(Received for publication, November 4, 1996, and in revised form, February 25, 1997)
From the Joseph J. Jacobs Center for Thrombosis, and Vascular Biology/FF-2, The Cleveland Clinic Foundation, Cleveland, Ohio 44195
Intercellular adhesion molecule-1 (ICAM-1) is a
cell surface ligand for L
2 and
M
2 integrins and has a key role in
leukocyte adhesion to the vascular endothelium. The plasma protein
fibrinogen has also been shown to interact with ICAM-1. We have
investigated the effect of fibrinogen binding to ICAM-1-expressing
cells on cell proliferation. The inclusion of 200-800 nM
fibrinogen but not fibronectin to the culture medium of Raji induced a
2-4-fold increase in [3H]thymidine incorporation after
8 h. Cell proliferation in cultures containing fibrinogen was also
confirmed by direct cell counting. The proliferative response in Raji
was abrogated by an anti-ICAM-1 mAb 84H10 which maps to the first Ig
domain of ICAM-1. A purified truncated form of ICAM-1 containing the
first two Ig-like domains and a peptide with amino acid sequence
corresponding to ICAM-1 (8-22) was also able to block the
proliferative action of fibrinogen on Raji. 200 nM
fibrinogen induced a 3-fold increase in [3H]thymidine
incorporation by 293 cells transfected with ICAM-1 cDNA but not
control non-transfected 293 cells. Comparable mitogenic effects were
achieved with fibrinogen fragments X and D100, and with a
synthetic peptide with an amino acid sequence matching fibrinogen
chain (117-133). These results indicate that interaction between
discrete sequences within ICAM-1 and fibrinogen result in cellular
proliferation.
Fibrinogen (Fg)1 and intercellular
cell adhesion molecule-1 (ICAM-1) are prominent ligands for several
integrin receptor subclasses (1) and have important roles in
facilitating cell-cell associations and adhesions (2, 3). ICAM-1
functions as an adhesive ligand for leukocytic 2
integrins and mediates leukocyte extravasation across vascular
endothelial barriers. The expression of ICAM-1 on endothelium is
controlled at least in part by inflammatory cytokines (2, 4). ICAM-1
has five extracellular Ig-like domains and numerous
N-glycosylations contributing to a molecular mass of 95 kDa.
The first two Ig-like domains are critical for binding to
L
2 (LFA-1) (5), whereas the third Ig-like
domain of ICAM-1 binds to
M
2 (Mac-1) (6).
Elevated levels of ICAM-1 have been detected on endothelium lining
regions of inflamed tissues, endothelium experiencing shear stress (7),
and endothelial cells present within atherosclerotic lesions (8,
9).
Fg is an abundant plasma protein with a well recognized role in blood
coagulation and hemostasis. Both Fg and fibrin are able to bind to a
variety of plasma and matrix proteins (10). Fg and Fg degradation
products have been localized in early and late stages of formation of
atherosclerotic lesions (11, 12). Fg is a homodimer of molecular mass
340 kDa, made up of two sets of ,
, and
polypeptide chains
linked through N-terminal regions by interchain disulfide bonds. Upon
the proteolytic action of plasmin, Fg is cleaved first to a 260-kDa
form of Fg termed fragment X and subsequently to smaller C-terminal Fg
fragments D (100 kDa) and a central Fg fragment E (50 kDa) (10). A
direct interaction between ICAM-1 and a discrete region of the Fg
chain has been demonstrated (13). The interaction of Fg with ICAM-1 has
been localized to the first Ig-like domain incorporating amino acids 8-21 of ICAM-1 (14), and Fg:ICAM-1 interactions have been shown to
mediate contact between cells (3, 13). However, the consequences of Fg
binding to ICAM-1 may be even more direct. A recent report has
described a Fg-mediated vasorelaxation of saphenous vein endothelium that was abrogated by treatment of the tissue with antibodies directed
against ICAM-1 (15), which suggests that Fg association with ICAM-1 is
able to effect a direct physiological response. Fg and fragments of Fg
are demonstrated to induce mitogenic activity in T and B lymphocyte
cell lines (16) and hematopoietic progenitor cells (17); however, the
mechanism through which Fg mediates this cellular response remains
unclear. This report provides evidence that ICAM-1 is a mitogenic Fg
receptor on B lymphocytes, and that the observed mitogenic response can
be attributed at least in part to discrete amino acid sequences within
the Fg
chain and the first Ig-like domain of ICAM-1.
