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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 3624-3631
Inhibition of Cell Adhesion by Antibodies to Arg-Gly-Asp (RGD) in
Normal Immunoglobulin for Therapeutic Use (Intravenous Immunoglobulin,
IVIg)
By
Tchavdar L. Vassilev,
Michel D. Kazatchkine,
Jean-Paul Duong Van
Huyen,
Medina Mekrache,
Emmanuelle Bonnin,
Jean Claude Mani,
Chantal Lecroubier,
Dirk Korinth,
Dominique Baruch,
Folke Schriever, and
Srini V. Kaveri
From INSERM U430 and the Université Pierre et Marie Curie,
Hôpital Broussais, Paris, France; INSERM U143, Hôpital de
Bicêtre, Bicêtre, France; CNRS UMR 9921, Montpellier, the
Laboratoire d'Hematologie, Hôtel Dieu, Paris, France; and the
Biomedical Research Centre Virchow Klinikum, Humboldt University,
Berlin, Germany.
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ABSTRACT |
Intravenous immunoglobulin (IVIg) therapy is associated with a broad
range of immunomodulatory activities. Several of the postulated
mechanisms of IVIg action relate to the presence of antibodies to
molecules relevant for regulation of the immune response.
This article reports that IVIg contains antibodies to the Arg-Gly-Asp
(RGD) sequence, and the attachment site of a number of adhesive
extracellular matrix proteins, including ligands for  1, 3, and
5 integrins. Anti-RGD antibodies were identified in IVIg by
enzyme-linked immunosorbent assay and by using the BIAcore (BIAcore,
Uppsala, Sweden) technology. The affinity of anti-RGD antibodies to a
synthetic RGD-containing peptide and to fibronectin (Fn) was found to
be in the micromolar range. F(ab')2 fragments specific
for RGD were purified from IVIg by affinity chromatography. Anti-RGD
F(ab')2 antibodies inhibited adenosine diphosphate
induced  IIb/3 integrin-mediated platelet aggregation and the
adhesion of activated 41 integrin-expressing B cells to Fn.
Adhesion of unstimulated platelets to fibrinogen (Fg) involving both
the  -chain dodecapeptide sequence and the RGD sequence was inhibited
by anti-RGD antibodies. In addition, adhesion of thrombin-stimulated platelets to von Willebrand factor or Fg was completely inhibited by
affinity-purified anti-RGD antibodies. Our results suggest that the
presence of natural IgG antibodies to the RGD motif may contribute to
the immunomodulatory and anti-inflammatory effects of therapeutic
preparations of normal IgG.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
INTRAVENOUS immunoglobulin (IVIg) is
pooled normal polyspecific IgG obtained from plasma of several thousand
healthy donors. It has been shown to be effective in the treatment of patients with a variety of different autoimmune diseases and
inflammatory disorders.1 Several mechanisms of action have
been postulated to account for the immunomodulatory effects of
IVIg.2,3 Of potential relevance to understanding the
effects of IVIg, is that it contains natural antibodies to cell surface
molecules that are essential for the induction and the control of the
immune response. Thus, IVIg was shown to contain antibodies reactive with human CD4,4 CD5,5 nonpolymorphic
determinants of HLA class I molecules,6 and determinants of
the human B-cell antigen receptor and  T-cell receptor
(TCR).7,8 Natural autoantibodies to these functionally
relevant molecules, purified from IVIg, exhibit immunomodulatory properties.
Integrins belong to a family of evolutionary-conserved heterodimeric
cell-surface glycoproteins that mediate divalent cation-dependent cell-to-cell and cell-matrix interactions.9-12 Integrins
play a critical role in cell differentiation and embryonic development, inflammation, immune responses, thrombosis, malignant transformation, and metastasis.13 Integrins consist of a distinct subunit noncovalently associated with a chain that is common to all integrin molecules of a particular family.9,14 Different
integrins may bind to one or more distinct site on the same ligand.
