CD93 Is Rapidly Shed from the Surface of Human Myeloid
Cells and the Soluble Form Is Detected in Human Plasma1
Suzanne S. Bohlson,2Richard Silva, Maria I. Fonseca, and Andrea J. Tenner
CD93 is a highly glycosylated transmembrane protein expressed on monocytes, neutrophils, endothelial cells, and stem cells.
Antibodies directed at CD93 modulate phagocytosis, and CD93-deficient mice are defective in the clearance of apoptotic cells from
the inflamed peritoneum. In this study we observe that CD93, expressed on human monocytes and neutrophils, is susceptible to
phorbol dibutyrate-induced protein ectodomain shedding in a time- and dose-dependent manner. The soluble fragment found in
culture supernatant retains the N-terminal carbohydrate recognition domain and the epidermal growth factor repeats after
ectodomain cleavage. Importantly, a soluble form of the CD93 ectodomain was detected in human plasma, demonstrating that
shedding is a physiologically relevant process. Inhibition of metalloproteinases with 1,10-phenanthroline inhibited shedding, but
shedding was independent of TNF-?-converting enzyme (a disintegrin and metalloproteinase 17). Phorbol dibutyrate-induced
CD93 shedding on monocytes was accompanied by decreased surface expression, whereas neutrophils displayed an increase in
surface expression, suggesting that CD93 shed from the neutrophil surface was rapidly replaced by CD93 from intracellular stores.
Cross-linking CD93 on human monocytes with immobilized anti-CD93 mAbs triggered shedding, as demonstrated by a decrease
in cell-associated, full-length CD93 concomitant with an increase in CD93 intracellular domain-containing cleavage products. In
addition, the inflammatory mediators, TNF-? and LPS, stimulated ectodomain cleavage of CD93 from monocytes. These data
demonstrate that CD93 is susceptible to ectodomain shedding, identify multiple stimuli that trigger shedding, and identify both
a soluble form of CD93 in human plasma and intracellular domain containing cleavage products within cells that may contribute
to the physiologic role of CD93. The Journal of Immunology, 2005, 175: 1239–1247.
ties of activated leukocytes (2) and releases bioactive ectodomain
products. For example, soluble TNF-?, TGF-?, TGF-?, and mem-
bers of the epidermal growth factor (EGF)3family are all derived
from shedding of transmembrane proteins (3). Another conse-
quence of shedding is the release of the intracellular domain (ICD)
fragment after regulated cleavage of the transmembrane domain
(4). The ICD fragment of Notch, CD44, and others relocates to the
nucleus and regulates gene transcription (5). Therefore, shedding
affects numerous aspects of cell biology and subsequent physiol-
ogy via both intracellular and extracellular pathways.
Shedding is largely mediated by zinc-dependent metalloprotein-
ases of the matrix metalloproteinase, membrane type-matrix met-
alloproteinase or a disintegrin and metalloproteinase (ADAM)
family. TNF-?-converting enzyme (TACE) (ADAM17) is the
rotein ectodomain cleavage, or shedding, is a common fea-
ture of adhesion molecules that is generally mediated by
metalloproteases (1). Shedding modulates homing proper-
most widely investigated sheddase and is responsible for cleavage
of numerous transmembrane proteins, including L-selectin,
TNF-?, TGF-?, TGF-?, members of the EGF family, and the amy-
loid precursor protein (6). The majority of mice made deficient in
TACE die between embryonic day 17.5 and the first day after
birth, illustrating the importance of ectodomain shedding in mam-
malian development (7). However, TACE is not a universal shed-
dase, because a variety of transmembrane proteins, including
CD44, are cleaved by distinct metalloproteinases (8).
