* Laboratoire Allergie, Centre de recherché Louis-Charles Simard, Pavillon Notre-Dame, Centre Hospitalier Université de
Montréal (CHUM), Montreal, Quebec H2L 4M1, Canada; Laboratory for Parasitic Diseases, National Institutes of Health,
Bethesda, Maryland 20892; § Immunex Research Development Corporation, Seattle, Washington 98101; and
Division of
Infectious Diseases, Washington University, St. Louis, Missouri 63110
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Abstract |
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The vitronectin receptor, v
3 integrin, plays
an important role in tumor cell invasion, angiogenesis,
and phagocytosis of apoptotic cells. CD47, a member of
the multispan transmembrane receptor family, physically and functionally associates with vitronectin receptor (VnR). Although vitronectin (Vn) is not a ligand of
CD47, anti-CD47 and
3 mAbs suppress Vn, but not fibronectin (Fn) binding and function. Here, we show
that anti-CD47, anti-
3 mAb and Vn, but not Fn, inhibit sCD23-mediated proinflammatory function
(TNF-
, IL-12, and IFN-
release). Surprisingly, anti-CD47 and
3 mAbs do not block sCD23 binding to
v+
3+ T cell lines, whereas Vn and an
v mAb (clone
AMF7) do inhibit sCD23 binding, suggesting the VnR
complex may be a functional receptor for sCD23.
sCD23 directly binds
v+
3+/CD47
cell lines, but coexpression of CD47 increases binding. Moreover, sCD23 binds purified
v protein and a single human
v
chain CHO transfectant. We conclude that the VnR
and its associated CD47 molecule may function as a
novel receptor for sCD23 to mediate its proinflammatory activity and, as such, may be involved in the inflammatory process of the immune response.
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Introduction |
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THE vitronectin receptor (v
3) is an ubiquitous receptor that interacts with several ligands, such as
vitronectin (Vn)1, fibronectin (Fn), osteopontin,
and metalloproteinase MMP-2 (for review see Felding-Habermann and Cheresh, 1993
; Brooks et al., 1996
). As a
consequence, this integrin plays a role in diverse biologic
processes such as cell migration, tumor invasion, bone resorption, angiogenesis, and immune responsiveness (Gladson and Cheresh, 1994
).
During the process of inflammation, circulating human
monocytes are able to leave the blood by attaching to, and
migrating through, endothelial and subendothelial matrices to the site of injury. The vitronectin receptor (VnR)
v
3, expressed on endothelial cells, is involved in the
transendothelial migration process (Brown and Lindberg,
1996
) together with PECAM (CD31) (Buckley et al., 1996
) and CD47 (Cooper et al., 1995
). When recruited at
the inflammatory or infected sites, phagocytes undergo a
number of physiological changes including increases in adhesiveness, production of reactive oxygen metabolites,
and augmentation in phagocytosis. Extracellular matrix
proteins containing RGD sequence peptides have been
shown to mediate some of these functions (Hynes, 1992
),
especially activation of phagocytic burst (Zhou and Brown,
1993
), and enhancement of ingestion of opsonized particles by monocytes (Gresham et al., 1989
).
The CD47 Ag is a widely expressed 50-kD multispan
transmembrane protein component of the v
3 and leukocyte response integrin signaling complex, since its expression was shown to enhance Vn, but not Fn, binding and
function to a variety of cells (Brown et al., 1990
; Rosales
et al., 1992
; Lindberg et al., 1993
). It was reported that
CD47 does not directly bind Vn, and CD47
cell lines, expressing
v
3, failed to bind Vn-coated beads (Lindberg et al., 1996b
). Moreover, CD47 deficient mice rapidly died
of Escherichia coli peritonitis, a phenomenon directly correlated with a reduction in leukocyte activation in response to
3, but not
2, integrin ligation (Lindberg et al.,
1996a
). The
v
3/CD47 trimolecular complex also participates in the resolution of inflammation by mediating phagocytosis of aging leukocytes undergoing apoptosis before
they disgorge their potentially harmful contents (Savill et
al., 1990
). This process is potentiated by the synthesis of proinflammatory cytokine such as GM-CSF, TNF-
, IL-1,
and IFN-
(Ren and Savill, 1995
).