Transferrin and bovine serum albumin were purchased from Sigma. 125I-Sodium iodide and [methyl-3H]thymidine were from Amersham Life Sciences. Fibrin-free fibrinogen (18) was provided by Dr. J. Shainoff (Cleveland Clinic Foundation, Cleveland, OH). Human fibronectin purified according to the method of Vuento and Vaheri (19) was a gift from Dr. T. Ugarova (Cleveland Clinic Foundation, Cleveland, OH).
AntibodiesAnti-ICAM-1 polyclonal antibodies R686 and R803
were prepared and purified in our laboratory from the serum of rabbits
injected with Escherichia coli-expressed ICAM-1. The
anti-ICAM-1 mAb 84H10 was from AMAC International (Westbrook, ME). This
antibody has been reported to block
L
2/ICAM-1-mediated lymphocyte
interactions (20-22), indicating that this antibody recognizes an
epitope within the first Ig domain of ICAM-1. The anti-ICAM-1 mAb QE2
(23) was provided by Dr. R. Faull (St. George Hospital, Kogarah,
Australia). The anti-
v
3 antibody LM 609 (24) was a gift from Dr. D. Cheresh (Scripps Research Institute, La
Jolla, CA). mAb1980, mAb1969, and mAb1965 mAbs directed against the
integrin subunits
v,
5, and
1, respectively, were obtained from Chemicon
International (Temecula, CA). MAb KB90 directed against integrin
subunit
X (CD11c, P150,95) and MHM23 against
2 integrin subunit were obtained from Dako Corp.
(Carpinteria, CA). Antibodies TS1/18 and TS1/22 against
2 and
L subunits, respectively, were from
the American Type Culture Collection (ATCC, Rockville, MD).
ICAM-1-expressing lymphoblastoid Raji cells and 293 cells of human kidney fibroblast origin were obtained from ATCC. Raji were grown in RPMI 1640 (BioWhittaker, Walkersville, MD) containing 7.5% FCS and 1.0 mM glutamine. 293 cells were maintained in Dulbecco's modified Eagle's medium/F-12 (BioWhittaker) containing 10% FCS and 1.0 mM glutamine. For transfection, 293 cells were washed and plated on 60-mm plastic Petri dishes in medium without FCS. Cells were stably transfected using the calcium-phosphate method with 10-20 µg of purified plasmid DNA isolated from E. coli transfected with pCDM8 containing ICAM-1 cDNA (25). Cells expressing ICAM-1 were detected by incubation with mAb QE2 and FITC-conjugated anti-mouse IgG and isolated in a fluorescence-activated cell sorter (FACS). Levels of ICAM-1 expression were routinely monitored by FACS analysis.
FACS Analyses of Cell Surface Expression of ProteinsControl and ICAM-1-expressing 293 cells were isolated
from culture flasks by brief trypsin treatment. Raji were isolated from culture medium by centrifugation. Each cell type was washed twice in
Dulbecco's phosphate-buffered saline, pH 7.4, and once in Hanks' balanced salt solution without divalent cations then resuspended in a
staining medium of Hanks' balanced salt solution containing 2.0 mM CaCl2, 2.0 mM MgCl2,
25 mM HEPES, pH 7.4, and 0.1% bovine serum albumin. Cells
were counted and resuspended at 106 cells/ml, and 0.1-ml
aliquots were incubated at 4 °C for 30 min with 5.0 µg/ml control
mouse IgG, anti-M mAb OKM1,
anti-
V
3 mAbs LM 609 or mAb1980,
anti-
X mAb KB90, anti-
5 mAb mAb1969, or
anti-ICAM-1 mAbs QE2 or 84H10. Cells were centrifuged through a cushion
of FCS and resuspended in staining medium containing 50 µg/ml
FITC-conjugated goat anti-mouse IgG antibodies (Zymed Laboratories,
South San Francisco, CA). Cells were incubated for 30 min at 4 °C
and then centrifuged and resuspended in 0.5 ml of staining medium.
Cell-bound antibodies were detected using a FACScan, and analysis was
performed using the LYSIS II program (Becton Dickinson, San Jose,
CA).