CD93 (C1qRP, AA4.1 (mouse)) is a 100,000 Mrtype 1 trans-
membrane glycoprotein that has been implicated in the regulation
of cell-cell interactions during development and in the efficiency of
phagocytosis (9–12). Mice deficient in CD93 display an impair-
ment in the uptake of apoptotic cells in vivo. In addition, an IgM
anti-CD93 mAb (R3) triggers phagocytosis when cells are plated
on the immobilized Ab and inhibits phagocytosis when cells are
treated in the fluid phase (13). However, the mechanisms respon-
sible for these effects on phagocytosis are unknown (10). CD93
has been implicated in regulating adhesive processes, because li-
gation of CD93 with the F(ab?)2of the mAb mNI-11 stimulates
homotypic aggregation of LPS-stimulated U937 cells, and homo-
typic aggregation is inhibited by Abs against LFA-1 or ICAM-1
(14). The same Ab triggers rapid spreading of HUVEC cells when
immobilized on a surface, and this spreading is inhibited by cy-
tochalasin D (such spreading is not detected with control samples
after HUVEC exposure to anti-CD44) (15). CD93 is expressed on
the endothelium and on circulating myeloid cells and platelets,
consistent with a role for CD93 in cellular homing to sites of
inflammation (12, 16). Furthermore, CD93 expression defines the
earliest human bone marrow stem cells because it is expressed on
CD34-negative and -positive hemopoietic and hepatic precursors,
and CD93 is a common marker for murine B cell development
Department of Molecular Biology and Biochemistry, Center for Immunology, Uni-
versity of California, Irvine, CA 92697
Received for publication November 30, 2004. Accepted for publication May 3, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by National Institutes of Health Grant AI41090 (to A.J.T.).
Support for obtaining human blood products used in this study was provided in part
by Public Health Service Research Grant M01RR00827 from the National Center for
2Address correspondence and reprint requests to Dr. Suzanne S. Bohlson, Depart-
ment of Molecular Biology and Biochemistry, University of California, 2419 Mc-
Gaugh Hall, Irvine, CA 92697. E-mail address: email@example.com
3Abbreviations used in this paper: EGF, epidermal growth factor; GalNAc, N-acetyl
galactosamine; HSA, human serum albumin; ICD, intracellular domain; PAF, plate-
let-activating factor; PDBu, phorbol dibutyrate; TAPI, TNF-? protease inhibitor;
PVDF, polyvinylidene diflouride; sCD93, soluble CD93; TACE, TNF-?-converting
enzyme; ADAM, a disintegrin and metalloproteinase.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc.0022-1767/05/$02.00
Previous studies demonstrated that glycosylation of CD93 stabi-
lized surface expression and that under conditions of induced hypo-
glycosylation, increased levels of CD93 were detected in culture me-
dium, suggesting that CD93 was proteolytically cleaved from the
surface (19). Because shedding has been implicated as an important
mechanism for regulation of other adhesion molecules with structural
and functional similarities to CD93 (e.g., L-selectin (2) and CD44
(8)), we chose to investigate cell stimuli that might induce CD93
shedding. The work described in this study shows that CD93 is sus-
ceptible to metalloproteinase-mediated shedding induced by the phor-
bol ester, phorbol dibutyrate (PDBu), independent of the glycosyla-
tion state. Interestingly, surface cleavage of CD93 occurs in both
human monocytes and neutrophils, but surface CD93 expression is
actually increased in neutrophils, possibly due to an intracellular store
of CD93 within neutrophil granules. Importantly, soluble CD93
(sCD93) was detected in normal human plasma, suggesting that the
cleavage event is physiologically relevant, and cytoplasmic tail cleav-
age products were detected in cell lysates, indicating that the liberated
CD93-ICD may also have biologic activity. In addition, CD93 shed-
ding was induced by cross-linking with mAbs and the inflammatory
ger this event. These data demonstrate a novel mechanism of regu-
lated expression of CD93 that may have implications in stem cell
development and inflammation.