Most integrins of the 1, 3, and 5 families bind to the RGD
(Arg-Gly-Asp) sequence that is expressed on a large number of cell
surface and matrix proteins.13,15-17
In the present study, we show that IVIg contains antibodies directed
against a 10-amino acid peptide containing the RGD motif, which inhibit
the adhesion of B lymphocytes to fibronectin (Fn). Further, the
affinity-purified anti-RGD antibodies inhibited several RGD-mediated
interactions such as adhesion of unstimulated platelets to fibrinogen
(Fg), adhesion of thrombin-stimulated platelets to Fg/von Willebrand
factor (vWF)-coated surface and adenosine diphosphate (ADP)-induced
platelet aggregation. These antibodies are relevant for the
immunomodulatory effects of IVIg in autoimmune and inflammatory
diseases and for understanding the role of normal IgG in immune homeostasis.
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MATERIALS AND METHODS |
Reagents.
The RGD sequence-containing peptide AVTGRGDSPA and the irrelevant
peptide ARTGVGSPDA containing the same residues in a shuffled order
were synthesized by Neosystems (Strasbourg, France). Human Fg, Fn,
vitronectin, laminin, and rabbit anti-Fn anti-serum were obtained from
Sigma (Sigma Chemical Co, St Louis, MO). Human vWF was from Rohrer
Biotechnology (King of Prussia, PA). The anti-CD19 monoclonal antibody
(MoAb) (clone J4.119) was from Immunotech, Marseille, France.
Sources of immunoglobulins.
IVIg (Sandoglobulin) was a gift of the Central Laboratory of the Swiss
Red Cross (Bern, Switzerland). Human IgG myeloma MC was a gift of D. Glotz (Hôpital Broussais). F(ab')2 fragments were
prepared from IVIg and from protein G-purified myeloma IgG by pepsin
digestion (2% wt/wt) (Sigma) in acetate buffer pH 4.1 for 18 hours at
37°C followed by chromatography on protein G-sepharose. F(ab')2 fragments were free of intact IgG and Fc
fragments as assessed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and enzyme-linked immunosorbent assay (ELISA).
Binding assays.
Binding of anti-RGD antibodies to the peptide and to proteins
expressing the RGD sequence was assessed by ELISA and by demonstration of complex formation using the BIAcore technology
(BIAcore, Uppsala, Sweden). For ELISA, the RGD-containing decapeptide,
Fn, vitronectin, fg, and vWF were bound to glutaraldehyde pretreated or
untreated MaxiSorp Immuno-Plates (Nunc, Roskilde, Denmark). The plates
were incubated with 1% bovine serum albumin (BSA) in
phosphate-buffered saline (PBS) before incubation with serial dilutions
of the antibodies to be tested for 1 hour at room temperature. Bound
antibodies were revealed by using peroxidase-conjugated anti-human
F(ab')2 antibodies (Jackson Immunoresearch Laboratories,
West Grove, PA). Optical density was recorded at 490 nm by using an
Emax ELISA reader (Molecular Devices, Menlo Park, CA). For competitive
binding experiments, affinity-purified anti-RGD F(ab')2
fragments were radiolabeled to a specific activity of 0.8 µCi/µg with 125I by using Iodogen (Pierce,
Rockford, IL). For real time analysis of complex formation between
anti-RGD F(ab')2 fragments and RGD-containing ligands by
using the BIAcore system, decapeptide (50 µg/mL in 10 mmol/L sodium
acetate buffer, pH 6.2) or Fn (50 µg/mL in the same buffer, pH 5),
were immobilized on a sensor chip surface that had been preactivated
with 100 mmol/L N-ethyl-N'-(3-dimethylaminopropyl) carbodiimidine
hydrochloride and 400 mmol/L hydroxysuccinimide. The surface was then
inactivated with 35 µL of ethanolamine hydrochloride 1 mol/L NaCl pH
8.5. F(ab')2 fragments to be tested were injected at
concentrations ranging between 1.75 to 14 µmol/L at a flow rate of 10 µL per minute. Regeneration of the sensor chip was performed by using
10 µL of 100 mmol/L hydrochloric acid. Controlled experiments were
performed by injecting F(ab')2 fragments into an uncoated
flow cell that had been activated and blocked with ethanolamine as
described above. Kinetic parameters of binding were determined by using
the BIAevaluation software (Biacore).