CD23 has been purported to play a role in inflammation
based upon its in vitro proinflammatory activity (Armant
et al., 1994; Lecoanet-Henchoz et al., 1995
; Bonnefoy et
al., 1996
; Sarfati, 1997
) and the observation that soluble
CD23 (sCD23) levels increased in various chronic inflammatory disorders, including rheumatoid arthritis and systemic lupus erythematosus (Ikizawa et al., 1993
; Bertero
et al., 1994
). sCD23 (Armant et al., 1994
; Leconaet-Henchoz et al., 1995) and CD23 ligation (Bonnefoy et al.,
1996
) can trigger monokine release by human monocytes.
Our studies have demonstrated that sCD23-induced TNF-
secretion costimulates IFN-
production by IL-2-activated T cells cocultured with syngeneic monocytes in the
absence of T cell receptor ligation (Armant et al., 1995
).
Here, we report a novel function for VnR and its associated CD47 molecule on monocytes, by demonstrating that
this trimolecular complex mediates proinflammatory cytokine synthesis via interaction with CD23. This may contribute to the perpetuation of the inflammatory process in
chronic disorders such as rheumatoid arthritis. In this disease, CD23, TNF-, and VnR expression are found to be locally elevated in the inflamed synovium (Ashton et al.,
1993
; Feldman et al., 1996
).
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Materials and Methods |
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Cell Lines and Reagents
Human recombinant IL-2, kindly provided by Dr. D. Bron (Institut Bordet, Brussels, Belgium), was used at 20 U/ml; IL-15 was obtained from Immunex and used at 200 ng/ml. Endotoxin-free (<15 pg/ml, as determined
by the chromogenic Limulus amebocyte lysate, QCL-1000, BioWhittaker
Inc.) affinity-purified sCD23 was prepared in our laboratory from CSN of
CHO cell line transfected with human cDNA encoding for aa 148-321 of
the CD23 molecule. The concentration of 25 ng/ml sCD23 used throughout this study was selected on the basis of previously reported dose-
response curves (Armant et al., 1994). Jurkat T (
v+
3+), THP-1 (
v
3
)
monocytic, Raji (
v+
3
), and Bowes melanoma (
v+
3+) cell lines were
obtained from the American Type Culture Collection (ATCC). K562 and
K562 transfected with the cDNA encoding the full-length CR2 (K562-CR2) were a generous gift from Drs. A. Masumoto and D. Fearon (Johns
Hopkins University, Baltimore, MD). CD47 deficient Jurkat T cell line
and 0V10 ovarian carcinoma cell line were generated in Drs. E. Brown
and F. Lindberg's laboratory (Washington University, St. Louis, MO).
cDNA encoding for CD51 (
v chain) was a generous gift from Dr. E. Ruoslahti (Burnham Institute, La Jolla, CA). 10G2 mAb (IgM class) was produced in our laboratory following immunization of mice with Jurkat T
cells. Hybridomas producing anti-CD47 (clone B6H12) and anti-
3
(CD61) mAbs (clone AP3) were purchased at the ATCC. Anti-
v
3
(CD51/CD61, clone LM609), and anti-
v (CD51, clone LM142) were
kindly provided by Dr. Cheresh (Scripps Research Institute, La Jolla,
CA). Anti-
v (CD51, clone AMF7) and anti-CD47 (clone BRIC126,
CKm1) were purchased from Immunotech, Serotec Ltd., and Accurate
Chemical and Scientific Corp., respectively. RGDS and RGES peptides,
Vn, Fn, and thrombospondin (TSP) were obtained from GIBCO BRL.
