidic molecule to
remain active on PMN. chain of the integrins is
involved in the process of binding of PMN onto type I collagen. The
main integrins already found in the membrane of PMN all belong to the
group of integrins and are
(also termed CD11a-CD18, LFA 1),
(CD11b-CD18 or Mac 1), and
(CD11c-CD18, p150-95)., that this
integrin mediates not only the binding but also transduction of the
stimulation message, and that this phenomenon depends on the
tyrosine-phosphorylation of both the and subunits. We also point out some new details on the system of
transduction operating intracellularly beyond the receptor.
Materials
Type I collagen was prepared from rat
tail tendon by 0.1 M acetic acid extraction(6) .
Ferricytochrome c (type VI), superoxide dismutase from bovine
erythrocytes, bovine serum albumin, fMet-Leu-Phe, pertussis toxin,
cholera toxin, cytochalasin B, lactoperoxidase, H-7
(1-(isoquinolenylsulfonyl)-2-methylpiperazine), staurosporine, were all
purchased from Sigma. Calphostin C was bought from Calbiochem (La
Jolla, CA). The following monoclonal antibodies were used: P4C10,
recognizing the 1 integrin subunit (Life Technologies, Inc.);
4-G7, raised toward the lymphocyte surface antigen Leu-12, D-12, recognizing CD11b, and 5-HCl-3, recognizing CD11c, all
from Becton-Dickinson (Mountain View, CA); MHM-24, raised against
CD11a, MHK-23, recognizing CD18 from Dakopatts (Glosdrup, Denmark);
MEM-25, Bear-1, FK-24, and MEM-48, recognizing CD11a, CD11b, CD11c,
CD18, respectively, from Monosan (Tebu, Paris, France). Monoclonal
antibodies to phosphotyrosine (clone 6G9, Life Technologies; and PY 20,
Calbiochem) were used.I and [
H]inositol were from
DuPont NEN.Preparation of PMN
Human blood was obtained on
consent from healthy subjects. PMN were isolated according to a
previously published method (4) involving a single-step
centrifugation procedure through a Metrizoate-Polyprep gradient
(Nycomed, Oslo, Norway) at 240 
g for 35 min at room
temperature. The PMN-rich layer was drawn, washed once in
Dulbecco's solution, pH 7.4, and centrifuged at 600 g for 15 min at room temperature. The contaminating erythrocytes
were eliminated by hypotonic lysis in 15 mM ammonium chloride.
The cells were finally suspended at 10
/ml in
Dulbecco's solution, pH 7.4, containing 1.3 mM Ca and 0.5 mM Mg
, at
4 °C. The percentage of PMN in cell preparations exceeded 95%, and
the cell viability, as determined by trypan blue exclusion, was over
98%.
Experiments of Cell Adhesion
Polystyrene 96-well
plates (Nunc, Copenhagen, Denmark) were coated with type I collagen
solubilized in 0.1 ml of 0.018 M acetic acid solution at a
concentration of 50 µg/well. The protein-containing plates were
washed three times with 150 mM NaCl prior to the addition of
1.5 10
cells in 100 µl of Dulbecco's
solution containing 1.3 mM Ca and 0.5
mM Mg
. After a 30-min incubation period, the
non-adherent cells were discarded. The adherent cells were fixed with
100 µl of a 1.1% (v/v) glutaraldehyde solution for 15 min. After
extensive washing with distilled water, 0.1 ml of crystal violet
solution (100 mg/100 ml in 0.2 M Hepes buffer, pH 6.0) was
added to every well, left for 15 min, and discarded. The adhesion of
PMN was evaluated spectrophotometrically as the absorbance of the
stained nuclei at 560 nm, according to Kueng(7) .
Measurement of Superoxide Ion
(O&cjs1138;
The
O&cjs1138;) release was measured from the
superoxide dismutase-inhibitable reduction of ferricytochrome c, according to English(8) . The PMN suspension
(10
cells in 100 µl) was added to a small glass test
tube containing 0.1 ml of 1 µM cytochrome c solution and 0.85 ml of Dulbecco's solution.