Platelet aggregation.
Aliquots of 200 µL of freshly prepared platelet-rich plasma (PRP)
from healthy donors were incubated with increasing amounts of
F(ab')2 fragments of IgG to be tested for 5 minutes at
37°C. After adding 2.5 6 mol/L ADP, platelet
aggregation was performed by using a four-channel computerized
aggregometer (REGULEST, Nancy, France).
Platelet adhesion assay.
Washed platelets were prepared as previously described.18
Briefly, platelets were isolated from PRP by centrifugation at 500g for 15 minutes at 37°C and washed twice with HEPES
buffer pH 6.7 (10 mmol/L HEPES, 136 mmol/L NaCl, 2.7 mmol/L KCl, 2 mmol/L MgCl2) containing 0.35% BSA in the presence of
apyrase (2 U/mL) and acid-citrate-dextrose (ACD) (1 mL for
40 mL). Unstimulated platelets (1 × 108 platelets per
milliliter) were suspended in HEPES buffer pH 7.5 containing 0.15% BSA
and 1 mmol/L CaCl2. Stimulation of platelets was performed
by adding 0.5 U/mL purified human thrombin (Diagnostica Stago,
Asnières, France) to platelet suspension for 10 minutes in the
absence of adhesive proteins. Adhesion of unstimulated or
thrombin-stimulated platelets was performed in 96-multiwell plastic
wells (Dutscher, Brumath, France) coated with human vWF (a gift from T. Hercend, les Ulis, France). vWF was purified by affinity chromatography
by using CNBr-activated Sepharose 4B (Pharmacia, Uppsala, Sweden)
coupled to anti-Fg and anti-Fn antibodies (Dako, Trappes, France).
The amount of vWF antigen was determined by ELISA.19 Fg was
purified from fresh plasma by exclusion chromatography20
and Fg concentration was determined by measurement at 280 nm. Depletion of vWF and Fn was performed by affinity chromatography.21
BSA (Calbiochem, La Jolla, CA) was heat denatured for 1 hour at 60°C before use.
Wells were coated for 2 hours at 37°C with 200 µL of a 10 µg/mL
solution of either Fg or vWF in PBS pH 7.4. BSA was used as a control.
After removal of the coating solution, wells were washed with PBS.
Platelets (107) were incubated in the protein-coated
wells at room temperature for 20 minutes. At the end of the adhesion
assay, the content of the wells was removed by aspiration and the
surface was washed twice with PBS. Adherent platelets were fixed by
paraformaldehyde 2% (Carlo Erba, Milano, Italy) for 30 minutes at room
temperature, washed twice with distilled water, and stained with
toluidine blue 5%. Microphotographs were taken by using a Yashica 108 camera (Kyocera, Tokyo, Japan) at 40-fold magnification.
When indicated, the substrate coated on the surface was treated before
the assay with 1.3 µmol/L of immunoaffinity-purified anti-RGD Ig
fraction or effluent fraction depleted of anti-RGD antibodies, for 1 hour at 37°C. The effect of the anti-IIb3 antibody AP2 (a
generous gift from Dr T.J. Kunicki, The Scripps Research Institute, La
Jolla, CA) was tested by incubating the platelets with this antibody
(100 µg/mL) or control antibody during 30 minutes at room temperature
before activation by thrombin and adhesion assay.
Binding of Raji cells to immobilized Fn.