Expression Cloning of 10G2 Antigen (CD47)
COS cells were transfected using the DEAE dextran method, with an expression library derived from Jurkat cells (4 × 105 clones). Cells expressing the molecule recognized by the 10G2 mAb were immunoselected by indirect panning using 10G2 and anti-mouse IgM-coated plates. Specifically bound cells were lysed, plasmid DNA was isolated, amplified, and retransfected into COS cells. Following an additional round of immunoselection and plasmid purification, pools of 150 clones each were transfected into COS cells, which were subsequently screened for positivity by incubation with radiolabeled 10G2 mAb. Two rounds of screening resulted in the isolation of a single 10G2-reactive clone.
Establishment of Stable CHO Transfectants
CHO cells were grown at 50% confluence in 150-cm2 culture flasks in 5%
heat decomplemented FCS containing DME (GIBCO BRL) supplemented with 2 mM glutamine, 100 IU penicillin, and 100 µg/ml streptomycin. Cells were harvested using versene solution (GIBCO BRL) and
washed twice with pure DME. Cells were then resuspended at 11 × 106 cells/
ml, and 107 cells were incubated in a 0.4-cm electrotransfection cuvette (BioRad Laboratories) for 10 min with 20 µg of pBJv (plasmid containing cDNA encoding the full-length
v molecule, CD51), in order to obtain
CHO cells expressing the
v molecules. Cells were then pulsed at 220 V
and 960 µF using a Gene Pulser (BioRad Laboratories). After another 10 min of incubation at room temperature, electroporated cells were grown
for 24 h in nonselective culture medium which was then replaced by complete DME containing 500 µg/ml active G418 (GIBCO BRL). After 14 d
of culture, the pool of survivors was analyzed by flow cytometry, and
CHO cell line expressing
v was subsequently enriched by FACS® for the
5% highest stained cells using anti-CD51 mAb as primary antibody. After
four rounds of sorting, we obtained stable cell lines, namely CHO-51, containing >99% of CD51+ cells.
Cell Separation and Culture Conditions
Monocytes.
PBMC were isolated by density gradient centrifugation of heparinized blood from healthy volunteers using Lymphoprep (Nycomed). Monocytes were prepared by cold aggregation as described in Armant et
al. (1995). Monocyte purity was shown to be >95% using phycoerythrin-conjugated anti-CD14 mAb and flow cytometry (Becton Dickinson and
Co.). Cellular viability was >90% using trypan blue exclusion.
T Cells. Enriched T cell populations were obtained from the monocyte-depleted PBMC by rosetting with AET-SRBC and treatment with ammonium chloride. To obtain highly purified T cells, rosette forming cells were washed and incubated for 20 min at 37°C in Lympho-Kwik T (One Lambda). Cell purity was assessed by flow cytometry (FACScan®; Becton Dickinson and Co.) using phycoerythrin-conjugated anti-CD3 mAb (Becton Dickinson and Co.) and shown to be >98% in all cases.
Cultures were performed in complete serum-free HB101 medium (Irvine Scientific) supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 10 mM Hepes, 100 IU penicillin, and 100 µg/ml streptomycin in the presence of polymyxin B 10 µg/ml (Sigma Chemical Co.). When cultured alone, monocytes were incubated in 5 ml sterile Falcon tubes (Becton Dickinson and Co.) at 2 × 105 cells/ml for cytokine measurement. For coculture experiments, T cells (106 cells/ml) were incubated with monocytes (2 × 105 cells/ml) in 24-well Falcon plates.Cytofluorometric Analysis
For sCD23 binding, cells or cell lines were washed in HBSS (Gibco Laboratories), resuspended in HB101 complete serum free medium containing
biotinylated sCD23 (B-sCD23; 50 ng/ml) in the absence or presence of
mAbs (CD47, CD51, CD51/CD61), soluble Vn or Fn (20 µg/ml), and
RGDS peptides (20 µg/ml). After 4-6 h of incubation at 22°C, cells were
washed with PBS containing 3% BSA, and further incubated with phycoerythrin-streptavidin (Becton Dickinson and Co.). Cell viability assessed
by trypan blue exclusion was >85% before staining with fluorochrome.