O&cjs1138; release was induced by adding 0.1 ml
of the activating agent solution (either fMet-Leu-Phe at the
concentration 0.1 µM in saline or 0.3 µM type
I collagen in 0.018 M acetic acid solution). The increase in
absorbance was followed spectrophotometrically as an index of the
amount of liberated superoxide. Test tubes supplemented with 0.05 ml of
superoxide dismutase solution in saline were taken as blanks assessing
the specificity of the reaction.Measurement of Granule Secretion
A 0.1-ml aliquot
containing 10 PMN was added to 0.8 ml of Dulbecco's
solution and 0.1 ml of collagen solution (final concentration 0.3
µM) already mixed in a glass test tube. The mixture was
incubated for 30 min at 37 °C. Cells were then removed by
centrifugation at 800 g for 5 min and the enzymatic
activities measured in the supernatant.-D-glucosaminidase activity was
measured according to the method of Troost et
al.(11) . Lactic dehydrogenase, whose evaluation permits
to verify the absence of cell lysis, was evaluated as described by Buhl et al.(12) . The enzymatic activities released in the
medium were expressed as percentage of the corresponding total activity
of the cell lysate.Effect of Monoclonal Antibodies on PMN
Treff
polypropylene microtubes (Polylabo, Strasbourg, France) were coated
with 1.5 ml of fetal calf serum (Life Technologies) by agitation at 37
°C for 1 h and then extensively washed with a 0.15 M NaCl
solution. A 0.1-ml volume of antibody solution, at the appropriate
titer, was added to each tube, followed by the PMN suspension in
Dulbecco's solution, in order to achieve a final concentration of
6 10 cells in 0.75 ml. Test tubes were shaken
horizontally in a mechanical shaker for 90 min at 150 cycles/min (13) .Affinity Chromatography
Ten mg of purified type I
collagen were covalently coupled to 1 ml of packed beads of
CNBr-activated Sepharose 4B (Pharmacia Biotech Inc.) according to the
manufacturer's instructions. In parallel, a suspension of 20
10 cells in 1.5 ml of phosphate-buffered saline was
iodinated using the lactoperoxidase method of Lebien et
al.(14) . Iodination was carried out on ice by addition of
200 µg of lactoperoxidase followed by 37 MBq of NaI
dissolved in 0.1 ml of water and two additions of 20 µl of 0.12%
(w/v) hydrogen peroxide at a 5-min interval. Reaction was terminated by
adding 2 ml of ice-cold phosphate-buffered saline, and the cells were
washed three times with this solution. The cells were then extracted in
1 ml of a 100 mM Tris-HCl buffer pH 7.4, containing 150 mM NaCl, 0.5% (w/v) Nonidet P-40, 0.1% (w/v) sodium dodecyl sulfate
(SDS), 1% aprotinin (w/v), 2 mM phenylmethylsulfonyl fluoride,
10 µg/ml leupeptin. Following clarification by centrifugation at
12,000
g for 15 min at 4 °C, the lysate was
diluted 1/5 with a 10 mM Tris-HCl buffer pH 7.4 containing 1.3
mM CaCl, 1 mM MgCl, 1 mM benzamidine, 2 mM phenylmethylsulfonyl fluoride, 10
µg/ml leupeptin. This dilution was necessary to reduce the
concentration of Nonidet P-40 and SDS. The extract was transferred onto
the collagen-Sepharose 4B beads in batch and incubated overnight in the
same conditions. The suspension was then packed into a 1.0-cm diameter
glass column and washed at 4 °C by buffer A at a flow of 5 ml/h. -D-glucopyranoside, 150 mM NaCl, 1 mM benzamidine, 2 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, at a rate
of 1 ml/h, then with the same buffer containing 1.0 M NaCl in
the place of 150 mM. In some instances, the proteins were
sequentially eluted by the specific peptides, first by 4 ml of a 3
mM solution of synthetic DGGRYY, then by 4 ml of a 3 mM solution of CGRGDSPC. Both peptides were dissolved in a 10 mM Tris-HCl buffer, pH 7.4, containing 0.1% (w/v) n-octyl
-D-glucopyranoside, 150 mM NaCl, 1 mM benzamidine, 2 mM phenylmethylsulfonyl fluoride, 10
µg/ml leupeptin. counter (Kontron MR-480)
in order to monitor the elution. For characterization of the eluted
substances, the proteins contained in 200-µl aliquots corresponding
to the peaks of radioactivity were precipitated by addition of ethanol
to 80% (v/v). The precipitates were redissolved in Laemmli sample
buffer (15) and submitted to electrophoresis in a 7.5%
polyacrylamide gel under reducing conditions. Radioactive bands were
revealed by exposure to a Hyperfilm MP (Amersham Corp.) for convenient
periods of time.
