Thy-1 (CD90) Is an Interacting Partner for
CD97 on Activated Endothelial Cells
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J Immunol 2012; 188:1442-1450; Prepublished online 30
December 2011;
doi: 10.4049/jimmunol.1003944
http://www.jimmunol.org/content/188/3/1442
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References
Elke Wandel, Anja Saalbach, Doreen Sittig, Carl Gebhardt
and Gabriela Aust
The Journal of Immunology
Thy-1 (CD90) Is an Interacting Partner for CD97 on
Activated Endothelial Cells
Elke Wandel,*,† Anja Saalbach,‡ Doreen Sittig,* Carl Gebhardt,‡ and Gabriela Aust*
L
eukocyte extravasation into perivascular tissue plays a
key role in inflammatory diseases. This recruitment requires leukocyte interaction with vascular endothelium
and consists of multiple consecutive steps, including the capture
of circulating leukocytes, subsequent leukocyte rolling, arrest,
firm adhesion, and ensuing diapedesis. The cascade occurs by
sequential activation-dependent interactions between endothelial
cell (EC) adhesion molecules and their specific ligands on leukocytes.
Thy-1 (CD90), a highly glycosylated GPI-anchored cell surface
protein with a molecular mass ∼35 kDa, is a receptor on EC,
belonging to the Ig superfamily, and is involved in arrest and firm
adhesion of leukocytes to the endothelium (1, 2). In humans, Thy1 expression is restricted to activated EC, fibroblasts, neuronal
cells, and a subset of peripheral CD34+ stem cells (3). Adhesion of
neutrophils to activated Thy-1+ EC, mediated by Thy-1/Mac-1
(CD11b/CD18) interaction, is one attachment mechanism facili*Research Laboratories, Department of Surgery, University of Leipzig, 04103 Leipzig, Germany; †Translational Center for Regenerative Medicine, University of Leipzig, 04103 Leipzig, Germany; and ‡Department of Dermatology, Venerology, and
Allergology, University of Leipzig, 04103 Leipzig, Germany
Received for publication December 2, 2010. Accepted for publication November 17,
2011.
This work was supported in part by a grant from the German Federal Ministry of
Education and Research (BMBF, Projektträger Jülich, 0315883, to E.W.) and by
grants from the German Research Foundation (AU132/7-1 to G.A. and SA863/2-1
to A.S.).
Address correspondence and reprint requests to Prof. Gabriela Aust, Research Laboratories, Department of Surgery, University of Leipzig, Liebigstraße 20, 04103
Leipzig, Germany. E-mail address: [email protected]
Abbreviations used in this article: CFDA, carboxyfluorescein diacetate succinimidyl
ester; EC, endothelial cell; EGF, epidermal growth factor; HDMEC, human dermal
microvascular endothelial cell; hFc, human Fc; mFC, murine Fc-tag; MFI, mean
fluorescence intensity; PMNC, polymorphonuclear cell; sCD97, soluble CD97;
sEMR2, soluble EMR2; TM7, seven-span transmembrane.
Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003944
tating their subsequent migration into lesions of psoriatic skin (4–
6). However, blocking of CD11b/CD18 did not result in complete
inhibition of Thy-1–mediated adhesion of myeloid cells to activated EC (6). This suggests the presence of an additional interacting partner of Thy-1 involved in Thy-1–mediated adhesion of
myeloid cells to activated EC.
CD97, a member of the epidermal growth factor (EGF)–sevenspan transmembrane (TM7) subfamily of adhesion (class B2) G
protein-coupled receptors (7), shows a hematopoietic expression
profile that merits its consideration as a potential ligand for
Thy-1 on activated EC. CD97 is a cell surface receptor present
in peripheral neutrophils, monocytes, and activated lymphocytes
(8). CD97 is expressed as a heterodimer of a noncovalently
bound extracellular a-chain, represented by tandemly arranged
EGF domains and a stalk, and a b-chain, composed of the TM7
and a short intracellular portion. Both chains result from intracellular autocatalytic cleavage (9). The CD97 a-chain has been
thought to be shed from the membrane of CD97-expressing cells.
It is very likely identical to soluble CD97 (sCD97), detected in
synovial fluid of rheumatoid arthritis patients (10). As the result of
alternative splicing in humans, three isoforms exist: CD97
(EGF1,2,5), CD97(EGF1,2,3,5), and CD97(EGF1–5). CD97 shows
remarkable homology to the EGF-TM7 receptor EMR2 (CD312)
(11), especially within the EGF-like domains. Although both receptors are present at high levels in immune cells, the overall
expression pattern, ligand binding, and, thus, function are dissimilar (12).
Signal transduction through CD97 and EMR2 is still unknown.
Because truncation of the TM7 region disrupted CD97-increased
single random cell migration (13), and binding of a specific Ab
to EMR2 regulated human neutrophil function (14), signaling
through EGF-TM7 receptors seems very likely.