Antibody-induced binding of B cells to Fn was performed as previously
described.22 Raji B-lymphoblastoid cells were incubated in
the presence of 10 µg/mL monoclonal anti-CD19 antibodies in RPMI-1640
medium containing 10% fetal calf serum with gentle rotation for 30 minutes at 4°C. Twenty-four-well plates (Falcon Labware, Oxnard, CA)
were coated with 10 µg/mL of Fn in PBS for 3 hours at 37°C and then
washed with PBS. The Fn-coated wells were then preincubated for 1 hour
at 37°C with either of the following reagents: F(ab')2
fragments of IVIg eluted from the RGD-affinity column, anti-RGD-depleted F(ab')2 fragments of IVIg,
F(ab')2 fragments from a human IgG myeloma, rabbit
anti-Fn serum (positive control, from Sigma), and rabbit anti-laminin
serum (negative control, from Sigma). After washing the plates, Raji
cells were incubated in triplicates at 1.105 cells per
Fn-coated well for 20 minutes at 37°C. Unbound cells were removed by
three washes with PBS and the adhering cells were fixed in 3%
glutaraldehyde/PBS for 10 minutes at 4°C. The number of bound cells
per well was calculated as the mean number of cells counted in five
high-power fields (×400). Percentage of inhibition of binding to Fn
by antibodies was calculated from the number of cells that bound on to
anti-laminin-treated, Fn-coated plates. Significance of differences of
the results was determined by using the Mann-Whitney U-test.
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RESULTS |
Anti-RGD antibodies in IVIg.
IVIg and F(ab')2 fragments of IVIg were subjected to
affinity chromatography on Sepharose-bound RGD-containing decapeptide to purify antibodies directed against RGD motif. The affinity column
was equilibrated with PBS pH 7.2, loaded with 30 mg of IVIg or
F(ab')2 fragments per milliliter of affinity matrix and the Ig were allowed to interact with the gel overnight at 4°C. The
column was washed with PBS pH 7.2 and then eluted by using 0.2 mol/L
glycine HCl, pH 2.8. The eluate was neutralized with 3 mol/L Tris and
dialyzed against PBS. The eluate represented approximately 0.15% of
the loaded Ig. Thus, in a typical experiment, on loading 30 mg of IVIg
or F(ab')2 fragments of IVIg per milliliter of affinity
matrix, a yield of 45 µg of anti-RGD antibodies was achieved.
Binding of different fractions of IVIg to the RGD-containing
decapeptide and to a panel of human extracellular matrix proteins was
assessed by ELISA. The affinity-purified anti-RGD fraction of IVIg
bound to Fn, Fg, vitronectin, vWF and laminin in a dose-dependent manner (Fig 1). Binding of anti-RGD
fractions to RGD-bearing molecules was approximately 10-fold higher
than that of unfractionated IVIg and F(ab')2 fragments of
IVIg (Fig 1; data not shown), indicating that the immunoaffinity matrix
specifically retained anti-RDG antibodies. The specificity of the
binding was confirmed by inhibition assays with the RGD-containing
decapeptide and a control peptide. Although the RGD-containing
decapeptide inhibited dose-dependent manner, the binding of
125I-labeled anti-RGD antibody enriched
F(ab')2 fragments to Fn; the control peptide showed no
effect (Fig 2). The interaction of anti-RGD
F(ab')2 fragments with the RGD-containing peptide and Fn
was further analyzed by using the BIAcore technology (Fig 3). The kinetics of association and
dissociation were assessed. The affinity constant of the binding at
equilibrium was 1.3 µmol/L and 1.1 µmol/L for the binding of
RGD-specific F(ab')2 fragments to the decapeptide and to
Fn, respectively.
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| Fig 1.
Binding of anti-RGD antibody-enriched
F(ab')2 fragments of IVIg to RGD-containing peptide and
RGD-containing extracellular matrix proteins. Depicted are the binding
of anti-RGD antibody-enriched F(ab')2 fragments of IVIg
( ) and unfractionated F(ab')2 fragments of IVIg ( )
as assessed by ELISA. The mean values ± SEM obtained in two
independent experiments conducted in duplicates are shown.
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| Fig 2.
