(Received for publication, October 15, 1994)
From the
In addition to its role in hemostasis, fibrinogen is
obligatorily required to mount competent inflammatory responses in
vivo. A molecular prerequisite of fibrinogen-dependent
inflammation may reside in its ability to associate with intercellular
adhesion molecule-1 (ICAM-1), and enhance monocyte adhesion to
endothelium by bridging the two cell types. Structure-function
characterization of the novel ICAM-1 recognition of fibrinogen was
carried out by synthetic peptidyl mimicry of the fibrinogen
chain. A novel peptide sequence,
N
NQKIVNLKEKVAQLEA
, designated
3,
dose-dependently inhibited (IC
20-40
µg/ml) binding of
I-fibrinogen to endothelial cells
or ICAM-1-expressing B lymphoblastoid Daudi cells. In contrast, none of
the previously identified vascular cell fibrinogen interacting
sequences was effective. Increasing concentrations of
3 completely
inhibited fibrinogen-mediated adhesion of peripheral blood mononuclear
cells or vitamin D
-differentiated monocytic HL-60 cells to
endothelium, but did not affect leukocyte-endothelium interaction in
the absence of fibrinogen.
I-Labeled
3 bound
specifically and saturably to genetically engineered ICAM-1
transfectants, but not to control non-transfected cells, and associated
with ICAM-1 on cytokine-activated endothelium with a K
of 34 µM. Consistent with
functional recognition of ICAM-1, immobilized
3 supported adhesion
of JY lymphoblasts in a dose-dependent reaction inhibited by monoclonal
antibodies to ICAM-1. We conclude that a novel fibrinogen
3
sequence N
NQKIVNLKEKVAQLEA
binds to ICAM-1
and modulates ICAM-1-dependent adhesion. These findings define the
structural basis of fibrinogen:ICAM-1 recognition and provide a
potential selective target for inhibiting fibrinogen-dependent
inflammatory responses.
Although primarily recognized for preserving
hemostasis(1) , fibrinogen plays a prominent role in
inflammation. Through the structurally diversified recognition of
disparate membrane receptors, regulated assembly of fibrinogen on
leukocytes participates in intracellular signal transduction,
transcription, and translation of activation-dependent genes, release
of inflammatory cytokines, and modulation of various leukocyte effector
functions (reviewed in (2) ). In this context, fibrinogen is
obligatorily required to organize competent inflammatory responses in vivo. Extending earlier observations on the pathogenetic
role of fibrinogen in delayed type hypersensitivity(3) , recent
studies have demonstrated that systemically defibrinogenated animals
fail to recruit monocytes/phagocytes after intra-abdominal piogenic
challenges(4) , or at the site of biomaterial
implant(5) , and are partially protected from acute tissue
damage mediated by platelet-leukocyte cooperation during immune complex
glomerulonephritis(6) . Adhesion of leukocytes to vascular
endothelium is one of the earliest molecular prerequisites of all
inflammatory responses(7, 8) . Although this process
is contributed by a redundant ``adhesion cascade'' maintained
by various classes of cell surface receptors(9) , recent
evidence suggests that fibrinogen may also participate in this
mechanism by acting as a bridging molecule between leukocytes and
endothelial cells through a novel recognition of
ICAM-1()(10) . In order to elucidate the molecular
requirements of fibrinogen-dependent inflammation, we have used
synthetic peptidyl mimicry and genetically engineered transfectants to
dissect the ICAM-1:fibrinogen recognition. The results indicate that a
novel fibrinogen
chain sequence 117-133 interacts with
ICAM-1 and modulates ICAM-1-dependent cell
adhesion(7, 8, 9) .