In this study, we identified Thy-1 as a potential new ligand
of CD97 and demonstrated that PMNC interact specifically, via
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Leukocyte recruitment in response to inflammatory signals is governed, in part, by binding to Thy-1 (CD90) on activated
endothelial cells (EC). In this study, we characterized the adhesion G-protein coupled receptor CD97, present on peripheral
myeloid cells, as a novel interacting partner for Thy-1. CD97 was upregulated on polymorphonuclear cells (PMNC) of patients
with psoriasis. In psoriatic skin lesions, CD97+ myeloid cells colocalized with Thy-1+ EC of small vessels in microabscesses,
suggesting an interaction between CD97 and Thy-1 that was further examined by adhesion and protein-binding assays. PMNC
and cell lines stably overexpressing CD97 adhered specifically to Thy-1+–activated human dermal EC, Thy-1+ CHO cells, and
immobilized Thy-1 protein. Binding of the CD97+ CHO clones correlated with their CD97 expression level. Soluble CD97 bound
specifically to immobilized Thy-1 protein, as well as Thy-1+–activated EC and CHO cells. In all assays, cellular adhesion or protein
binding was blocked partially by CD97 and Thy-1–blocking mAb. Our data suggested that CD97 interacts via its stalk with Thy-1
because mAb directed to the stalk of CD97 showed stronger blocking compared with mAb to its epidermal growth factor-like
domains, and binding was calcium independent. Moreover, soluble CD97 without the stalk and soluble EMR2, containing highly
homologous epidermal growth factor-like domains but a different stalk, failed to bind. In summary, binding of leukocytes to
activated endothelium mediated by the interaction of CD97 with Thy-1 is involved in firm adhesion of PMNC during inflammation
and may play a role in the regulation of leukocyte trafficking to inflammatory sites. The Journal of Immunology, 2012, 188: 1442–
1450.
The Journal of Immunology
CD97, with Thy-1 expressed in activated EC, thus mediating leukocytic adhesion.
Materials and Methods
Ab
FIGURE 1. Expression of CD97 is elevated in
PMNC of psoriatic patients. A, Upper left panel, CD97
was strongly expressed on infiltrating or existing myeloid cells in psoriatic skin lesions (arrows). Smooth
muscle cells are known to be CD97+ (arrowhead). Upper middle and right panels, Compared with CD31,
Thy-1 was found on EC (arrows) and, to some extent,
on fibroblasts. Scale bar, 50 mm. Lower panels, Infiltrating leukocytes located in epidermal microabscesses
of psoriatic skin lesions were CD97+. Microvascular EC adjacent to these leukocytes expressed Thy-1
and CD31, as shown by double immunofluorescence
staining. Scale bar, 30 mm. B, PMNC of psoriatic
patients (n = 15) adhered more strongly to activated
HDMEC compared with PMNC of healthy subjects
(n = 12; median, 5th/95th percentile). **p , 0.01. C,
PMNC of psoriatic patients (n = 15; pso) showed
higher CD97 expression compared with PMNC of
healthy controls (n = 12; ctr). Expression of EMR2
and CD11b on PMNC was comparable between both
groups. Expression was quantified by flow cytometry
as MFI (median: solid line, mean: dotted line; 5th/95th
percentile). *p , 0.05. D, TNF-a increased the expression of CD97 but not of EMR2 and CD11b on
PMNC of healthy donors (n = 5, median: solid, mean:
dotted; 5th/95th percentile). *p , 0.05. E, TNF-a–
activated PMNC adhered more strongly to stimulated
EC compared with untreated PMNC (n = 5). *p ,
0.05, **p , 0.01.
Patients
Patients aged $18 y (n = 15; 11 males; mean age, 49.7 6 20 y) with
plaque-type psoriasis that had been refractory to topical treatment with
external glucocorticoids or vitamin D3 analogs within the last 4 wk were
included in the study. Age- and sex-matched normal subjects (n = 12; 10
males; mean age, 47.7 6 14 y) were used as controls. The study was
approved by the University of Leipzig Ethics committee. All persons gave
their written consent prior to their enrollment into the study. The study was
conducted in accordance with the guidelines of the World Medical Association’s Declaration of Helsinki.
Immunohistology
Cryostat sections of patients with psoriasis were stained for double immunofluorescence and analyzed by laser-scanning microscopy or for simple
immunohistology, as described (20).
Cell separation and cell culture
Human dermal microvascular EC (HDMEC) and HUVEC were prepared, as
described (21). HDMEC were cultured in EGM-MV media (Promocell,
Heidelberg, Germany). Only preparations with .95% CD31+ EC were used
(21). For induction of Thy-1, EC in the first or second passage were
stimulated with 10 ng/ml PMA (Invitrogen, Karlsruhe, Germany) for 24 h.