Inhibition of the binding of 125I-labeled
anti-RGD antibody-enriched F(ab')2 fragments of IVIg to
Fn by soluble RGD-containing peptide AVTGRGDSPA () and the control
ARTGVGSPDA peptide ( ). The amount of anti-RGD F(ab')2
fragments (0.084 µmol per well) was chosen to obtain the binding of
1,000 cpm per well in the absence of competitor peptide. The mean
values ± SEM obtained in two independent experiments conducted in
duplicates are depicted. The difference between the levels of
inhibition obtained was significant and calculated by using the
Mann-Whitney test.
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| Fig 3.
Real-time analysis of complex formation between anti-RGD
antibody-enriched F(ab')2 fragments of IVIg and the
RGD-containing ARTGVGSPDA decapeptide (A) and human plasma Fn (B).
Shown are the overlayed sensorgrams obtained after the injection of
1.75, 3.5, and 7 µmol/L concentrations of the F(ab')2
fragments. The signal corresponding to the binding of the same
concentrations of F(ab')2 to a sham-treated uncoated
sensor chip was deduced from total recorded binding by using the
BIAevaluation software (Pharmacia).
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Inhibition of platelet aggregation by anti-RGD-IVIg.
To test the ability of anti-RGD antibodies to interfere with cell
adhesion in vitro, we examined whether the anti-RGD-enriched fraction
of IVIg inhibits IIb/3 integrin-dependent ADP-induced platelet
aggregation. As shown in Fig 4, aggregation
of platelets in platelet-rich plasma was suppressed by micromolar
amounts of anti-RGD F(ab')2 fragments of IVIg in a
dose-dependent fashion. At the highest concentration tested, (1.2 µmol/L), the effluent of the RGD affinity chromatography column, used
as a negative control, showed less than 15% of inhibition of platelet
aggregation (data not shown). A similar concentration of
F(ab')2 fragments obtained from a human IgG myeloma had
no inhibitory effect (Fig 4). In contrast, anti-RGD-enriched
F(ab')2 fragments of IVIg had no effect on arachidonic
acid-induced platelet aggregation, which is not dependent on integrins
(data not shown).
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| Fig 4.
Inhibition of the IIb/3 integrin-dependent
aggregation of human platelets by affinity purified anti-RGD antibodies
from IVIg. The aggregation was induced in platelet-enriched plasma by
ADP and was monitored in the presence of different concentrations of
F(ab')2 fragments of IVIg, eluted from an RGD peptide
affinity column or of F(ab')2 fragments of a human IgG
myeloma.
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Suppression of adhesion of B lymphocytes to Fn.
We further examined whether anti-RGD antibodies interfere also with the
in vitro adhesion of B cells to Fn. For this purpose, we used our
observation that anti-CD19 antibodies induce an integrin 4-mediated
adhesion of B cells to Fn22 as an experimental model. Pretreatment of the plates with the anti-RGD-enriched fraction of
F(ab')2 fragments of IVIg, but neither with the anti-RGD
antibody depleted fraction, with F(ab')2 fragments of a
human IgG myeloma, nor with control serum resulted in dose-dependent
inhibition of Raji cell adhesion to immobilized Fn (Fig
5). Anti-Fn antibodies also exhibited
significant inhibitory effect on the adhesion of Raji cells to
immobilized Fn.
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| Fig 5.
Inhibition of adhesion of B lymphocytes to Fn by affinity
purified anti-RGD antibodies from IVIg. Fn-coated wells were
preincubated with rabbit anti-Fn serum, control rabbit antibodies,
F(ab')2 fragments of IVIg eluted from the RGD-affinity
column and anti-RGD-depleted F(ab')2 fragments of IVIg.
Raji cells that were pretreated with anti-CD19 MoAb was incubated in
triplicates at 1.105 cells per Fn-coated well for 20 minutes at 37°C. After washing, the number of bound cells per well
was calculated as the mean number of cells counted in five high-power
fields (×400) as described in Materials and Methods.
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Adhesion of human platelets to Fg/vWF-coated surface.