Indirect immunofluorescence staining of cells or cell lines with different
mAbs was performed according to standard techniques (Armant et al.,
1995).
Lymphokine Determinations
IFN-, TNF-
, IL-10, and IL-12 were measured exactly as described in
Armant et al. (1994
, 1995
). IL-1
, IL-8, and PGE2 were measured by
ELISA kits purchased from R & D Systems, Inc.
Immunoprecipitation and Western Blot Analysis
Cells were lysed in PBS containing 1% NP-40 (NP-40/PBS) supplemented
with protease inhibitors. The lysate was purified on anti-3 affinity column, and the eluate was separated by SDS-PAGE (5%) under nonreducing conditions and transferred to PDVF membrane (Millipore Corp.).
Nonspecific binding sites on the membrane were blocked with PBS containing 5% milk. The membrane was incubated with milk containing
B-BSA, B-sCD23, B-CD51 mAbs (clone AMF7 and LM142), or B-CD61
mAb. After overnight incubation, membrane was treated with avidin-DH
and biotinylated peroxidase complex (Vectastain, ABC kit, Vector Labs,
Inc.) followed by ECL detection reagent (Nycomed Amersham).
Statistical Analysis
Paired t tests have been used to assess levels of significance (*P < 0.05; **P < 0.01; ***P < 0.001).
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Results |
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10G2 mAb, Which Inhibits sCD23 Biological Activities, Recognizes CD47 Ag
sCD23 displays potent proinflammatory activity by directly triggering monokine release from purified monocytes in the absence of costimulatory signals, such as bacterial antigens (LPS or SAC) or T cell-dependent signal
(sCD40L; Armant et al., 1994, 1995
; Lecoanet-Henchoz
et al., 1995
). Although CD21 (CR2) and CD11b (CR3)
were previously described as novel CD23 counterreceptors (Aubry et al., 1992
; Leconaet-Henchoz et al., 1995),
we also detected binding of sCD23 to several T cell lines
lacking CR2 or CR3 expression (Ishihara et al., 1995
; Table I). In an effort to identify sCD23 binding component,
we generated mAbs to Jurkat T cells. We identified one
mAb, clone 10G2, which neutralized sCD23 biological activities (Fig. 1 A), and displayed similar cell reactivity as
sCD23 (Table I). Specifically, 10G2 mAb, like sCD23, did
not stain the CD11b+ (CR3) and CD11c+ (CR4) THP-1
cell line. It recognized weakly, but with similar intensity,
K562 and K562-CR2 cell lines. 10G2 mAb also stained peripheral blood T, B cells, and monocytes (Table I). 10G2
mAb significantly suppressed sCD23 costimulation of
IFN-
production by IL-2-stimulated T cells cocultured
with autologous monocytes (mean inhibition of 10 experiments, 66%, P < 0.03; Fig. 1 A).
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|
The molecule recognized by 10G2 mAb was identified by immunoaffinity panning of COS cells transfected with a cDNA library prepared from Jurkat T cells. After several rounds of selection, a single clone was selected which was found by DNA sequencing to encode CD47 Ag (data not shown). Results in Fig. 1 B demonstrate binding of 10G2 to COS cells transfected with CD47 cDNA clone.
The effects of several anti-CD47 mAbs on sCD23 biological activity were examined (Fig. 1 A). Besides the
10G2 mAb (IgM isotype), three other anti-CD47 mAbs of
different Ig isotype subclasses, C1Km1 (IgG1), BRIC126
(Ig2b), and B6H12 (IgG1) inhibited IFN- production induced by sCD23 plus IL-2. These results support the notion that CD47 is part of the signaling complex triggered
by sCD23.