Fg
was purified from fresh human plasma by cryoethanol precipitation (26,
27). Using electrophoretic conditions that allow the separation of
fibrin from fibrinogen monomer and subsequent visualization using
Coomassie violet R-250 (18), the isolated material was estimated to
comprise greater than 95% fibrinogen. Preparations of Fg were also
analyzed for the presence of free fibrinopeptides A and B by elution on
a Sep-Pak C18 high pressure liquid chromatography column
using standard preparations of each of the fibrinopeptides (Sigma). At
protein concentrations of at least 50-fold greater than those used in
these experiments, amounts of fibrinopeptides A and B were below
detectable levels. Fg was radiolabeled with 125I-Na using
IODO-BEADS (Pierce) (28, 29) and extensively dialyzed against 0.3 M NaCl. The specific activity of 125I-labeled
Fg was 0.45-0.52 mCi/mg. Radiolabeled Fg was aliquoted and stored at
80 °C until use.
For the preparation of plasmin-digested Fg fragments (26), purified Fg
(5.0 mg/ml) was incubated at 37 °C with 8.3 µg/ml plasmin for
1 h (fragment X) and 0.1 mg/ml plasmin for 2 h (fragment D100). Each fragment was purified by ion-exchange
chromatography on a column of DEAE-cellulose (Pharmacia, Uppsala,
Sweden) using an elution buffer of 50 mM Tris/HCl, pH 7.4, containing 0.4 M NaCl and dialyzed extensively against
phosphate-buffered saline. Fg fragment E was isolated as a by-product
of fragment D preparations by subjecting these preparations to size
exclusion chromatography on a column of Sephacryl S200 (Pharmacia,
Uppsala, Sweden) equilibrated with a solution of 50 mM
Tris/HCl, pH 7.4, containing 0.15 M NaCl. Fractions
containing Fg fragment E were dialyzed extensively against distilled
water and lyophilized. The molecular weights of plasmin digestion
products of Fg were verified by electrophoresis on SDS gels. Protein
concentrations were adjusted to 1 mg/ml using Iscove's modified
Dulbecco's Eagle's medium containing penicillin, streptomycin, 1.0 mM glutamine, and 5 µg/ml transferrin (IMDM) (Life
Technologies, Inc.), and stored at 20 °C.
The full-length five-domain form of
ICAM-1 was ligated into the viral vector PVL1392 (Invitrogen, San
Diego, CA) and allowed to recombine with baculoviral genomic DNA (14,
30-32). Truncated ICAM-1 cDNA was generated by polymerase chain
reaction using specific primers which annealed to the extreme 5 region
of the cDNA and a region 3
to cDNA encoding the second
IgG-like domain. The 3
reverse primer contained appropriate sequences
for a stop codon and a NotI restriction enzyme site which
was unique for cDNA encoding the first two Ig domains of ICAM-1.
The nucleotide sequence of the polymerase chain reaction-amplified DNA
was confirmed, and the DNA was ligated into the baculovirus vector
pBlueBacIII using XbaI and NotI restriction
sites. Recombinant virus containing vector DNA was purified by repeated
plaque purification and used for infection of Spodoptera
frugiperda (Sf9) insect cells. Sf9 cells were grown in Grace's
insect cell media (Life Technologies, Inc.) for 4 days, then washed
with phosphate-buffered saline, pH 7.4, and lysed in 1% (w/v) octyl
glucoside in phosphate-buffered saline, pH 7.4. Expression of intact
ICAM-1 or the two domain form of ICAM-1 (D1D2
ICAM-1) was confirmed by enzyme-linked immunosorbent assay and
immunoblotting using ICAM-1-specific antibodies as described previously
(14).
Full-length ICAM-1 and D1D2 ICAM-1 were
purified from Sf9 cell lysates on a Rotofor preparative isoelectric
focusing apparatus (Bio-Rad) using a pH gradient of 6-8. Portions of
SDS gels containing similar, unstained material were excised, and
ICAM-1 proteins were extracted in a buffer of 0.2 M
Tris/HCl, pH 7.4, containing 2% (w/v) SDS and precipitated with 80%
(v/v) acetone. The purity of D1D2 ICAM-1 was
greater than 90%, as judged by silver staining of SDS gels, and
portions of purified D1D2 ICAM-1 were
resuspended in IMDM in 1 µM aliquots and stored at
20 °C. In adhesion assays, this protein preparation was able to
support the adhesion of 51Cr-labeled peripheral blood
lymphocytes and adhesion could be blocked by the anti-
2
antibody MHM23.