Immunoprecipitations
Aliquots of 200 µl of the
eluted fractions were coupled to the monoclonal antibodies and to
protein A-Sepharose by incubation at 4 °C for 2 h. The
Sepharose-adsorbed material was washed three times with 3 ml of
Tris-buffered saline. The eluted proteins were analyzed by SDS-PAGE
under reducing conditions and detected by autoradiography as described
above(16) .Preincubation of PMN with Specific or Competitive
Inhibitors
PMN were incubated for 10 min at 37 °C either
with pertussis toxin (final concentration 100-500 ng/ml), with
cholera toxin (2.5 µg/ml), with cytochalasin B (2.5 µg/ml),
with the protein kinase C inhibitors staurosporine (0-500
nM), H-7 (0-1000 µM), calphostin C
(0-500 nM), or with the inhibitor of tyrosine kinase
genistein (0-2.0 µg/ml). After this preincubation, they were
stimulated by addition of 0.1 µM fMet-Leu-Phe or of 0.3
µM collagen I. The production of superoxide was evaluated
by spectrophotometry of the superoxide dismutase-inhibitable reduction
of cytochrome c as indicated above. production and enzyme-rich granule
release were evaluated as described above.Intracellular Calcium Ion Measurement
The
intracellular Ca concentration was measured according
to Grynkiewicz et al.(17) . A suspension of PMN
(10
/ml), in a Dulbecco's solution containing 1.3
mM CaCl, 0.5 mM MgCl and 10
mM glucose, was loaded with 0.5 µM Fura-2-AM for
30 min at 37 °C, rinsed twice with Dulbecco's solution, and
sedimented. The cells were resuspended in Dulbecco's solution at
an amount of 10 cells/ml. Two ml of this suspension were
transferred into the cuvette of a Shimadzu RF 5000 spectrofluorometer.
The apparatus was set up at two excitation wave lengths of 340 and 380
nm, and the emitted fluorescence monitored at 510 nm for 3 min in order
to determine the basal level, the stimulating agent (final
concentration 0.1 µM fMet-Leu-Phe or 0.3 µM type I collagen) was added and fluorescence recorded for 10 min.
At the end of each experiment, cells were lysed by adding a 0.1% (w/v)
Triton X-100 solution and the maximal fluorescence measured. Minimal
fluorescence was also determined after addition of 2.0 mM MnCl solution. Results were expressed as nanomoles of
Ca/10
cells.Intracellular Inositol Trisphosphate
Measurement
PMN were suspended at a concentration of 10 cells/ml in Dulbecco's solution containing 0.0025% (w/v)
bovine serum albumin and were incubated with 1.11 MBq of
[H]inositol for 120 min at 37 °C. Ten minutes
before the end of this incubation, 1 ml of a solution of 10 mM
LiCl was added in order to inhibit the hydrolysis of
inositol phosphate. Finally, the cells loaded with
[H]inositol were rinsed twice with
Dulbecco's solution containing 10 mM LiCl and resuspended at 2 10 cells/ml in fresh
Dulbecco's solution. g for 15
min at 4 °C. The supernatants were neutralized with a 9.0 M KOH solution and deposited at the top of columns of Dowex AG-1X8
(200-400-mesh) equilibrated under the formate form. The column
was eluted sequentially with 3 ml of distilled water (for free
[H]inositol), 10 ml of 5 mM sodium
tetraborate/60 mM sodium formate (for
glycerophospho[H]inositol), 14 ml of 0.1 M formic acid/0.2 M ammonium formate (for
[H]inositol phosphate), 18 ml of 0.1 M formic acid/0.5 M ammonium formate (for
[H]inositol bisphosphate), 18 ml of 0.1 M formic acid/1.0 M ammonium formate (for
[H]inositol trisphosphate), according to the
method of Berridge(18) . The radioactivity of the eluted
fractions was measured in a Packard 1900 TR liquid scintillation
counter. Results were expressed as percentage of total inositol
phosphate.Measurement of Protein Kinase C Activity
Protein
kinase C activity was evaluated in the particular or in the cytosolic
fractions of PMN stimulated with fMet-Leu-Phe, PMA, or type I collagen,