Unstimulated platelets were able to adhere and spread onto Fg (Fig
6A), whereas no measurable adhesion of
unstimulated platelets to vWF was found (data not shown). In the
presence of the anti-RGD IgG fraction, platelet adhesion to Fg was
significantly inhibited. Interestingly, we observed a complete
inhibition of platelet spreading (Fig 6C). In contrast,
anti-RGD-depleted Ig fractions had no effect on both adhesion and
spreading (Fig 6B). Thrombin-activated platelet adhesion to vWF and Fg
was characterized by adhesion and spreading of isolated platelets as
well as formation of aggregates, consecutive to platelet activation and
release of adhesive proteins from -granules (Fig 6D and G).
Adhesion, spreading, and aggregation to either vWF and Fg were
completely blocked by the anti-RGD IgG fraction (Fig 6F and I). In
contrast, no inhibitory effect was observed with the anti-RGD-depleted
fraction (Fig 6E and H). Furthermore, thrombin-stimulated platelet
adhesion to vWF and Fg was completely inhibited by the anti-IIb3
AP2 (data not shown) showing the specificity of this interaction.
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| Fig 6.
Inhibition of platelet adhesion to vWF and Fg. Wells were
coated for 2 hours at 37°C with 10 µg/mL of vWF or Fg. The
protein-coated wells were incubated with buffer (A, D, G), anti-RGD
depleted Ig fraction (1.3 µmol/L) (B, E, H) or anti-RGD Ig (1.3 µmol/L) (C, F, I) for 1 hour at 37°C. Unstimulated platelets (A-C)
and thrombin-stimulated platelets (D-I) were allowed to adhere to
Fg-coated wells (A-F) or vWF-coated wells (G-I) for 20 minutes at room
temperature. After removal of nonadherent platelets, adherent platelets
were fixed, stained, and microphotographed at a 40-fold
magnification.
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DISCUSSION |
Integrins are a major group of adhesion molecules that serve both
adhesive and signaling functions. Many of the integrins share affinity
toward the RGD recognition sequence in their extracellular matrix
ligand and are able to discriminate between different RGD-containing proteins.23 In the present study, we show that therapeutic
preparations of normal polyspecific IgG (intravenous immunoglobulin,
IVIg) contain antibodies that bind to human RGD-containing integrin ligands. The presence of anti-RGD antibodies in IVIg was shown by
ELISA, radioimmunoassay (RIA), and real-time analysis of
complex formation with RGD-expressing molecules by using the BIAcore
system. The biological relevance of anti-RGD antibodies in IVIg was
demonstrated by their ability to inhibit integrin-dependent platelet
aggregation and B-lymphocyte adhesion to Fn.
Natural antibodies of the IgG isotype directed against self components
are present in normal human serum.24-28 IVIg has been shown
to recognize several surface molecules of homologous lymphocytes and
antigen presenting cells involved in immunoregulation, including CD5,5 CD4,4 idiotypes of
immunoglobulins,29,30 HLA class I-derived
peptides,6 as well as framework and clonotypic determinants of the human TCR.7 Here we show that IVIg contains
antibodies specific for the RGD adhesion motif expressed by ligands of
many integrins.
RGD-specific fractions of antibodies affinity purified from IVIg, in
contrast to unfractionated IVIg were shown to be at least 10-fold more
effective in binding to RGD-containing matrix proteins, including Fg,
Fn, vitronectin, laminin, and vWF. The interaction of
anti-RGD-containing IgG with RGD-expressing proteins was selectively inhibited by free RGD-containing peptide. Real-time analysis of immune
complex formation between immunopurified anti-RGD antibodies and an RGD
sequence-containing peptide as well as Fn, by using BIAcore, similarly
showed an association binding constant for these interactions in
the micromolar range. These data are consistent with previously
reported affinities of natural autoantibodies toward self antigens,
including cytokines, ABO blood group antigens, HLA class I antigens,
Fas molecule, and intracellular proteins.6,28,31-35 The
affinities calculated for anti-RGD antibodies are, thus, within the
range of those of natural antibodies previously shown to exert potent
biological activities.6,31,36
We addressed the question of whether natural anti-RGD antibodies can
interfere with RGD-mediated biological functions. Therefore, we
examined the effect of immunopurified anti-RGD antibodies on platelet
IIb3 integrin-dependent functions. A direct effect of anti-RGD
antibodies present in IVIg on RGD-mediated platelet functions was
assessed by studying three different properties, adhesion, spreading,
and aggregation. The latter was studied by using either washed
platelets or PRP.