However, it appears that 10G2 was preferentially binding to a particular epitope on CD47. As depicted in Fig. 1
C, THP-1 cells express CD47 Ag but are not recognized by
10G2 mAb. Clone B6H12, a well-defined anti-CD47 mAb,
but not 10G2, stained THP-1 cell line in a dose-dependent
manner, while Jurkat T cells were recognized with similar
intensity by both anti-CD47 mAbs. There was no cross-inhibition of Jurkat staining by the two clones (data not
detailed). Our unpublished data also indicated that erythrocytes which expressed CD47 in the absence of integrins
(Rosales et al., 1992) were stained by B6H12 but not 10G2.
Anti-CD47 mAbs Suppress CD23 Costimulation of
IL-12 and IFN- Production Via
Fc-independent Pathways
We investigated the mechanisms underlying the suppression of IFN- production by anti-CD47 mAbs. In addition
to 10G2 mAb of the IgM isotype, the F(ab)'2 fragments of
B6H12 or intact BRIC126 mAb suppressed, in a dose-dependent manner, the ability of IL-2 and sCD23 to augment the production of IFN-
by T cells cocultured with
monocytes, demonstrating that the inhibition of IFN-
secretion by anti-CD47 mAb was not Fc-mediated (Fig. 2
A). Interestingly, IFN-
secretion by IL-2-stimulated T
cells cocultured with monocytes and graded numbers of
CD23-transfected CHO cells, was also abrogated by anti-CD47 mAbs (Fig. 2 B).
|
We previously reported in the presence of IL-2 or IL-15,
low levels of CD40L, expressed by unstimulated T cells,
were sufficient to engage CD40 on monocytes and trigger
IL-12 release. This monocyte-derived IL-12 production
synergized with IL-2 or IL-15 to augment IFN- production by T cells (Armant et al., 1996
; Avice et al., 1998
). The
IFN-
response could be further amplified by sCD23-induced TNF-
release (Armant et al., 1995
). Although
sCD23 did not trigger IL-12 production by purified monocytes (Armant et al., 1995
), it costimulated IFN-
and IL-12
release in this coculture system, and the secretion of both
cytokines was strongly inhibited by anti-CD47 mAbs (Fig.
2, C and D).
Anti-CD47 mAb Suppresses sCD23-induced Monokine Release
Because sCD23 directly triggers TNF- release by purified
monocytes (Armant et al., 1995
), and monocytes express
CD47 (Table I), we examined the effect anti-CD47 mAb
had on sCD23-induced monokine release by monocytes.
Anti-CD47 mAb significantly suppressed the induction by
sCD23 of TNF-
, IL-1
, and PGE2 without affecting IL-8
secretion (Fig. 3). The data (i.e., inhibition of TNF-
and IL-12 production) provide a mechanism by which anti-CD47 mAb can suppress sCD23 costimulation of IFN-
production.
|
However, anti-CD47 mAb, in the absence of sCD23 or
in the presence of LPS, did not modulate TNF- production (Fig. 3 A). Our unpublished observations revealed
that anti-CD47 mAb, used alone or in combination with
sCD23, did not induce the production of monocyte deactivators (such as IL-10 or TGF-
), nor did it modulate SAC-induced TNF-
, IL-6, or IL-10 release. These results support the hypothesis that anti-CD47 mAbs interfere with
the sCD23 signaling pathway without impairing general
monocyte function.
sCD23 and Vitronectin Share the Same Receptor: VnR/CD47 Complex
Several studies indicate that CD47 is physically and functionally associated with the vitronectin receptor, v
3.
Both anti-CD47 and anti-
3 (CD61) mAbs can block binding of Vn, but not Fn, to
v
3, even though Vn does not
bind to CD47 (Brown et al., 1990
; Lindberg et al., 1993
).