according to the method described by Kikkawa et
al.(19) .Tyrosine Phosphorylation of the
The suspension of PMN (10Integrin
cells/ml) in Dulbecco's solution containing 1.3 mM CaCl, 0.5 mM MgCl, was first
preincubated for 30 min at 20 °C in the presence of 2.5 mM phenylmethylsulfonyl fluoride. Then temperature was raised to 37
°C for 5 min prior to the addition of the stimulating agent
(Dulbecco's solution as a control, 0.1 µM fMet-Leu-Phe peptide in Dulbecco's solution, or 0.3
µM type I collagen in Dulbecco's solution). The
incubation was stopped after 2 min by centrifugation at 1000 g for 4 s at 4 °C. The supernatants were discarded and the
cells lysed by addition of 150 µl of a 0.05 M Hepes
buffer, pH 7.5, containing 150 mM NaCl, 5 mM EGTA, 10
mM sodium pyrophosphate, 10 mM sodium fluoride, 5
mM orthovanadate, 1% (w/v) SDS, 1% (w/v) Nonidet P-40, 10%
(w/v) glycerol, and a mixture of proteinase inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10
µg/ml leupeptin, and 1 µM pepstatin A)(20) .
Cell lysis took place within 10 min at 4 °C.L2 integrin subunits, PMN (5 10 cells)
were labeled with 18.5 MBq of PO
H
for 30 min and then incubated with 0.3 µM type I collagen
(pepsinized or not). The membrane extracts were immunoprecipitated with
anti-CD18 or anti-CD11a antibodies, and the immunoprecipitated material
was analyzed by SDS-PAGE. The integrin subunits were submitted to an
alkaline treatment prior to 5.7 M HCl hydrolysis. Phosphoamino
acids were derivatized with 9-fluorenylmethylchloroformate (Fmoc)
according to (21) , and the Fmoc-derivatives were analyzed by
reverse phase high performance liquid chromatography on an Hypersil RP
18 (5 µm) column.
Identification of the Sequences of Type I Collagen
Responsible for Adhesion and Stimulation
Preparations of PMN
were submitted to the effect of solutions of synthetic peptides
containing sequences RGD and DGGRYY. After a 15-min treatment, an
aliquot of cells was added onto type I collagen molecules layered to
the bottom of the wells of culture polystyrene plates. Adhesion to
collagen and production of O&cjs1138; by these
cells were measured (Table 1). The linear peptide GRGD and the
control peptide RFDS did not exert any significant action. On the other
hand, the peptide CGRGDSPC, which contains RGD in a loop formed under
the influence of a disulfide bridge, exerted an inhibiting effect of
about 50% on both adhesion and superoxide production, whereas peptide
CGRGESPC did not. This effect was slightly dose-dependent. Peptide
DGGRYY did not inhibit the binding of PMN to type I collagen but
inhibited the formation of O&cjs1138; in a
dose-dependent manner. The secretion by PMN of lytic enzymes such as
elastase or gelatinase was found to be inhibited by the peptides
CGRGDSPC or DGGRYY in parallel to superoxide liberation (Table 2).
Identification of LFA 1 as the Receptor for Type I
Collagen on PMN Membrane
In experiments similar to the previous
one, monoclonal antibodies to the various subunits of the
integrins were incubated with PMN, then the adhesion
of the cells was measured as well as superoxide secretion (Table 3) and granule exocytosis (Table 4). Two monoclonal
antibodies recognizing the chain of integrins, MHM 23
and MEM 48, were found to inhibit both adhesion of PMN to collagen and
secretions of superoxide and lytic enzymes. On the other hand, one of
the monoclonal antibodies recognizing the chain, MEM
25, inhibited both adhesion and stimulation of the cells, whereas the
second monoclonal antibody to the chain did not
induce any inhibition. The secretions of lytic enzymes by PMN, such as
elastase or gelatinase induced by type I collagen, were found to be
inhibited by the same monoclonal antibodies as superoxide liberation.