Raji or 293 cells were maintained
in appropriate medium without FCS for 24 h prior to commencement
of an experiment, to achieve cellular quiescence. Cells were washed
twice in IMDM and counted with a hemocytometer. Some cells were
preincubated for 30 min with control antibodies or antibodies directed
against ICAM-1. Aliquots of 0.2 ml of cells (4 × 105
cells/ml) were mixed with 0.2-ml protein solutions diluted with IMDM
and 4 µl of [3H]thymidine (1.0 µCi/µl). Cell
suspensions (0.1 ml) were aliquoted into four replicate wells of a
96-well flat-bottomed plate (Becton Dickinson, Franklin Lakes, NJ), and
plates were stored at 37 °C and 5% CO2 for 4-24 h. To
measure uptake of [3H]thymidine, the contents of each
well were transferred to a 96-well plate with v-shaped wells. Cells
were isolated from incubation medium by centrifugation (200 × g/10 min) and washed twice in phosphate-buffered saline, pH
7.4. [3H]Thymidine that had been incorporated into
cellular DNA was precipitated by addition of 0.1 ml of 20% (v/v)
trichloroacetic acid for 2 h at 4 °C. Trichloroacetic acid
pellets were rinsed once and resolubilized in 2% (w/v) SDS solution
and assayed for radioactivity in a -counter.
Raji were centrifuged and resuspended
three times in Hanks' balanced salt solution and finally in Hanks'
balanced salt solution containing 0.1% bovine serum albumin. The
number of cells isolated was ascertained using a hemocytometer, and
some cell suspensions were preincubated with 25 µg/ml TS1/18
anti-2 integrin antibody or an anti-ICAM-1 antibody
84H10 for 30 min. Increasing amounts of 125I-labeled Fg
were mixed with 0.1 ml of Raji (5 × 106 cells/ml) in
a final volume of 0.2 ml. Some cell mixtures also containing a
10-50-fold excess of either unlabeled Fg or transferrin. All cell
suspensions were incubated for 30 min at 22 °C. Following incubation, free radioactivity was separated from radioactivity bound
to the cell surface by centrifugation at 11,300 × g for 2.5 min through an upper phase of 8% sucrose (0.2 ml) and a lower phase
(0.1 ml) containing dibutyl phthalate and dimethyl phthalate (Aldrich)
mixed at a proportion of 10:1.05 (33). Radioactivity associated with
the cell pellet was assayed using a
-counter.
Peptides with amino acid sequences
corresponding to regions of ICAM-1 and fibrinogen were synthesized by
the Fmoc (N-(9-fluorenyl)methoxycarbonyl) method on an
Applied Biosystems ABI-66 instrument. Specific sequences were ICAM-1
(8-22) KVILPRGGSVLVTCS, ICAM-1 (130-139) REPAVGEPAE, Fg
A(571-576) GRGDSP, Fg
(117-133) NNQKIVNLKEKVAQLEA, Fg
(117-133) scrambled ALENAEVQNLVKKIQKN, and Fg
(124-133)
LKEKVAQLEA. Peptides were cleaved from the resin and deprotected using
crystalline phenol and thioanisole as described in the instruction
manual. Peptides were purified by high pressure liquid
chromatography.