Therefore, we examined the biological effects of anti-
3
mAb and the natural ligands of VnR (Vn and Fn) on
sCD23 function. It is important to note that all cultures were performed in HB101 serum-free medium to eliminate FCS as a source of Vn and Fn. The results (Fig. 4)
suggested that Vn and sCD23 might share the same receptor, namely VnR/CD47 complex. Anti-
3 mAb suppressed
sCD23 function, as defined by TNF-
secretion and IFN-
production (Fig. 4 A, and data not shown). Furthermore, soluble Vn, but not Fn, suppressed sCD23-induced TNF-
release by purified monocytes (Fig. 4 B). sCD23 and Vn
most likely bound to distinct epitopes on
v
3, since an
anti-
v
3 mAb (clone LM609), which specifically inhibited
Vn binding and function (Gao et al., 1996a
), and RGDS
peptide had no suppressive activity (Fig. 4 A, and data not
shown).
|
We next investigated the ability of mAbs directed to the
VnR complex, anti-CD47, CD61 (3) and
v
3 (LM609)
mAbs, and natural ligands of VnR, Vn, Fn, and RGDS
peptides, to alter sCD23 binding to the Jurkat T cell line.
Unexpectedly, anti-CD47 mAbs, alone or in combination
(Fig. 5 b), anti-CD61 (Fig. 5 c), or anti-
v
3 clone LM609
(not shown) did not inhibit sCD23 binding to Jurkat or
monocytes (Hermann, P., unpublished observations). Note
that sCD23 binding was specifically suppressed by anti-CD23 mAb (Fig. 5 a). The data suggested ligation of the
CD47 or
3 chain of the trimolecular complex by mAbs
could indirectly inhibit sCD23 function by providing a
negative signal to the target cells, or by modifying CD47
complex configuration without displacing the sCD23 molecule. We examined whether engagement of VnR/CD47
complex by its natural ligands would modify sCD23 binding. As shown in Fig. 5 d, Vn, but not Fn or RGDS peptide
(not shown), significantly inhibited sCD23 binding, strongly
suggesting the VnR complex was involved in the secretion
of proinflammatory cytokine via interaction with sCD23.
|
The possible role of the v chain (CD51) of VnR in
sCD23 binding was explored. Three anti-CD51 mAbs
were tested, and we found one anti-CD51 mAb (clone
AMF7) inhibited the interaction between sCD23 and
v+
3+ Jurkat T (Fig. 5 e), as well as
v+
3
Raji B cell
lines (Fig. 5 f). Note that the Raji B cell line expresses the
5 integrin (data not shown). We postulated that sCD23
was binding to
v and not
3 chain of the VnR, whereas
3 and CD47 chains were likely to be involved in the signaling of the trimolecular complex in monocytes because
anti-CD47 and anti-
3 inhibited sCD23 function, but not binding.
sCD23 Interacts with v/CD51 and CD47 Coexpression
Increases its Binding
Next, we selected a melanoma cell line, strongly expressing v
3 to purify this integrin by affinity chromatography
using anti-
3 (CD61) immobilized AFFi-gel. Western blot
analysis (Fig. 6) shows sCD23 (lane 6) reacted with a single band of ~135 kD, displaying a similar migration pattern as molecular species recognized by a cocktail of anti-CD51 mAbs (lane 2). However, sCD23 did not appear to
bind purified
3 (CD61) chain which was identified by anti-CD61 mAb (lane 4), or purified recombinant CD47
(not shown).