PMNC of normal subjects and psoriatic patients were isolated, as described
(5). The purity was .95% CD15+ cells. PMNC were labeled with 0.1 mM
carboxyfluorescein diacetate succinimidyl ester (CFDA; Invitrogen) for 15
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The CD97EGF mAb (clone BL-Ac/F2 (8) detects a glycosylation-dependent
epitope within the first two EGF-like domains of CD97 and EMR2 (15). The
CD97stalk mAb (clone CLB-CD97/3) (16) binds to the stalk region of the
CD97 a-chain. The rabbit polyclonal CD97 Ab was purchased from SigmaAldrich Chemie (Munich, Germany). The mAb 1B5 binds to the fourth
EGF-like domain present only in the largest isoform of CD97 and EMR2
(17). The Thy-1 mAb clone AS02 does not block Thy-1–dependent cell
adhesion, whereas clone BC9 does (3, 18). The CD55 mAb (clone BRIC
216), binding to the short consensus repeat 3 domain of CD55, was purchased from the International Blood Reference Group laboratory (Bristol,
U.K.). The CLB-CD97L/1 mAb, binding to the short consensus repeat 1 domain of CD55 (19), and the EMR2 mAb (clone 1A2) (11) were kind gifts of
J. Hamann (University of Amsterdam, Amsterdam, The Netherlands). Both
CD55 mAb inhibit binding of erythrocytes to COS-7 cells transfected with
CD97(1,2,5) cDNA (19). The mAb to avb3 integrin (clone LM609) and the
polyclonal Ab to a5b1 integrin were purchased from Millipore (Schwalbach, Germany). MAb directed to ICAM-1 (CD54; clone R6.5.D6), CD11b
(clone X-5), and CD18 (clone IB4) were purchased from the American Type
Culture Collection (LGC Standards, Wesel, Germany), BMA Biomedicals
(Augst, Switzerland), or Calbiochem (Darmstadt, Germany).
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1444
min on ice. In some experiments, CFDA-labeled PMNC were activated with
10 ng/ml TNF-a (Immunotools, Friesoythe, Germany) for 30 min.
Generation of stably transfected clones overexpressing Thy-1
or CD97
CD97(EGF1,2,5) and CD97(EGF1–5) cDNA cloned into the pcDNA3.1
Zeo(+) vector (22) were used as basic templates for the generation of new
constructs. pEGFP-N1 (BD Biosciences, Heidelberg, Germany) generates
a fusion protein consisting of CD97 with enhanced GFP, allowing the
direct monitoring of transfected cells without further labeling.
Wild-type HT1080, WiDr, and CHO cells (American Type Culture
Collection) were stably transfected with 1 mg construct DNA by electroporation (290 V, 1500 mF; Multiporator; Eppendorf, Hamburg, Germany).
A total of 7.5 mg/ml geneticin (Invitrogen) was added after 24 h to select
stable clones. From each transfection, 3 clones of .10 were randomly
selected by high CD97 mean fluorescence intensity (MFI) in flow cytometry. Control cells contained either the empty plasmid (empty) or the
inverted construct (mock). CHO clones stably expressing human Thy-1
were generated as described (18).
Preparation of sCD97 and soluble EMR2
Cell-to-cell adhesion assays
In PMNC-to-cell–adhesion assays, EC or Thy-1– or mock-transfected
CHO cells were cultured in a 96-well plate. Confluent EC were stimulated for 24 h to induce stronger Thy-1 expression. A total of 2 3 104
CFDA-labeled unstimulated or TNF-a–activated PMNC were added to the
wells. After incubation for 45 min at 37˚C, unbound PMNC were removed
by washing with PBS. Adherent PMNC were lysed by the addition of 50
ml 10% SDS to the wells. Fluorescence was quantified using a fluorimetric
plate reader (SpectraMax; Molecular Devices, Sunnyvale, CA). Fluorescence and adherent cell number correlated in a linear fashion (data not
shown).
In cell line to cell-adhesion assays, 2 3 105 HT1080, WiDr, or CHO
cells stably overexpressing CD97 as an enhanced GFP-fusion protein were
added to a well of an eight-chamber slide precultured with Thy-1– or
mock-transfected CHO cells to confluence. After incubation for 30 min at
37˚C, unbound cells were removed by several washes with PBS. Adherent
cells were fixed with ice-cold methanol for 10 min at 220˚C, rinsed in
PBS, and mounted. The number of adherent cells was counted in 20 fields
at 403 magnification under a fluorescence microscope.
In blocking experiments, cells were preincubated with a specific or
control IgG mAb (10 mg/ml) for 30 min at 37˚C in the case of endothelial
or CHO monolayers or at 4˚C in the case of PMNC. EC were preincubated
with Ab directed to Thy-1, CD55, avb3 or a5b1 integrin, or ICAM-1.
PMNC were preincubated with mAb to CD18 or CD97.
In the sCD97-to-cell–binding assay, 1 3 105 Thy-1–transfected or
control CHO cells and unstimulated or activated HUVEC were incubated
with 0.1 mg sCD97(EGF1,2,5), sCD97(EGF1–5), or mFc-control protein
for 60 min at room temperature, followed by PE-labeled goat anti-mouse
(Dako) to detect the Fc-tag in flow cytometry. Alternatively, biotinylated
sCD97 could also be detected by PE-streptavidin (Dako).
To block binding of sCD97 to EC, cells were preincubated with 10 mg/ml
the respective Ab for 1 h at 37˚C prior to sCD97 addition. Nonbound
protein was washed away several times with PBS. EC were analyzed by
flow cytometry.