Materials and Methods
Reagents and Abs
R139 and R3 were generated by immunization with C1q-binding proteins
isolated from U937 cell extracts as previously described (13, 20). The
polyclonal anti-CD93 cytoplasmic tail Ab 1150 was generated against the
C-terminal 11 aa of CD93 as previously described (21). Polyclonal anti-
CD93 1157 was generated by immunization with sCD93 produced in insect
SF9 cells as previously described (16). The anti-L-selectin mAb, Dreg 56,
was purchased from eBiosciences. FITC- or PE-conjugated secondary
F(ab?)2Abs were obtained from Jackson ImmunoResearch Laboratories.
TNF-? was purchased from PeproTech. C5a was a gift from R. DiScipio
(La Jolla Institute for Molecular Medicine, San Diego, CA). TNF-? pro-
tease inhibitor (TAPI) 1 and -2 were obtained from Peptides International.
R0-31-9790 and R0-32-7315 were obtained from Hoffmann La Roche.
Ultra Pure LPS was purchased from List Biological Laboratories. All me-
dia were obtained from Invitrogen Life Technologies unless otherwise
noted. All other reagents were purchased from Sigma-Aldrich at the high-
est quality unless stated otherwise.
Cells and plasma
Human monocytes and lymphocytes were purified by counterflow elutria-
tion using a modification of the technique of Lionetti et al. (22) as de-
scribed previously (23). Plasma from normal healthy donors was saved at
the time of elutriation and centrifuged at 16,000 ? g for 10 min to remove
platelets before use. In some experiments, IgG was depleted from plasma
by overnight incubation at 4°C with 120 ?l of GammaBind G-Sepharose
(Amersham Biosciences) to 200 ?l of plasma. Neutrophils were purified
from human blood as previously described (24) and resuspended in
HBSS?/0.25% human serum albumin (HSA; American Red Cross, distrib-
uted by FFF Enterprises) with 20 mM HEPES. ldlD cells were a gift from
Dr. M. Krieger (Massachusetts Institute of Technology, Cambridge, MA)
and were transfected with CD93 (ldlD-CD93) as previously described (19).
ldlD-CD93 were maintained in serum-free medium, Chinese hamster ova-
ry-serum-free medium II, supplemented with 100 U/ml penicillin G sodi-
um/100 ?g/ml streptomycin sulfate (Invitrogen Life Technologies) and 400
?g/ml G418 sulfate (Cellgro). To reconstitute wild-type glycosylation, 20 ?M
galactose and 400 ?M N-acetylgalactosamine were added to culture medium
48 h before experiments were conducted. HEK293T cells were cultured in
DMEM with 10% FBS (HyClone), 100 U/ml penicillin G sodium/100 ?g/ml
streptomycin sulfate (Invitrogen Life Technologies), 1? nonessential amino
acids (Invitrogen Life Technologies), and 10 mM HEPES, pH 7.4.
For analysis of surface expression by flow cytometry, monocytes were
resuspended at 2 ? 106/ml in RPMI 1640/10% heat-inactivated FBS, 10
mM HEPES and stimulated as described in the figure legends. For ELISA
studies, monocytes were resuspended at densities ?8 ? 106/ml before
activation, and neutrophils were resuspended at ?3 ? 107/ml. For immu-
noprecipitation studies, monocytes were treated in serum-free HL1 me-
dium (BioWhittaker). For inhibitor treatment, 5 mM 1,10-phenanthroline
(dissolved in methanol to 2 M) was added immediately before addition of
PDBu. For TACE inhibitor studies, inhibitors were added 30 min before
addition of PDBu. All TACE inhibitors were dissolved in DMSO, except
for TAPI-2, which was dissolved in HL1 medium (BioWhittaker). For Ab
cross-linking experiments, Abs were coated on two-well Lab-Tek chamber
slides (Nalge Nunc International) at 50 ?g/ml for 2 h in 0.1 M carbonate
buffer, pH 9.5. Chamber slides were washed with sterile PBS, and 2 ? 106
cells in 1 ml of RPMI 1640/10% FCS/10 mM HEPES were added, cen-
trifuged at 70 ? g for 3 min, and incubated at 37°C for various time
periods. Monolayers were washed with ice-cold PBS, and adherent cells
were harvested by scraping in 0.5 ml of extraction buffer (0.3% Nonidet P-40,
10 mM triethanolamine, 1 mM CaCl2, 1 mM MgCl2, 0.15 M NaCl, 0.1 M
PMSF, and 1/200 protease inhibitor mixture set III (Calbiochem)) on ice. Cell
supernatants and wash containing nonadherent cells were spun down at 1,000
rpm for 10 min, washed with ice-cold PBS, and added to the cell lysates.
