(Received for publication, April 11, 1995; and in revised form, July 31, 1995)
From the
Monoclonal antibodies to the 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.
The
transduction pathways depending on the 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.
Acrylamide gel electrophoresis of the protein bands
corresponding to the integrin ,
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.
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 ,
, 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) .
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 (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.
By using monoclonal antibodies, we were able
to demonstrate that the 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).
The
aim of this paper is to demonstrate that the receptor of type I
collagen is the integrin , 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.
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).
NaI and [
H]inositol were from
DuPont NEN.
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--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.
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 -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.
Fractions of 1 ml were collected and their
radioactivity counted with an automatic 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.
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; production and enzyme-rich granule
release were evaluated as described above.
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 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.
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
L
2 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.
Figure 1:
Dose-dependent inhibiting effect of
monoclonal antibodies to the integrin subunits 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.
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 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.
Figure 5:
Increase of the cytoplasmic free
Ca 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.
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 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.
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 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.
Figure 9:
Tyrosine phosphorylation of the
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).
The 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) .
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 (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) .
Previously
we found that a 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%.
Two monoclonal antibodies (clones MEM 25
and MHM 24) had been used to immunoprecipitate the 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) .
The properties of the inhibiting antibody MEM 25
suggest that the I domain of 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) .
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 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.
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 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
L
2 integrin; one can question at which point the transduction
pathways for both systems become identical.
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 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.
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 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.
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 ), 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.