Statistical analysis
Statistical analysis was performed using the Student t test or Mann–
Whitney U test; p values , 0.05 were regarded as significant.
Results
PMNC of patients with psoriasis express elevated levels of
CD97
Adhesion of PMNC to activated Thy-1+ EC, mediated by the interaction of Thy-1 and Mac-1, facilitates their migration into
lesions of psoriatic skin (4–6). The presence of an additional
interacting partner for Thy-1 in PMNC has been suggested, because blocking of Mac-1 did not result in complete inhibition of
Thy-1–mediated adhesion of PMNC (6). In this study, we examined whether interaction of Thy-1 and CD97 is involved in the
adhesion of PMNC to activated EC.
Infiltrating leukocytes located in epidermal microabscesses and
in dermal inflammatory infiltrates of psoriatic skin lesions were
CD97+. Microvascular EC adjacent to these leukocytes expressed
Thy-1 (Fig. 1A).
According to Wetzel et al. (6), we confirmed that psoriatic
PMNC adhered more to activated EC compared with PMNC
of normal subjects (Fig. 1B). However, expression of the Thy-1
counterreceptor Mac-1 was not different on psoriatic and healthy
PMNC (Fig. 1C). In contrast, psoriatic PMNC showed higher
expression of CD97 than did normal PMNC, whereas expression
of EMR2, the close subfamily member of CD97, was comparable
between psoriatic and healthy PMNC (Fig. 1C).
Stimulation of healthy PMNC with TNF-a, resulting in an
enhanced adhesion of these PMNC to Thy-1–transfected cells,
Adhesion and binding assays with purified proteins
In cell-to-protein adhesion assays, 96-well MaxiSorb plates (Fisher Scientific, Schwerte, Germany) were coated with 500 ng human Fc (hFc)–
Thy-1 or hFc-control protein/well in TBS, 2 mM CaCl2, 0.1 mM MgCl2
overnight. Plates were washed with TBS and blocked with TBS/1% BSA
for 1 h at room temperature. A total of 5 3 105 CFDA-labeled PMNC,
preincubated with IgG ctr-mAb or mAb to CD97, were added to the coated
wells. Fluorescence was measured as described above. In other experiments, cells of three CHO clones, differing in the expression level of
CD97, were added to the coated wells.
In protein-binding assays, 1 mg purified sCD97(EGF1,2,5), sCD97
(EGF1,2,5) d stalk, sCD97(EGF1–5), sEMR2(EGF1,2,5), sEMR2(1,2,3,5),
or sEMR2(EGF1–5) was added to immobilized hFc–Thy-1 or hFc-control
protein-coated wells and incubated for 2 h at room temperature. Plates
were washed with TBS, 0.1% BSA, 0.05% Tween 20, 2 mM CaCl2, and
0.1 mM MgCl2. CD97 binding to immobilized Thy-1 was performed in the
absence or presence of 10 mg/ml mAb to Thy-1 or CD97, respectively.
Bound proteins were detected by addition of the biotinylated CD97EGF
mAb and HRP-labeled streptavidin (DakoCytomation, Hamburg, Germany), followed by tetramethylbenzidine substrate (Fisher Scientific).
Color reaction was measured at 450 nm. To evaluate whether binding
depends on the presence of Ca2+, binding was performed with buffers
containing Ca2+ or 1 mM EDTA without Ca2+.
FIGURE 2. Thy-1 expression is increased on activated HUVEC and
HDMEC. Unstimulated HUVEC (A) and HDMEC (B) were slightly
Thy-1+. Activation increased the percentage of Thy-1+ EC and the expression level of Thy-1 in both EC types. **p , 0.01, ***p , 0.001.
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Constructs encoding sCD97(EGF1,2,5), sCD97(EGF1,2,5) d stalk (without
the stalk), sCD97(EGF1–5), soluble EMR2 (sEMR2)(EGF1,2,5), sEMR2
(EGF1,2,3,5), and sEMR2(EGF1–5) fused to a murine Fc-tag (mFc) were
used (17, 23). mFc-sCD97 and mFc-control protein were biotinylated with
the RTS AviTag E. coli Biotinylation Kit from Roche Diagnostics (Mannheim, Germany).
THY-1 IS AN INTERACTING PARTNER OF CD97
The Journal of Immunology
thus imitating characteristics of psoriatic PMNC, increased the
expression of CD97 1.5-fold. The expression of EMR2 and
CD11b was unchanged (Fig. 1D). Psoriatic PMNC showed no
additional increase in CD97 levels after TNF-a treatment (data
not shown). Accordingly, TNF-a–activated PMNC adhered more
strongly to stimulated micro- and macrovascular EC compared
with control PMNC (Fig. 1E). In summary, our data suggested
the possible involvement of CD97 in adhesion of PMNC to activated EC.