The monoclonal antibodies inhibited the secretions by PMN in a
dose-dependent manner (Fig. 1). None of the monoclonal
antibodies recognizing or chains
inhibited adhesion or activation of PMN.
and
on the PMN adhesion to collagen (A) and on
the superoxide production triggered by type I collagen (B). A
first incubation of 6 10 PMN was performed for 90
min in the presence of monoclonal antibodies anti-CD11a (clone MEM 25,
) or anti-CD18 (clone MEM 48, &cjs2108;), and then an aliquot of
these cells (1.5 10) was layered on a type I
collagen-coated plate and their adhesion measured by nuclei staining
with crystal violet. Another aliquot (1 10) was
incubated for 15 min with type I collagen in a test tube in order to
measure the production of superoxide by the superoxide
dismutase-inhibitable reduction of ferricytochrome. Bars represent 1 S.D. from the mean.
Isolation of the Receptor
The isolation of the
receptor membrane protein was undertaken by the method of affinity
chromatography on a column of type I collagen-Sepharose. The elution
diagram is shown on Fig. 2. Two peaks were found, one eluted
with buffer A containing 5 mM EDTA and 150 mM NaCl
and the second with the same buffer A containing 1 M NaCl.
Further Steps of Transduction of the Message beyond the
Receptor: G Proteins
The necessity of a G
protein
for transduction of the message was checked by addition of cholera
toxin to the preparation of PMN and measurement of the production of
superoxide in the presence of type I collagen. No influence of this
toxin was noticed either on collagen-stimulated PMN or on control
fMet-Leu-Phe-stimulated PMN (data not shown).Effect of Cytochalasin B and Colchicine on the Production
of Superoxide
These inhibitors of cytoskeleton assembly were
added to PMN at concentrations 2.5 µg/ml and 10M, respectively, 10 min prior to stimulation by addition of
type I collagen or control fMet-Leu-Phe. The result of this addition on
the production of superoxide by the cells was measured (Table 5).
While cytochalasin B or colchicine increased the production of
superoxide in the presence of fMet-Leu-Phe, they suppress this effect
in the presence of type I collagen. Similar results were obtained when
measuring granule exocytosis (data not shown).
Inositol Trisphosphate
The results of the
evaluation of the inositol trisphosphate in the cytoplasm of PMN after
various stimulations are shown on Fig. 4. There was a fast rise
in the concentration of inositol trisphosphate, with a peak about 0.5
min after the onset of stimulation in the case of the use of type I
collagen or peptide fMet-Leu-Phe, whereas in the case of treatment with
pepsinized type I collagen the amount of inositol trisphosphate
remained comparable to the base-line levels obtained with
Dulbecco's solution as a control. Preincubation of PMN with
monoclonal antibodies anti-CD11a or anti-CD18 inhibited the rise in the
concentration of inositol trisphosphate triggered by collagen I,
without affecting that induced by fMet-Leu-Phe.
10 cells/ml) were preincubated for 120 min at 37 °C in the
presence of [H]inositol (1.11 MBq/ml), and then
aliquots of 10 cells were stimulated either with control
Dulbecco's solution, with 0.1 µM fMet-Leu-Phe, 0.3
µM type I collagen, or 0.3 µM pepsinized type
I collagen. Inositol trisphosphate was evaluated as described under
``Experimental Procedures.'' Data represent the mean of
quadruplicate determinations.
Calcium Ion
The results of the evaluation of
calcium ion concentrations in the cytoplasm of PMN are shown on Fig. 5. Once again, type I collagen elicited a sudden rise of
the calcium concentration, comparable to that elicited by the control
peptide fMet-Leu-Phe, followed by a decrease within 2 min after the
onset of stimulation. Pepsinized collagen was inactive. On the other
hand, preincubation of PMN with monoclonal antibodies anti-CD11a or
anti-CD18 inhibited the calcium mobilization triggered by collagen I
without affecting that induced by fMet-Leu-Phe (data not shown).
concentration. PMN were loaded for 30 min with
Fura-2. The vertical arrow shows the instant of addition of
stimulating agent. A, Dulbecco's solution as a control. B, 0.1 µM fMet-Leu-Phe solution. C, 0.3
µM pepsinized type I collagen. D, 0.3 µM type I collagen.