These studies were carried out using the Raji B lymphocyte
cell line as these cells demonstrate a constitutive and stable expression of ICAM-1, and are readily grown in suspension culture. FACS
analysis of Raji using the anti-ICAM-1 mAb QE2 revealed a constitutive
and stable expression of ICAM-1 (Fig. 1A, panel
a) which represented approximately 75% of the levels of ICAM-1
found on endothelial cells upon treatment with tumor necrosis
factor- using mAb QE2 (14). Raji also demonstrated a constitutive
expression of
L (panel d); however, binding
of Raji by antibodies directed against the
subunits of integrins
reported to bind Fg including
V (panel b),
5 (panel c),
M (panel
e), and
X (panel f) could not be
detected, suggesting negligible levels of the integrin heterodimers
M
2,
X
2,
V
3, or
5
1
on Raji. As levels of these Fg-binding proteins (34-37) were below
detectable levels, Raji B lymphocytes provided a suitable means of
exclusively studying Fg:ICAM-1 interactions. Fg binding to Raji has
been previously reported by others (16), and we have recently
demonstrated that Raji adhesion to Fg was ICAM-1-dependent
(14). This finding was confirmed using a soluble Fg binding assay. A
specific and saturable binding of 125I-labeled Fg to Raji
was observed (Fig. 1B). 125I-Labeled Fg
associated with Raji with a Kd of 3 × 10
7 M and this association could be
competitively blocked by an excess of unlabeled Fg but not an
equivalent amount of transferrin. This Kd value is
comparable to other Kd values reported for ICAM-1/Fg
interactions (3) and Fg interactions with Raji cells (16). Analysis of
the 125I-labeled Fg binding data suggested a single class
of binding sites on Raji which bound a total of approximately 40,000 molecules/cell. Inclusion of 25 µg/ml anti-ICAM-1 antibody 84H10
completely abrogated specific 125I-labeled Fg binding to
Raji, while
2 integrin mAb TS1/18 failed to block
125I-labeled Fg binding to Raji.
Experiments were designed to investigate consequences of soluble Fg
binding to Raji. When purified human Fg was included in the culture
medium of serum-depleted Raji, DNA synthesis was stimulated in a time-
and dose-dependent manner as measured by incorporation of
[3H]thymidine into macromolecules (Fig. 2,
A and B). This effect was specific to Fg as
inclusion of equivalent amounts of purified fibronectin or transferrin
did not induce any proliferative response in Raji (Fig. 2B).
Medium containing 200 nM Fg consistently induced a
2-3-fold increase in DNA synthesis of Raji after 8 h as compared with control Raji cultures incubated in
[3H]thymidine-containing medium alone (n = 15). This stimulation represented approximately 120% of the maximal
stimulation achieved by incubation of Raji in medium containing 10%
FCS after 8 h. After 24 h the Fg-mediated stimulation of Raji
was approximately 80% of that observed in cultures of Raji grown in
medium containing 10% FCS. A 2-fold increase in Raji cell numbers
after 8 h in culture with medium containing Fg was verified by
direct cell counting and by measurement of levels of intracellular
enzymes (data not shown), confirming that Fg had induced a cellular
proliferation. Similar amounts of fibrin-free Fg induced a response in
Raji identical to results presented in Fig. 2, A and
B, confirming that Fg was the mitogenic component.
A putative mitogenic Fg receptor of approximately 92 kDa was isolated
from Raji but not conclusively identified (38). As fibrinogen and
ICAM-1 are known to interact, and ICAM-1 is present on Raji cell
surfaces, we investigated whether the mitogenic Fg receptor on Raji was
ICAM-1. Fig. 2C shows that preincubation of Raji with 20 µg/ml anti-ICAM-1 mAb 84H10 before the addition of 200 nM
Fg resulted in a 91% inhibition of Fg-induced incorporation of
[3H]thymidine by these cells. The mAb 84H10 has been
reported to block
L
2-dependent binding (20,
21), and the epitope for 84H10 is located within the first Ig domain of
ICAM-1 (22). Inclusion of normal mouse IgG (not shown) or
anti-
2 integrin mAb TS1/18 (20 µg/ml) had no effect on
the Fg-induced uptake of [3H]thymidine by Raji.
To determine whether Fg could invoke a proliferative
response in another ICAM-1-expressing cell type, a stably transfected cell line was used in proliferation assays. Fibroblast 293 cells that
had been stably transfected with ICAM-1 cDNA and cloned by FACS as
described under "Materials and Methods," were maintained for
24 h in serum-free medium. As non-transfected 293 cells have detectable levels of integrin 5
1 (39),
which has been reported to mediate fibrinogen binding to endothelial
cells (37), we included non-transfected 293 cells for comparison of
fibrinogen-mediated cell proliferation. FACS analysis of cell surface
levels of ICAM-1 on transfected cells using either control IgG or the
anti-ICAM-1 mAb QE2 and an FITC-conjugated disclosing antibody as
described under "Materials and Methods" confirmed the stable
expression of ICAM-1 (Fig. 3A, panel b) with
mean fluorescence intensities approaching levels observed on Raji. No
measurable amounts of ICAM-1 (Fig. 3A, panel a; Ref. 39)
could be detected on non-transfected 293 cells.