MAb to CD97 and Thy-1 inhibit adhesion of PMNC to
activated EC
PMNC showed stronger adhesion to activated, compared with
unstimulated, micro- and macrovascular EC, indicating the upregulation of an endothelial receptor that mediates PMNC adhesion
(Fig. 2). Unstimulated EC slightly expressed Thy-1. Activation
increased the percentage of Thy-1+ HUVEC (Fig. 2A) and
HDMEC (Fig. 2B), as well as the expression level of Thy-1 in
these cells.
1445
Because adhesion of PMNC to activated Thy-1+ HDMEC and to
Thy-1–transfected cells can be blocked only partially with Ab to
CD18 (6), we suggested additional interaction partners for endothelial Thy-1 on PMNC. To examine whether CD97 is involved in
binding of PMNC to Thy-1+ cells, the adhesion of PMNC to
HUVEC and HDMEC was determined upon blocking by specific
mAb.
First, PMNC were pretreated with different mAb to CD97 (Fig.
3A, 3C). As a positive control, PMNC were preincubated with
mAb to CD18, which indeed partially blocked the binding of
PMNC to HUVEC (Fig. 3A) and HDMEC (Fig. 3C). Interestingly,
both CD97 mAb directed to the stalk (CD97stalk) or to the EGFlike domains (CD97EGF) blocked PMNC binding. The CD97stalk
mAb showed a stronger blocking compared with the CD97EGF
mAb in both EC. In a parallel assay, adhesion of CD55+ erythrocytes to CD97-transfected HT1080 cells, performed as described by Hamann et al. (24), was strongly inhibited by the
CD97EGF mAb, thus demonstrating the blocking function of this
mAb (data not shown). Consequently, our data indicated that the
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FIGURE 3. Adherence of PMNC to activated HUVEC (A, B) and HDMEC (C–E) is mediated, in part, by CD97 and Thy-1. Either PMNC (A, C, E) or EC
(B, D) were pretreated with the various Abs, as indicated, and incubated together. The number of adherent PMNC was determined. PMNC adhered more
strongly to activated EC compared with unstimulated EC. Binding of PMNC could be inhibited, in part, by mAb directed to CD97 on PMNC and to Thy-1
on activated EC. The combination of mAb to CD18 and CD97 for blocking PMNC (C) and the combination of mAb to Thy-1 and ICAM-1 for blocking EC
(D) caused additional inhibition. E, The increased adhesion of TNF-a–stimulated PMNC to activated HDMEC was prevented by mAb to CD18 and CD97.
The CD97stalk mAb decreased the adhesion of TNF-a–activated PMNC compared with the level of unstimulated PMNC, which were set as 100%. Data are
shown as mean 6 SD (n = 5). *p , 0.05, **p , 0.01, ***p , 0.001, compared with ctr; #p , 0.05, compared with the respective mAb alone. bl, blocking;
ctr, control; mAb CD55/1, clone BRIC 216; mAb CD55/2, clone CLB-CD97L1; nbl, nonblocking.
1446
FIGURE 4. CD97+ cells adhere to Thy-1–overexpressing CHO cells. A,
CD97-overexpressing clones of various cell lines were strongly CD97+ in
flow cytometry. CD97 MFI of one typical experiment of one clone is
shown. B, Adhesion of CD97(EGF1,2,5) or mock-expressing CHO, WiDr,
and HT1080 clones to Thy-1–overexpressing or vector-transfected CHO
clones was quantified. The number of adherent cells was counted/observation field (mean 6 SEM, n = 4). Strongest binding was seen between
CD97+ clones of the various cell types and Thy-1–overexpressing CHO
cells. **p , 0.01, ***p , 0.001.
cell-to-protein adhesion assay. PMNC showed strong adhesion
to hFc–Thy-1–coated wells but only weak binding to an irrelevant
immobilized hFc-control protein (Fig. 5A, 5B). This adhesion
could be partially inhibited by mAb to CD97. The CD97stalk mAb
showed the strongest inhibition.
To verify whether the number of adherent cells depends on the
expression level of CD97, we examined three CHO clones with
different CD97 expression levels (Fig. 5C). Clearly, the number of
CD97-overexpressing clones adhere specifically to
Thy-1–transfected cells
Next, we examined whether CD97-overexpressing cells adhered to
Thy-1+ CHO cells more strongly compared with the corresponding wild-type or CD97 mock-transfected cells. CHO, WiDr, and
HT1080 clones stably overexpressing CD97 were generated to
rule out that the measured effects depend on the parental cell line
transfected. Wild-type WiDr and HT1080 cells showed basal expression of CD97. However, the selected CD97-overexpressing
clones showed higher levels of CD97 compared with wild-type
or CD97 mock cells (Fig. 4A).
Irrespective of the cell line, CD97-overexpressing clones adhered
strongly to Thy-1–transfected CHO cells (Fig. 4B). The number of
adherent cells was comparably low in all control combinations:
adhesion of CD97 mock-transfected clones to vector-transfected
CHO cells, CD97-overexpressing clones to vector-transfected
CHO cells, and CD97 mock-transfected CHO or WiDr clones
to Thy-1–transfected CHO cells. A greater number of wild-type
HT1080 cells (data not shown) or CD97 mock-transfected HT1080
cells adhered to Thy-1–transfected CHO cells, because HT1080
wild-type cells express CD97 at a significant level. However, binding of CD97-overexpressing HT1080 clones to Thy-1–transfected
CHO cells was significantly greater compared with binding of
HT1080 mock-transfected cells (Fig. 4B).