When platelets are activated by the potent thrombin agonist, a
conformationally active IIb3 is generated and is involved in
adhesion, spreading, and aggregation to RGD-containing ligands. We show
a complete inhibition by anti-RGD antibodies of adhesion and spreading
to vWF and Fg. The specificity is illustrated by the lack of inhibition
in the presence of Ig fractions depleted of anti-RGD antibodies. These
functions involve distinct sites of IIb3 integrin, which are
located between amino acids 109-180 of 3 subunit, which binds to RGD
sequence,37 and between amino acids 294-314 of the IIb
subunit, which interacts with the Fg dodecapeptide
sequence.38 There are several studies that define the
primary effect of RGD peptides in the interaction with activated IIb3 integrin. All snake venom disintegrins are potent inhibitors of ADP-stimulated platelet adhesion to Fg. These antagonists are more
potent inhibitors of cell adhesion than synthetic linear or cyclic RGD
peptides. For example, platelet adhesion of ADP-stimulated platelets to
Fg was completely inhibited by kistrin at dose of 1 µmol/L, whereas
adhesion was completely inhibited by RGD peptides at 40 µmol/L.39 In our experimental conditions, platelet
adhesion was completely blocked with 1.33 µmol/L of anti-RGD IgG fraction.
Further, aggregate formation was observed after thrombin stimulation
induced secretion of adhesive proteins. The anti-RGD antibodies were
able to partially inhibit aggregate formation, showing their ability to
interact with the RGD-mediated binding of endogenous secreted proteins
to activated platelets. In addition, a complete inhibition of spreading
of unstimulated platelets by the anti-RGD IgG fraction underlines the
role of RGD in stabilization of the Fg interaction with platelet
cytoskeleton, thus leading to platelet spreading. Furthermore, we found
that unstimulated platelet adhesion to immobilized Fg was partially
inhibited by anti-RGD IgG fraction. These data confirm that adhesion of
unstimulated platelets to Fg occurs via both the dodecapeptide sequence
and the RGD sequence, as shown by Fg fragments corresponding to the dodecapeptide (fragment D) or the RGD sequence (fragment
E).40
Micromolar amounts of anti-RGD F(ab')2 fragments
inhibited ADP-induced platelet aggregation in a dose-dependent fashion.
Under these conditions, platelet aggregation involves the platelet
IIb3 integrin (glycoprotein IIb/IIIa) that binds to RGD. These
antibodies are powerful inhibitors of adhesion of both resting and
activated platelets and may be relevant in inhibition of irreversible
platelet adhesion and thrombus formation. Peptides or mimetics that
block the IIb3 binding sites are efficacious in the treatment of
arterial thrombosis in animal models and are being evaluated in human
disease.41 Agents modeled after the RGD cell-binding motif
that hold promise in other thrombotic disorders, including unstable
angina and myocardial infarction are in development. In this context,
the beneficial effect of IVIg in the thrombotic complications
associated with thrombotic thrombocytopenic purpura or with the
antiphospholipid syndrome could possibly be explained, at least, in
part, by inhibition of platelet aggregation.42-44
The biological relevance of the natural anti-RGD antibodies is further
demonstrated by our observation that these antibodies blocked the
anti-CD19-triggered adhesion of Raji cells to Fn. This binding to Fn
was shown to be mediated specifically by integrin 4.22
Integrin 41 is the receptor for the HepII domain and CS-1 site in
Fn, but binds also to the RGD sequence of Fn after activation via
1.45,46 These findings support our interpretation that
anti-CD19 is required to induce a RGD-specific binding to Fn mediated
by integrin 4.