Figure 1:
A, effect of 3 on
I-fibrinogen binding to Daudi. Serum-free suspensions of
CD11b/CD18
lymphoblastoid Daudi cells at 1.5
10
/ml were preincubated with the indicated increasing
concentrations of
3 (
, fibrinogen
chain
117-133), or control peptides P1 (
, 100 µg/ml), GPRP
(
, 100 µg/ml), or L10 (
, 50 µg/ml) for 10 min at
22 °C. Cells were mixed with 50 µg/ml
I-fibrinogen for 30 min at 22 °C in the presence of 1
mM CaCl
and 1 mM MgCl
, and
free from cell-associated radioactivity was separated by centrifugation
through mixture of silicone oil, before calculation of specific
binding.
I-Fibrinogen binding to Daudi in the absence of
competitors (100% binding) was 25,532 ± 3,150 molecules/cell.
Data are the mean ± S.E. of three independent experiments. B, effect of
3 on fibrinogen-mediated
leukocyte-endothelium interaction. Suspensions of PBMC (closed
symbols;
,
3;
, L10) or vitamin
D
-differentiated monocytic HL-60 cells (open
symbols;
,
3;
, L10) were preincubated with the
indicated increasing concentrations of
3 or with 400 µg/ml
control peptide L10, stimulated with 10 µM fMet-Leu-Phe,
and equilibrated with 150 µg/ml fibrinogen for 20 min at 22 °C,
in the presence of 2.5 mM CaCl
. Leukocyte adhesion
under the various conditions tested was quantitated after a 30-min
incubation at 22 °C. Data are the mean ± S.E. of two
independent experiments.
Figure 2:
A, characterization of ICAM-1
transfectants. CHO cells were transfected with a full-length ICAM-1
cDNA, selected in 1 mg/ml G418, and cloned by limiting dilution.
Quantitation of ICAM-1 surface expression on WT CHO cells or on ICAM-1
transfectants (clone 1) was carried out by flow cytometry with
anti-ICAM-1 mAbs LB-2 or 2D5 (Footnote 2). Similar results not shown in
the figure were obtained with anti-ICAM-1 mAbs 1G12 and 6E6 (Footnote
2). Background fluorescence was determined in the presence of control
mAb OKM1. B and C, binding of I-
3
to ICAM-1 transfectants. Increasing concentrations (6-150
µg/ml) (B) or 50 µg/ml (C) of
I-
3 were added to ICAM-1 transfectants, or control
WT CHO cells, for 45 min at 22 °C in the presence of 1 mM CaCl
and 1 mM MgCl
. Nonspecific
binding was assessed in the presence of a 100-fold molar excess of
unlabeled
3 and was subtracted from the total to calculate net
specific binding. Data are the mean ± S.E. of the replicates of
a representative experiment.
Figure 3:
Scatchard plot analysis of ligand binding
to ICAM-1. Confluent HUVEC monolayers were stimulated with 100 units/ml
tumor necrosis factor- for 6 h at 37 °C, and incubated with
increasing concentrations of
I-fibrinogen (1-50
µg/ml) or
I-
3 (6-150 µg/ml) for 45 min
at 22 °C. After washes, specific binding was calculated as
described in the legend to Fig. 2. Scatchard plot analysis and
quantitative parameters of binding isotherms were generated with the
Ligand program based on the algorithm of MacPherson(16) . Data
are representative of at least four independent
experiments.
The functional effect of 3 on
ICAM-1-dependent adhesion was investigated. As shown in Fig. 4,
immobilized
3 specifically supported adhesion of JY lymphoblasts
in a concentration-dependent reaction (Fig. 4A), that
was abrogated by cell preincubation with mAbs 1G12 and 2D5, previously
selected for inhibiting ICAM-1-fibrinogen interaction
(Fig. 4B).
Figure 4:
A, 3-mediated lymphoblast adhesion.