PMNC and CD97-overexpressing CHO cells attach to
immobilized Thy-1 protein
Next, we examined whether PMNC and CD97-overexpressing
CHO clones bind specifically to immobilized Thy-1 protein in a
FIGURE 5. CD97+ cells adhere to Thy-1 protein. A and B, PMNC adhered specifically to immobilized recombinant hFc–Thy-1 but only slightly
to the unrelated hFc-control protein (Fc-ctr). MAb to CD97 inhibited this
adherence; CD97stalk mAb showed the strongest blocking (n = 4, mean 6
SEM). Scale bar, 100 mm. *p , 0.05, ***p , 0.001. C, CD97 expression
level of three CHO clones was determined by flow cytometry. CD97 MFI
of one typical experiment is shown. D, The number of cells adhering to
hFc–Thy-1 correlated with the CD97 expression level of these CHO clones
(n = 4, mean 6 SEM). *p , 0.05.
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stalk of CD97 is involved in the interaction of CD97 with Thy-1.
The combination of mAb to CD18 and CD97stalk caused a further
significant decrease in adhesion of PMNC compared with blocking with either mAb alone (Fig. 3C). This indicated that both
molecules mediate the adhesion of PMNC to activated EC.
Second, EC were pretreated with mAb directed to EC-specific
Ags (Fig. 3B, 3D). As expected, adherence of PMNC to HUVEC
was decreased by the Thy-1–blocking mAb (Fig. 3B). Thus,
blocking of HDMEC (Fig. 3D) was expanded to ICAM-1, a known
ligand of Mac-1 (CD11b/CD18), as well as to the other potential
interacting partners of CD97 on EC, such as CD55 and avb3 and
a5b1 integrin. Strongest blocking was demonstrated with the Thy1–blocking and the ICAM-1 mAb. Using two CD55 mAb, we
demonstrated slight blocking with the mAb BRIC 216. In a preassay, we confirmed that both CD55 mAb used, BRIC 216 and
CLB-CD97/L1, nearly completely blocked adhesion of erythrocytes to CD97-transfected HT1080 cells (data not shown). Adhesion of PMNC to activated HDMEC was also blocked by avb3
and a5b1 integrin Ab, but not as strongly as by the Thy-1
blocking mAb. The combination of the Thy-1–blocking mAb
and the ICAM-1 mAb caused an additive effect, whereas the
combination of the Thy-1–blocking mAb and the CD55 mAb
(BRIC 216) did not.
The slight adhesion of PMNC to unstimulated EC could not be
inhibited by mAb directed to CD18 or CD97 on PMNC or by mAb
to the various Ags in EC (Fig. 3A–D).
To clarify whether CD97 mediates the increased binding of
activated PMNC to Thy-1+ EC, we performed blocking experiments using TNF-a–stimulated PMNC (Fig. 3E). After preincubation of activated PMNC with the CD18, CD97EGF, or CD97stalk
mAb, adhesion of activated PMNC was blocked significantly.
Blocking of CD97 by the CD97stalk mAb decreased adhesion of
activated PMNC to the level of adhesion of unstimulated PMNC
to activated EC.
THY-1 IS AN INTERACTING PARTNER OF CD97
The Journal of Immunology
adherent CD97-overexpressing CHO cells correlated with the MFI
of the different clones (Fig. 5D).
sCD97 binds to immobilized Thy-1 protein
sCD97 binds to Thy-1+ cells
Finally, we examined binding of sCD97 to Thy-1+ cells. Binding
of sCD97(EGF1,2,5) and sCD97(EGF1–5), but not mFc-control
protein, to EC and to Thy-1–transfected CHO cells was detected
via the mFc-tag or the biotin-tag in flow cytometry (Fig. 7A).
FIGURE 6. sCD97 binds to Thy-1 in a cell-free
protein-binding assay. A, mFc-sCD97(EGF1,2,5) and
mFc-sCD97(EGF1–5) bound, in a concentrationdependent manner, specifically to immobilized hFc–
Thy-1. No binding was seen with the hFc-control protein. B, Binding of 20 ng/ml mFc-sCD97(EGF1–5) to
immobilized hFc–Thy-1 is shown in the presence of
10 mg/ml blocking and nonblocking Thy-1, CD97EGF,
or CD97stalk mAb (n = 4, mean 6 SEM). C, Twenty
nanograms per milliliter of sCD97(EGF1,2,5), but not
sCD97(EGF1,2,5) d stalk, the various sEMR2 isoforms, or the mFc-control protein bound to hFc–Thy-1
(n = 4, mean 6 SEM). D, Binding of 20 ng/ml sCD97
(EGF1,2,5) and sCD97(EGF1–5) to hFc–Thy-1 did
not depend on Ca2+. ***p , 0.001.