ICAM-1-transfected 293 cells and control non-transfected cells were removed from tissue culture flasks, washed, and placed in culture medium containing 0-800 nM Fg and [3H]thymidine for 8 h. Fig. 3B shows that in the presence of increasing amounts of Fg, levels of incorporation of [3H]thymidine in ICAM-1-transfected 293 cells increased over levels of [3H]thymidine in non-transfected cells. The pattern of the mitogenic response induced by Fg on 293-transfected cells resembled that observed in Raji. Maximal proliferation was noted at 400 nM Fg, while at levels of 800 nM Fg the response was reduced. Inclusion of equivalent amounts of fibronectin or transferrin had no effect on the incorporation of [3H]thymidine in incubations of either cell type. However, levels of incorporated [3H]thymidine increased in cultures containing either non-transfected 293 cells or ICAM-1-transfected 293 cells in response to basic fibroblast growth factor as compared with cells incubated in medium alone (data not shown).
The First Ig-like Domain of ICAM-1 Mediates Fg-induced Raji ProliferationIn our previous report (14), a truncated form of
ICAM-1 containing the first two Ig domains
(D1D2 ICAM-1) was determined to be able to
interact with Fg. Because of the importance of this segment of ICAM-1
for associations with Fg, we investigated whether this region of ICAM-1
was important for the Fg-mediated proliferative response of Raji. The
cDNA encoding for a truncated form of ICAM-1 that contained the
first two Ig-like domains was expressed in Sf9 cells and purified as
described under "Materials and Methods." D1D2 ICAM-1 migrated on an SDS gel with an
apparent molecular mass of 35 kDa and was recognized by R686
anti-ICAM-1 polyclonal antibody by immunoblotting (Fig.
4A) and enzyme-linked immunosorbent assay
(data not included). In addition, D1D2 ICAM-1
supported the adhesion of purified peripheral blood lymphocytes. This
adhesion was 2-integrin-dependent as it could be blocked
by inclusion of antibody MHM23. When Fg was preincubated at 37 °C
with increasing amounts of D1D2ICAM-1,
Fg-induced proliferation was blocked in a dose-dependent
manner (Fig. 4B). Approximately 350 nM
D1D2 ICAM-1 abolished the proliferative action
of Fg (Fig. 4B). Control incubations containing Fg treated
with equivalent amounts of material purified from Sf9 cell lysates that
contained no anti-ICAM-1 immunoreactivity, had no effect on Fg-induced
Raji cell proliferation. The association of a 125I-labeled
D1D2ICAM-1 preparation with Raji cells could
not be detected in cell binding assays suggesting that this fragment of
ICAM-1 did not influence Fg-mediated proliferation by binding directly
to Raji; rather, D1D2 ICAM-1 competed with cell
surface ICAM-1 for binding sites on Fg. Furthermore, the inability of soluble ICAM-1 to bind to integrin
L
2 has been reported (40, 41).
Previous work from our laboratory has demonstrated that Fg is able to
interact with amino acids 8-22 of ICAM-1, which lie within the first
Ig domain of ICAM-1 (14). To further define the regions of ICAM-1 that
mediated the proliferative action of Fg, Fg was preincubated with
synthetic peptides with sequences corresponding to specific regions of
ICAM-1 and used in proliferation assays. Fig. 5 shows
that the mitogenic action of 200 nM Fg could be reduced by
up to 80% by preincubation of Fg with 300-500 µM amounts of ICAM-1 (8-22) but not ICAM-1 (130-139). Concentrations of
100 µM ICAM-1 (8-22) were ineffective in blocking
mitogenesis; however, we have noted that such concentrations of this
peptide were effective in blocking Raji adhesion to Fg (14). Incubation of Raji with medium containing 500 µM ICAM-1 (8-22)
alone did not affect the viability of Raji as judged by uptake of
[3H]thymidine by the cells and by trypan blue exclusion
(results not shown), suggesting that the peptide was acting externally. In summation these results suggest that the Fg-induced proliferative response in Raji is mediated by ICAM-1, specifically by at least one
region within the first IgG-like domain of ICAM-1 containing amino
acids 8-22.