Protein Kinase C
The participation of protein
kinase C in the process was tested with three inhibitors: H-7,
staurosporine, and calphostin C. The first inhibitor had no effect on
the production of superoxide by PMN stimulated by collagen (whereas
under the same conditions it decreased this formation when induced by
contact with peptide fMet-Leu-Phe) (Fig. 6). Staurosporine and
calphostin C decreased the stimulation exerted by type I collagen in a
concentration-dependent manner parallel to that induced by the control
peptide fMet-Leu-Phe but in greater proportion (Fig. 6). The
stimulation of PMN with collagen I triggered the translocation of
protein kinase C from cytosolic to particular fraction as fMet-Leu-Phe
or PMA did (Fig. 7).
cells was preincubated for 10
min at 37 °C in the presence of the protein kinase C inhibitor and
then stimulated with 0.3 µM collagen () or 0.1
µM fMet-Leu-Phe (). Data represent mean of
quadruplicate determination. Bars represent 1 S.D. from the
mean.
Effect of Genistein
The stimulation of the
production of superoxide by type I collagen and by the peptide
fMet-Leu-Phe was found to be decreased by this inhibitor of protein
tyrosine kinase (Fig. 8).
cells) were
preincubated for 10 min with genistein and then stimulated with 0.3
µM type I collagen () or with 0.1 µM peptide fMet-Leu-Phe (). The release of superoxide was
measured through the superoxide dismutase-inhibitable reduction of
ferricytochrome c. Data represent mean of quadruplicate
determinations. Bars represent 1 S.D. from the
mean.
Detection of the Tyrosine Phosphorylation of the Subunits
of Integrin
After stimulation
of PMN by type I collagen, the proteins extracted from plasma membrane
have been precipitated by anti-CD18 or anti-CD11a monoclonal
antibodies. Fig. 9shows that the precipitated proteins,
analyzed by SDS-PAGE, migrate with electrophoretic mobilities identical
to that of the and subunits.
Immunoblotting by an antibody to phosphotyrosine was inhibited by an
excess of free phosphotyrosine. The electrophoregram showed that a
tyrosine-phosphorylation of the subunit of the
integrin was taking place in case of adhesion of PMN to pepsinized type
I collagen, whereas stimulation by complete collagen induced the
tyrosine-phosphorylation of both and subunits. Phosphoamino acid analysis confirmed the presence of P-labeled phosphotyrosine in the
-integrin subunit in PMN incubated with pepsinized
type I collagen, and in both and subunits in PMN stimulated with complete type I collagen.
integrin subunits after PMN
stimulation. SDS-PAGE of the membrane proteins precipitated by
anti-CD18 (lanes 1-5) or with anti-CD11a (lanes 6 and 7) and stained by an antibody to phosphotyrosine.
Incubation of PMN for 2 min with Dulbecco's solution alone (lane 1), bovine serum albumin (lane 2), 0.1
µM fMet-Leu-Phe (lanes 3 and 7), 0.3
µM acid-soluble collagen I (lanes 4 and 6), and 0.3 µM pepsinized collagen I (lane
5).
1(I) chain of type I collagen stimulates PMN
functions such as respiratory burst and degranulation through two
distinct sequences, an RGD sequence corresponding to residues
915-917 and a DGGRYY sequence corresponding to the C-terminal
extremity of this polypeptide chain, residues
1034-1039(4) .(I) chain of
collagen I, which does not contain the sequence DGGRYY, still binds
PMN, but this binding is not followed by stimulation. It must be noted
that the binding of PMN onto type I collagen seems necessary for the
stimulation to occur but that a specific inhibition of the stimulation
may occur independently of the binding. Finally, this binding also
necessitates the presence of Mg and Ca
ions, as we have already demonstrated(5) .