The RGD motif has a central role in mediating cell-to-cell and
cell-matrix adhesion in a variety of immunological and inflammatory processes.47 For instance, cyclic RGD peptides have been
shown to inhibit 41-dependent adhesion of T cells to
cytokine-activated endothelial cells48 and to decrease the
severity of ischemic acute renal failure in rats.49,50
Furthermore, RGD-containing peptides effectively block phorbol
ester-induced adhesion of monocytes to Fn51 and reduce
polymorphonuclear leukocyte (PMN) adhesion to
Fn.52 These data are further extended by our present
observations that affinity-purified anti-RGD antibodies block the
adhesion of Raji cells to Fn. By inhibiting leukocyte adhesion,
antibodies in IVIg that recognize the RGD adhesion motif may contribute
to the anti-inflammatory effects of IVIg.2,3,53,54 The
plasma concentration of IgG reached in a recipient of IVIg is in the range of 20 to 35 mg/mL. Because the amount of anti-RGD antibodies in
IVIg is approximately 0.15%, the concentration of anti-RGD antibodies
achieved in vivo is within the range of concentrations required to
exert the biological activities in the in vitro experiments described herein.
Recently, we have observed that the infusion of IVIg in apoE-knockout
mice prevents the progression of atherosclerotic lesions in this model
of accelerated inflammatory vascular disease.55 At least
part of the protective effect of IVIg the in apoE knockout mouse of
atherosclerosis may involve antibodies that suppress cell adhesion,
such as antibodies to RGD. The latter hypothesis may be addressed
experimentally. Another area in which inhibition of cell adhesion by
anti-RGD antibodies may be critical is the Fn matrix formation
involving 5/1 integrins and the subsequent cell adhesion in the
progression of metastasis.56-58 Metastasis formation was
shown to be suppressed by agents interfering with RGD-dependent
adhesion in several animal models and in vitro models by using human
tumoral cells.23,59,60 MoAbs to integrins and adhesion-blocking peptides have been used in experimental models of
autoimmune and inflammatory diseases as well as in the treatment of
patients with solid organ allograft rejection.61-63 Because human IgG autoantibodies recognizing the same target molecules as these
MoAbs are present in IVIg, we speculate that IVIg may have similar in
vivo effects.
Finally, our observations suggest that the cell/cell and
cell/extracellular matrix integrin-mediated interactions take place physiologically in the presence of natural anti-integrin-ligand autoantibodies. Our findings extend previous observations on the presence of a broad spectrum of IgG antibodies to self antigens in
disease-free individuals.64 The data are compatible with the suggestion that natural autoantibodies play a role in the control
of receptor-ligand interactions in the maintenance of immune
homeostasis.64
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ACKNOWLEDGMENT |
The authors thank Marie-Françoise Bloch for technical assistance
and Dr M. Kyurkchiev for useful discussions.
| |
FOOTNOTES |
Submitted April 17, 1998; accepted January 18, 1999.
Supported by the Institut National de la Santé et de la Recherche
Médicale (INSERM; Grant No. 5 REW 03); the Central Laboratory of
the Swiss Red Cross, ZLB, Switzerland; the Bulgarian National Science
Foundation (Grant No. L-508/95), the Deutsche Forschungsgemeinschaft, Germany (Grant No. Schr 318/4-1), and by the NATO Scientific and Environmental Division (Grant No. HTECH.EV 960287). M.M. is a recipient
of a Sanofi fellowship. F.S. is a recipient of a fellowship from the
Deutsche Forschungsgemeinschaft, Germany (Grant No. Schr 318/3-1).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Srini V. Kaveri, PhD, INSERM U430,
Hôpital Broussais, 96, rue Didot, F-75014 Paris, France; e-mail:
kaveri{at}hbroussais.fr.
| |
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ABSTRACT
INTRODUCTION