A Discrete Region of Fg Contains Mitogenic Activity
We
investigated whether the ICAM-1-mediated proliferative property of Fg
could be attributed to a portion of Fg. A preparation of Fg was treated
with plasmin as described under "Materials and Methods," and the
resultant Fg degradation products were purified by ion-exchange
chromatography and used in Raji proliferation assays. A similar
induction of Raji proliferation could be demonstrated using purified Fg
plasmin-degradation products fragment X or fragment D but not fragment
E (Fig. 6), suggesting that at least one mitogenic site
resided within a region of Fg common to fragments X and D. This is in
good agreement with previous studies that report an association between
Fg and Raji cell surfaces (16), and a mitogenic proliferation of Raji
cells mediated by Fg fragment D (38, 42).
At least one region of Fg has been reported to be important for
interactions with ICAM-1. The Fg chain sequence 117-133 was
reported to block the Fg-mediated adhesion of leukocytic cells to
endothelial cell monolayers and was shown to interact directly with
ICAM-1-expressing transfectants (13). We investigated whether this
region of Fg encompassing amino acids 117-133 of the
chain could
induce a proliferative response in Raji. The peptide
(117-133) was
compared with Fg for mitogenic activity in Raji proliferation assays.
Fig. 7 shows that increasing concentrations of
(117-133) in Raji incubation medium were able to increase
incorporation of [3H]thymidine by Raji after 8 h.
Concentrations of 100 µM
(117-133) induced a response
similar to the observed proliferation of Raji in medium containing 200 nM Fg over the same period of incubation. Control peptides
(117-133) scrambled and
(124-133) had no discernible proliferative activity, suggesting that the amino acid sequence and in
particular the NH2-terminal portion of
(117-133) is
important for the proliferative activity of this peptide. Inclusion of
either the RGD-containing peptide Fg A
(571-576) (Fig. 7) or
fibrinopeptides A or B (data not included) also had no effect on
proliferation of Raji.
This paper documents evidence that ICAM-1 is the receptor
responsible for the Fg-mediated mitogenic response in Raji. The mitogenic effect of Fg and Fg fragment D on Raji has been reported (16,
42); however, the receptor responsible for this effect has not been
clearly defined. The present studies identify ICAM-1 as the candidate
protein and delineate the sites within ICAM-1 and Fg that mediate this
effect. In addition, our results indicate that Fg mediates a similar
mitogenic response in ICAM-1-transfected 293 cells, indicating that
other non-lymphoid cells have the internal signaling machinery required
for Fg-induced mitogenesis. Previously, the exposure of the B
(15-42) region of fibrin was demonstrated to enhance proliferation of
fibroblasts and endothelial cells (43) and the A
and B
chains of
fibrinogen have been reported to induce proliferation in fibroblasts
(44, 45). A 60-kDa protein resembling calreticulin was demonstrated to
be a receptor for the B
chain of Fg, and antibodies against
calreticulin could inhibit the observed mitogenic response (45). In a
separate report, a 130-kDa protein was isolated from endothelial cell
lysates by elution from a Sepharose-Fg B
(15-42) affinity column
(46). This candidate B
receptor was not identified; however, it was apparently not related to PECAM-1 or any known endothelial cell integrin
subunits. Fibrinopeptide A has been reported to induce proliferation in pleural mesothelial cells (47); however, under experimental conditions used for our studies, levels of fibrinopeptides A and B comparable to those used in studies by others did not induce a
mitogenic response in Raji. Interestingly, in a report of proliferation
of phorbol ester-stimulated tonsillar B lymphocytes induced by mAbs
against CD11c/CD18 (
X
2), occupancy of the
receptor by Fg blocked the mitogenic response in these cells (48).
We have previously reported that the first two Ig domains of ICAM-1
bind directly to Fg (14), and this portion of ICAM-1 contains sites
necessary for interaction with L
2,
rhinovirus (49-51), and Plasmodium falciparum-infected
erythrocytes (22, 52). Results presented here demonstrate that mAb
84H10 which maps to the first Ig domain of ICAM-1 blocked Fg binding to
Raji (Fig. 1B) and blocked Fg-induced mitogenesis (Fig.
2C). Consistent with these results,
D1D2 ICAM-1 protein specifically blocked
Fg-mediated mitogenesis in Raji (Fig. 4B), suggesting that
this region of ICAM-1 is also important for initiation of the cellular
response to Fg binding. In previous studies, the region of ICAM-1
spanning amino acids 8-21 was determined to be involved in Fg
recognition (14). We used ICAM-1 (8-22) in the present studies as this
peptide has superior solubility properties and has Fg-binding
properties similar to ICAM-1 (8-21). At a concentration of 300 and 500 µM, comparable to amounts required to block Raji adhesion
to Fg, ICAM-1 (8-22) also blocked Fg-mediated Raji proliferation (Fig.