Plastic assay plates were coated with the indicated increasing
concentrations of
3 or control peptide L10, blocked with 3%
gelatin, and incubated with
Cr-labeled JY lymphoblasts at
1
10
/ml for 45 min at 37 °C, before
quantitation of attached cells. B, ICAM-1-dependent
lymphoblast adhesion to
3. The experimental conditions are the
same as in A, except that JY lymphoblasts were preincubated
with or without the various anti-ICAM-1 mAbs 2D5, 1G12, 6E6 (Footnote
2) at 25 µg/ml before addition to peptide-coated plates and
quantitation of cell attachment. Anti-ICAM-1 mAb 6E6 does not inhibit
ICAM-1-fibrinogen interaction (Footnote 2).
In this study we have identified a novel fibrinogen
chain sequence (N
NQKIVNLKEKVAQLEA
) that
mediates ligand binding to ICAM-1 and potentially contributes to
fibrinogen-dependent
inflammation(3, 4, 5, 6) . As
anticipated in previous studies(10) , the ability of fibrinogen
to enhance monocyte adhesion to endothelium by bridging the two cell
types (10) may represent one of the earliest molecular
prerequisites for its obligatory role in preserving competent
inflammatory responses in
vivo(3, 4, 5, 6) .
Initial
screening of a library of fibrinogen-derived peptides synthesized and
characterized in previous studies(11) , identified the 3
sequence for its ability to block binding of
I-fibrinogen
to HUVEC and other ICAM-1-expressing cells, i.e. Daudi, and to
inhibit monocyte-endothelium interaction mediated by
fibrinogen(10) . While none of the other previously identified
fibrinogen interacting motifs with vascular cell receptors was
effective in this system, the
3 sequence may act in concert with
the previously described CD11b/CD18 recognition on the fibrinogen
chain Gly
-Val
(11) , to facilitate
leukocyte anchoring to endothelium in the bridging model of
intercellular adhesion(10) . Physical association of the
3
sequence with ICAM-1 (10) was demonstrated in direct
I-labeled peptide binding studies to HUVEC and
genetically engineered ICAM-1 transfectants. As judged by Scatchard
plot analysis of binding isotherms,
I-
3 association
with ICAM-1 was regulated by a K
of 34
µM, in excellent agreement with the IC
concentrations of 20-40 µg/ml required for peptide
inhibition of fibrinogen binding and leukocyte-endothelium bridging.
This suggests that
3-mediated inhibition of ICAM-1 adhesive
recognitions is mediated by genuine peptidyl mimicry, with disruption
of the ligand binding groove as a consequence of receptor
occupancy(19) . It is noteworthy that
3 had no inhibitory
effect in experiments that directly investigated the molecular
association between ICAM-1 and CD11a/CD18, as exemplified by HL-60 cell
adhesion to cytokine-activated HUVEC or to monolayers of ICAM-1
transfectants in the absence of fibrinogen. This suggests that the
fibrinogen binding site on ICAM-1 may be spatially and topographically
distinct from the CD11a/CD18 recognition on the first Ig-like domain of
the receptor(9) .
While other regions in fibrinogen may
cooperate in ligand docking with ICAM-1, the data reported here suggest
that the 3 motif functions as a dominant recognition sequence for
ICAM-1 in cell adhesion reactions. In this context, similar to the
ability of other linear peptidyl sequences in fibrinogen(11) ,
CS1 fibronectin(20) , or ICAM-2 (21) to function as
competent cell adhesion-promoting ligands irrespective of their
conformational state, immobilized
3 supported attachment of JY
lymphoblasts in a reaction inhibited by anti-ICAM-1 mAbs.
In
summary, the identification of the 3 sequence as a minimal ligand
recognition for ICAM-1 offers a novel potential target for highly
selective inhibition of fibrinogen-dependent cellular inflammatory
responses in
vivo(3, 4, 5, 6) . In this
context, peptidomimetics derived from the
3 sequence and structure
would have the required specificity to interfere with
fibrinogen/ICAM1-1-dependent leukocyte-endothelium bridging,
without affecting the hemostatic functions of other vascular cell
fibrinogen receptors in vivo(2) .