|
To further support the hypothesis that sCD23 may directly interact with v chain of the trimolecular complex,
we prepared CHO transfectants singly expressing human
v chain (CHO-CD51). The results in Fig. 7 demonstrated
sCD23 strongly bound to CHO-CD51 compared to untransfected cell line. Although CHO-CD51 transfectant did not express human
3 (CD61) chain, CHO cell lines
were reported to express rodent
chain integrins (Lindberg et al., 1993
) which likely associated with the human
v chain underlying the successful stable expression of
a single human integrin chain. Nevertheless, CHO cells
also expressed hamster CD47 which might contribute to sCD23 binding to
v/CD51 on live cells as reported for Vn
binding to untransfected CHO cells (Lindberg et al.,
1993
). To directly assess whether CD47 expression was required for sCD23 interaction with
v/CD51, we examined
sCD23 binding to human CD47 deficient cell lines. As
shown in Fig. 8, sCD23 bound to OV10 ovarian carcinoma VnR+/CD47
cell line demonstrating that CD47 was dispensable for sCD23 binding. Coexpression of CD47 further
increased its binding. A similar effect was seen on transfection of CD47
Jurkat with CD47 (data not shown).
|
|
Given that sCD23 was not binding to CD47+/v
(THP-1
cell line; Fig. 8), but reacted with
v+
3
(Raji cell line;
Fig. 5 f), we concluded that sCD23 ligated
v/CD51. The
VnR complex (
v
3/CD47 and/or
v
x/CD47) was used as
a functional receptor for sCD23 to mediate its proinflammatory activity and, as such, may be involved in the inflammatory process of the immune response.
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Discussion |
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Our results can be summarized in the schematic model
presented in Fig. 9. We first postulate (Fig. 9 A) that
sCD23 and Vn have distinct recognition sites on the v
3/
CD47 complex. sCD23 binds to
v (CD51) and Vn to
v
3
(CD51/CD61) conformational site. Binding of Vn may induce structural changes in the integrin complex leading to
the masking of sCD23 binding sites, and vice versa. This
hypothesis is based on observations that Vn, anti-
v mAb (clone AMF7), but not clone LM609 (which specifically
recognizes Vn binding site), nor RGDS peptide inhibited
sCD23 binding and function (Figs. 4 and 5), and sCD23 directly bound to
v chain (Figs. 6-8). However, CD47 was
not a direct ligand for sCD23 while its coexpression improved sCD23 binding to
v/CD51. sCD23 bound CD47
deficient cell lines, but failed to bind CD47 in the absence of
v/CD51 integrin (THP-1 cell line, erythrocytes and recombinant CD47 protein; Fig. 8, and data not shown).
|
Secondly, (Fig. 9, B and C) CD47 or CD61 engagement
by mAbs prevents Vn binding (Lindberg et al., 1993) without displacing sCD23 molecule (Fig. 5) while these mAbs
inhibit both sCD23 (Figs. 2-4) and Vn function perhaps by
modifying
3 conformation or signaling pathway. Lindberg
et al. (1993)
indicated that anti-CD47 and anti-
3 (CD61)
mAbs (directed against an epitope of
3 located outside the Vn binding site) inhibited Vn-opsonized particle binding and function. They also proposed the CD47 molecule
participated in the appropriate folding of
v
3, and modulated the affinity of
v
3 for Vn (Brown et al., 1990
; Lindberg et al., 1993
). Vn-coated particles failed to bind to a
CD47 deficient cell line (Lindberg et al., 1996b
) or to cells
isolated from CD47 deficient mice (Lindberg et al., 1996a
),
while these cells expressed
v
3, demonstrating that CD47
is dispensable for VnR expression, but required for Vn
binding and function. Using truncated forms of CD47,
they also reported the interaction between the extracellular domain of CD47 (IgV) and
v integrins was sufficient
for Vn binding (Lindberg et al., 1996b
).
It has been reported that adhesion of integrins to their
counterreceptors is a dynamic phenomenon regulated by
intracellular signal transduction pathways (Diamond and
Springer, 1994). Structural changes in the extracellular domains of integrins following mAb ligation may modify
ligand adhesiveness in inducing or inhibiting ligand binding (inside-out signaling), or may directly provide a negative signal to the cell (outside-in signaling), as proposed here for anti-CD47 and anti-CD61 mAb-mediated inhibition of sCD23 function.