6). Lower concentrations of ICAM-1 (8-22) did not inhibit Fg-mediated
mitogenesis, but were able to block adhesion of Raji to Fg bound to
plastic (14), implying that ICAM-1-binding sites are more readily
accessible within immobilized Fg. Therefore ICAM-1 (8-22) is
implicated in the Fg-mediated response of Raji. The control peptide
with amino acid sequence corresponding to ICAM-1 (130-139) was chosen
as it contains a charged, hydrophilic NH2-terminal region
similar to ICAM-1 (8-22). Computer modeling of this region of ICAM-1
suggests that several amino acid residues in this defined region are
accessible to solvents (14), and ongoing studies expressing recombinant ICAM-1 with mutations in this region will confirm the involvement of
this region in mitogenesis.
The physiological degradation of Fg and fibrin by plasmin is key to the
process of fibrinolysis and breakdown of a thrombus (10). Purified Fg
degradative products fragment X and fragment D100 also
induced a mitogenic response in Raji (Fig. 6). It is important to note
that even though the precise affinities of fragments X and D to ICAM-1
have not been reported, the estimated affinity of fragment X for ICAM-1
(8-21) appears comparable to that of Fg and that fragment D is only
5-fold lower than Fg (14). This is in contrast to the reported
affinities of fragments X and D for IIb
3,
which are 10- and 100-fold less than Fg, respectively (53). Consistent
with the apparent binding affinity of ICAM-1 for Fg and Fg fragments X
and D, we observed that the extent of mitogenesis induced by fragment X
was at least equal to that by Fg while mitogenesis induced by fragment
D was approximately 80% of that observed using intact Fg.
The Fg chain sequence
(117-133) is reported to interfere with
Fg binding to ICAM-1, and this region of Fg is directly involved in the
transmigration of leukocytes across endothelial cells (13). In our
previous studies,
(117-133) blocked Fg binding to ICAM-1 (8-21),
suggesting that
(117-133) and ICAM-1 (8-21) may be an important
reactive pair in Fg-ICAM-1 interactions (14). The
(117-133) peptide
was able to induce a mitogenic response in Raji at concentrations of 50 µM (Fig. 7), suggesting that this region of the Fg
chain contains a novel mitogenic capacity. This sequence resides within
the CS region of Fg and is likely to be at least partially shielded by
the C-terminal portion of the A
chain, A
(242-611). Support for
this model comes from the observation that the region immediately
adjacent,
(112-119), is inaccessible to mAb 9F9 in soluble Fg but
becomes exposed by proteolytic removal of the C-terminal aspect of the
A
chain (54). Other groups have reported an association between
soluble Fg and ICAM-1 expressing cells (13), and in this report we
demonstrate an association between soluble Fg and Raji that was blocked
by an anti-ICAM-1 antibody. It is possible that the association between soluble Fg and ICAM-1 through
(117-133) forms part of a multistep process. However, it remains to be determined whether some proteolytic processing of soluble Fg is required for complete exposure of the
(117-133) sequence.
Elevated levels of ICAM-1 are found on inflamed tissues and ICAM-1 is present in atherosclerotic lesions, and within these regions fibrin, Fg, and Fg degradation products have been detected. As levels of Fg circulating in human plasma normally approach 3.0 mg/ml, it may be anticipated that the ICAM-1-mediated mitogenic activity of Fg in vivo is modulated by levels of expression of ICAM-1 on cells and therefore would not occur under normal physiological conditions. However, under conditions of both acute and chronic inflammation, elevated levels of ICAM-1 on endothelial cells and smooth muscle cells (8, 55) may mediate a Fg-dependent proliferation of cells. This remains to be determined experimentally; however, a combined role for ICAM-1 and Fg in the adhesion and arrest of infiltrating inflammatory cells within such sites which contributes to the observed pathology of these lesions has been proposed (3).
We thank Vicky Byers-Ward for expert technical assistance, and Jane Rein for assistance in preparation of the manuscript. We also acknowledge and thank Dr. Edward Plow and Dr. John Shainoff for helpful discussions as well as critical review of the manuscript.