integrin is involved in the
stimulation of PMN by type I collagen(5) , but we had not yet
identified the chain. In this paper, we demonstrate that the
membrane receptor of type I collagen on PMN is constituted by integrin
(LFA 1). The adhesion of PMN to type
I collagen is abolished by a preliminary treatment of the cells with
two monoclonal antibodies to the chain (clones MHK 23
and MEM 48) by about 95%. chain and the recombinant I domain(23) . Surprisingly, in
our experiments, the MHM 24 antibody did not inhibit the adhesion of
PMN to collagen I, whereas MEM 25 did. This discrepancy may depend on
the fact that the epitope recognized by the MHM 24 antibody is too
distant from the domain of collagen binding, as documented in (24) . chain is involved in the
adhesion of PMN to collagen. The I domain is present in the
chains of the group of integrins as well as in the
chain of the integrin, which is a receptor for collagen in various
cells(25) . This domain may represent a common marker for the
adhesion of type I collagen, despite the restricted level of identity
(36%) between the three chains of integrins(26) . Other authors suggested the adhesion of
PMN to collagen I through the integrin(27) . and chains (16) . The identity of the two chains was assessed
by immunoprecipitation of the fractions with anti- and
anti- monoclonal antibodies. Anti- and anti- monoclonal antibodies were devoid of
effect. In addition, peak 1 contained traces of two other proteins,
whose apparent molecular masses, as estimated by SDS-PAGE, corresponded
to 31 and 35 kDa. We have no information on their nature and
relationship with the integrin. nor G
proteins were involved (absence of effect of cholera and
pertussis toxins). The final result of PMN stimulations by the
bacterial peptide fMet-Leu-Phe and by type I collagen is the same, but
the former depends on a G protein associated to a
seven-transmembrane domain receptor, whereas the second depends on the
L2 integrin; one can question at which point the transduction
pathways for both systems become identical. integrins (28) . Inositol trisphosphate stimulates the
output of calcium ions from the endoplasmic reticulum. Calcium
signaling through Mac 1 has been described(29, 30) ,
in the case of cross-linking of this integrin by monoclonal antibodies.
The stimulation depends on the presence of Ca in the
medium surrounding cells. This requirement is at present unexplained.
It may be related to the binding of collagen to the receptor. The
effect of Ca
on enzyme-containing granules probably
depends on the actomyosin system responsible for the exocytosis of
these granules. The effect of calcium on the activation of the
NADPH-oxidase system of PMN is difficult to explain and may be due to a
calcium-dependent isoform of protein kinase C able to phosphorylate the
cytoplasmic protein p47, a preliminary step for the assembly of the
NADPH-oxidase at the membrane and to its activation.
integrin,
when liganded by type I collagen, is phosphorylated on tyrosyl residues
as demonstrated by the use of monoclonal antibodies to phosphotyrosine
reacting with the subunits of the integrin separated by PAGE. PMA or
fMet-Leu-Phe treatments of monocytes induce the phosphorylation of
CD18, mainly on seryl residues, and to a lesser extent on threonyl and
tyrosyl residues(16, 31) . Phosphoserine was also
detected on CD11a (16) . On which residues of the and peptide chains does this reaction take
place? The chain contains 1 tyrosyl residue in its
intracellular domain(32) . Four seryl residues but no tyrosyl
are present in the intracellular domain of . However,
in the transmembrane domain, the 6th residue closer to the cytoplasmic
domain is a tyrosine(23) . Our results suggest that it might be
accessible to an intracellular tyrosine kinase. Tyrosine
phosphorylation of several intracellular proteins is a major
transduction pathway described for integrins (33) . It also occurs by stimulating PMNs by fMet-Leu-Phe or
TNF(19, 34) . In this study, we show tyrosine
phosphorylation of the two chains of an integrin upon its reaction with
one of its ligands, opening new horizons on the way this integrin is
able to transmit information to cytoplasm by binding specific proteins
through SH domains.), the integrin is
phosphorylated on tyrosine residues belonging to both subunits, there
is no G protein involved, and cytoskeleton participates in the
stimulation pathway, which involves inositol trisphosphate, calcium
ion, and protein kinase C.
)
We express our thanks to C. Perreau for beautiful
technical work, to Dr. G. Bellon for initiating the studies on the
chromatographic fractionation of receptors, to Dr. L. Martiny for
showing us the technique of purification of inositol phosphates, and to
Dr. Legendre for improving the syntax of this paper.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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