Unstimulated EC, slightly expressing Thy-1, showed weak binding of both sCD97(EGF1,2,5) and sCD97(EGF1–5). Activated EC
showed an increase in sCD97 binding compared with unstimulated EC.
Both sCD97 isoforms also bound to Thy-1–transfected CHO
cells (Fig. 7B, 7C). However, we observed significant binding of
sCD97(EGF1–5), but not sCD97(EGF1,2,5), to vector-transfected
CHO cells. Because wild-type CHO cells express chondroitin
sulfate B, a potential ligand of the longest, but not the shortest and
middle, CD97 isoform (17), it is very likely that sCD97(EGF1–5)
bound to chondroitin sulfate B. We tested this hypothesis by
blocking the interaction site for chondroitin sulfate B on sCD97
(EGF1–5). Pretreatment of sCD97(EGF1–5) with either chondroitin sulfate B or the mAb 1B5 abolished the binding to vectortransfected CHO cells, whereas binding to Thy-1–transfected
cells was diminished but still detectable (Fig. 7C). Binding of
sCD97(EGF1–5) to Thy-1+ EC could be partially inhibited by the
blocking mAb to Thy-1 and by the CD55 mAb BRIC 216 (Fig.
7D).
Discussion
The adhesive interaction of PMNC with activated EC is an essential
step in the process of PMNC accumulation at sites of inflammation
in vivo. In this study, we demonstrated that CD97 is involved in the
adhesion of PMNC to activated EC. Binding of CD97 and Thy-1
was shown at various levels. On the one hand, PMNC, CD97overexpressing cells, and sCD97 bound to activated EC, Thy-1–
overexpressing cells, as well as immobilized Thy-1 protein. On
the other hand, adherence and binding could be blocked partially
and specifically with the corresponding Ab.
CD97 belongs to the receptors that are able to interact with ligands different in structure and expression profiles. The long extracellular a-chain, probably identical to naturally occurring sCD97,
provides many possibilities for binding different cellular and extracellular matrix ligands.
Downloaded from http://www.jimmunol.org/ by guest on July 12, 2017
To further examine the interaction between CD97 and Thy-1, we
analyzed binding of the purified proteins hFc–Thy-1 and sCD97
(EGF1,2,5) or sCD97(EGF1–5) in a cell-free in vitro ligandbinding assay. Control binding to an irrelevant hFc-control protein was used to exclude unspecific binding of the Fc-tag. Both
sCD97 isoforms bound to immobilized Thy-1 but not to the hFccontrol protein (Fig. 6A). Binding was concentration dependent
and saturable.
The blocking, but not the nonblocking, Thy-1 mAb inhibited
binding up to 20% (Fig. 6B). Both the CD97EGF and CD97stalk
mAb also disturbed Thy-1 binding. The CD97stalk mAb showed
the strongest inhibition of Thy-1 binding, suggesting the involvement of the stalk of CD97 in Thy-1–CD97 interaction.
To clarify this point in more detail, we compared binding of
sCD97(EGF1,2,5) and sCD97(EGF1,2,5) d stalk to hFc–Thy-1.
sCD97 without the stalk did not bind hFc–Thy-1 (Fig. 6C), indicating that the stalk is essential for CD97 binding to Thy-1. Furthermore, we examined whether the three isoforms of sEMR2 bind
to hFc–Thy-1. EMR2 shows 97% amino acid sequence identity to
CD97 within the EGF-like domains but only 46% amino acid
sequence identity in the stalk region. None of the sEMR2 isoforms
bound hFc–Thy-1 (Fig. 6C). Furthermore, withdrawal of Ca2+
did not disturb Thy-1–CD97 interaction (Fig. 6D). Because
binding to the EGF-like domains depends on Ca2+, this result
also suggested the involvement of the CD97 stalk in binding
Thy-1.
1447
1448
THY-1 IS AN INTERACTING PARTNER OF CD97
The first identified ligand was CD55 (decay-accelerating factor)
(19), which binds to the first two EGF domains of CD97 (24).
CD55 is present in resting EC at a high level, whereas Thy-1 is
only slightly expressed on unstimulated EC but is increased after
activation. In our study, CD97+ cells, either PMNC or CD97transfected cells, bound much more to activated, compared with
unstimulated, EC, which did not indicate binding via CD55.
Moreover, pretreatment of activated EC with Thy-1– or CD55blocking mAb resulted in stronger inhibition of adherence by the
Thy-1–blocking mAb. Additionally, in normal human skin, CD55
is present in vascular structures, but its expression is decreased
in nonlesional psoriatic skin and virtually abolished in lesional
psoriatic skin (25). Furthermore, CD97–CD55 interaction is not
involved in human leukocyte adhesion to porcine EC in transplantation models (26). All of the data indicated that, although one
of the examined CD55 mAb slightly blocked adhesion of PMNC
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FIGURE 7. sCD97 binds to Thy-1+
cells. A, sCD97(EGF1,2,5) and sCD97
(EGF1–5), but not mFc-control protein,
showed stronger binding to activated
HUVEC compared with unstimulated
HUVEC, measured as CD97 MFI in
flow cytometry (n = 4, mean 6 SEM).