In agreement with this hypothesis, it was reported that
antibodies recognizing CD81, another member of multispan transmembrane receptors family, inhibited FcRI-mediated mast cell degranulation without affecting IgE
binding, receptor-mediated Ca2+ release, or tyrosine phosphorylation (Fleming et al., 1997
).
Therefore, we propose a novel function for VnR/CD47
complex in the regulation of inflammatory response. In
the absence of pathogen, ligation of VnR by sCD23 mediates monokine release such as TNF- which enhances the
inflammatory process by triggering the cascade of proinflammatory cytokine secretion (IL-1, IL-6, GM-CSF . . .;
Feldman et al., 1996
), and facilitates the elimination of
apoptotic cells (Ren and Savill, 1995
). Both effects are
negatively regulated by the presence of Vn (Fig. 4 B and
Savill et al., 1990
). Vn is a glycoprotein which is synthesized in the liver and circulates in plasma at high concentration (200-400 µg/ml). The insoluble form is localized
extravascularly and is associated with granulation tissue
areas in rheumatoid arthritis synovia (Seiffert et al., 1993
).
It has been reported that 3 complex signaling via CD47
affected
2 (CD18/CD11b and CD18/CD11c) integrins
binding to their ligands (Van Strijp et al., 1993
; Ishibashi
et al., 1994
). Previous studies identified CD11b and CD11c
as novel ligands for sCD23 (Lecoanet-Henchoz et al.,
1995
). Our study shows that sCD23 bound CD11
cell
lines (Jurkat and CHO cells; Figs. 1 and 8) failed to stain the THP-1 (CD11+) cell line (Fig. 8), and anti-CD47 mAb
did not alter CD11b expression, or sCD23 binding on
monocytes (data not shown), indicating that sCD23 does
not interact with
2 integrins. We currently have no explanation for these contradictory results.
Interestingly, our unpublished data indicating TSP, a
newly discovered CD47 ligand (Gao et al., 1996b), also suppressed sCD23 function, without directly triggering monokine release, further supporting our present model. The
absence of VnR-mediated monokine secretion following
engagement by Vn, Fn, or TSP does not exclude the possibility that other ligands (see review Felding-Habermann and Cheresh, 1993
; Gladson and Cheresh, 1994
) would
share, with sCD23, its proinflammatory activity. Finally,
the ability of the anti-CD47 mAbs examined to inhibit the
function of sCD23 without interfering with the phagocytosis of senescent cells (Savill et al., 1990
) may help in the
design of novel therapeutic strategies for chronic inflammatory disorders, such as rheumatoid arthritis, in which
CD23 and TNF-
are implicated (Plater-Zyberk and Bonnefoy, 1995
; Feldman et al., 1996
).
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Footnotes |
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Address all correspondence to Dr. M. Sarfati, University of Montreal, Laboratoire Allergie (M4211-K), Centre de recherché Louis-Charles Simard, Pavillon Notre-Dame, 1560 Sherbrooke St. East, Montreal, Quebec H2L 4M1, Canada. Tel.: (514) 281-6000 ext. 6632. Fax: (514) 896-4753.
Received for publication 11 June 1998 and in revised form 2 December 1998.
P. Hermann and M. Armant contributed equally to this study.
We thank Dr. Y. Ohshima and Dr. C.E. Demeure for their help and criticism of the manuscript. The secretarial assistance of Norma Del Bosco is greatly appreciated.
This work was supported by a grant from the Medical Research Council of Canada (MRC). Dr. M. Sarfati is supported by a MRC Scientist Scholarship.
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Abbreviations used in this paper |
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B, biotinylated; Fn, fibronectin; sCD23, soluble CD23; TSP, thrombospondin; Vn, vitronectin; VnR, vitronectin receptor.
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References |
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