B and C, sCD97(EGF1,2,5) and sCD97
(EGF1–5), but not mFc-control protein,
bound more strongly to hFc–Thy-1
than to vector-transfected CHO cells.
sCD97(EGF1–5) bound also to wildtype CHO cells expressing chondroitin
sulfate B. C, Preincubation of sCD97
(EGF1–5) with chondroitin sulfate B or
the mAb 1B5 prevented binding to
vector-transfected cells but not to Thy1–transfected CHO cells. One representative of three independent experiments is shown. D, Binding of sCD97
(EGF1,2,5) and sCD97(EGF1–5) to
HUVEC was inhibited by the Thy-1–
blocking (bl) and CD55 mAb (clone
BRIC 216) but not by the Thy-1–nonblocking (nbl) and a5b1 and ICAM-1
Ab (n = 5, mean 6 SEM). **p , 0.01,
***p , 0.001.
on HDMEC, it is unlikely that CD97 in myeloid cells interacts
with CD55 in EC in psoriatic lesions.
Subsequently, a5b1 and avb3 integrins were identified as interacting proteins for human sCD97(1,2,5) and sCD97(EGF1–5)
on macrovascular EC (27). These sCD97 forms chemoattracted
HUVEC in a migration, as well as a Matrigel-based invasion,
assay. The Arg-Gly-Asp tripeptide present only in the stalk region
of human, but not mouse, CD97 was partially involved in these
effects (27). avb3 integrin is present in EC in psoriatic lesions
(28). In our study, binding of CD97 to a5b1 and avb3 integrins
may be partially involved in the adhesion of PMNC to activated
microvascular EC, because blocking Ab to these integrins slightly
inhibited the attachment of PMNC. We used the same Ab that
inhibited binding of sCD97 to HUVEC, as shown recently (27). In
contrast to our study, Wang et al. (27) used only sCD97 to evaluate
binding to a5b1 and avb3 integrins in EC. We performed cell-
The Journal of Immunology
structs, failed to bind Thy-1, although the EGF-like domains of
both proteins are almost identical. Moreover, binding did not need
calcium that is necessary for binding to the CD97 EGF-like
domains. However, both CD97 mAb used prevented binding of
Thy-1 protein only partially, indicating that they did not bind to
the specific interaction site of CD97 and Thy-1.
Second, are there discrepancies between the binding affinities or
properties of CD97 expressed at the cell surface and in soluble
forms of CD97? In our adhesion assays using PMNC in activated
EC, we observed a clear inhibiting effect of the Thy-1–blocking
mAb but only a weak effect of the CD55 mAb. However, binding
of sCD97 to activated EC was equally well blocked by the Thy-1–
blocking and CD55 mAb. Overall, our data agree with those
of Hamann et al. (24) and Kwakkenbos et al. (34): cell surfaceassociated CD97(EGF1–5) showed only a very weak binding to
CD55, whereas binding of sCD97(EGF1–5) to HEK293 cells was
clearly mediated by CD55, as shown by blocking of this interaction with a CD55-specific mAb.
Third, does CD97–Thy-1 binding play a significant role in vivo?
The interaction of CD97 with Thy-1 in EC is probably restricted
to sites of inflammation, because Thy-1 is expressed in EC at
a significant level only in regions in which cell activation and
inflammation occurred (35). Several murine experimental studies
indicated the involvement of CD97 in PMNC accumulation at
inflammatory sites. Targeting mouse CD97 by mAb inhibited the
accumulation of neutrophils at sites of inflammation, thereby affecting antibacterial host defense (36) and inflammatory disorders
(37). Otherwise, accumulation of PMNC at sites of inflammation
was not affected in CD97-deficient mice (20, 38) that display no
overt phenotype at steady-state, except for a mild granulocytosis, which increases under inflammatory conditions. Interestingly,
application of CD97 mAb blocked neutrophil trafficking after
thioglycollate-induced peritonitis in wild-type, but not in CD97
knockout, mice (20). Consequently, CD97 mAb induced an inhibitory effect that disturbed normal granulocyte trafficking, which
was not perturbed in the absence of the molecule (20). Overall,
comparison of the consequences of mAb treatment and gene targeting implied that CD97 mAb actively inhibited the innate response, presumably at the level of granulocyte or macrophage
recruitment to sites of inflammation in mice.
In summary, we identified Thy-1 as a potential new ligand of
CD97. PMNC interact specifically via CD97 with Thy-1 upregulated on activated EC. Thus, Thy-1–CD97 is probably involved,
together with other receptor–ligand pairs, in mediating leukocyte
adhesion at sites of inflammation.
Acknowledgments
The expression constructs for the various sCD97 and sEMR2 isoforms were
kindly provided by M. Stacey (Sir William Dunn School of Pathology, Oxford, U.K., now University of Leeds, Leeds, U.K.).
Disclosures
The authors have no financial conflicts of interest.
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