Cells were lysed for 1 h on ice and centrifuged at 14,000 rpm for 15 min
at 4°C, and soluble material was analyzed by ELISA or Western blot.
Cells (1 ? 106) were washed twice in ice-cold FACS buffer (HBSS?, 0.2%
BSA, and 0.2% sodium azide) and incubated with primary Abs (3 ?g of
IgM/R3 or 1 ?g of IgG1/Dreg 56) for 30 min on ice with shaking every 10
min. Cells were washed twice in ice-cold FACS buffer and incubated with
2 ?l of FITC- or PE-conjugated secondary Abs (F(ab?)2) for 30 min on ice,
with shaking every 10 min. Cells were washed three times and either an-
alyzed immediately or fixed overnight in 1% formaldehyde (prepared
fresh) before analysis. Analysis was performed using the FACSCalibur
apparatus (BD Biosciences) and the CellQuest program.
CD93 sandwich ELISA using anti-CD93 mAbs R139 and R3 was per-
formed as previously described (19). To estimate the amount of CD93 in
picomolar concentrations, values were compared with a standard curve of
U937 cell extract. U937 cells were previously shown to contain 8.1 ? 104
molecules of CD93/cell (19). In some experiments, values in picomolar
concentrations were divided by the total number of cell equivalents. To
detect recombinant CD93 fusion proteins anti-V5 (Invitrogen Life Tech-
nologies) was used at a 1/1000 dilution to capture recombinant CD93
ectodomain fragments, and R3 was used to detect. To determine whether
sCD93 contained a cytoplasmic tail, an ELISA was used in which 1150
(rabbit polyclonal anti CD93 cytoplasmic tail) was used at 20 ?g/ml to
capture, and R3 was used to detect.
Immunoprecipitation and Western blot
Tissue culture supernatants were precleared for 2 h with 10 ?g of IgG2b
(ICN Pharmaceuticals) and 25 ?l of a 1/1 slurry of GammaBind G-Sepha-
rose (Amersham Biosciences). CD93 was immunoprecipitated from pre-
cleared tissue culture supernatants by incubation with 10 ?g of R139 for
1 h at 4°C, followed by a similar incubation with 25 ?l of a 1/1 slurry of
GammaBind G-Sepharose. The beads were washed three times in extrac-
tion buffer (described above) and boiled in nonreduced SDS-PAGE sample
buffer. For Western blot analysis, proteins were resolved by SDS-PAGE
and transferred to polyvinylidene difluoride (PVDF). After blocking in 5%
milk/TBST (20 mM Tris, 150 mM NaCl, and 0.1% Tween 20), blots were
probed for 2 h at room temperature with primary Abs. Blots were washed
and probed with secondary HRP-conjugated Abs for 1 h at room temper-
ature and developed using ECL (Amersham Biosciences).
Expression of rCD93 extracellular domains
The CD93-CRD was amplified from pcDNA3.1-CD93 (11) using the fol-
lowing primers: 5?-GGAATTCAGAGGGCCACACAGAGACCG and 3?-
GATATCTGAAGCT-GAACTTGCACAC. The CD93 CRD-EGF5was
amplified using the same 5? primer and 3?-GATATCTGGTGCAAG
AGACCCC. The endogenous CD93 signal sequence was included in the
constructs. The PCR products were subcloned into pGEM-T (Promega)
and sequenced at the University of California-Irvine core facility. The
products from pGEM-T were cloned into a modified pENTR-11 (Invitro-
gen Life Technologies) vector that had been engineered to remove its start
codon. Target genes were transferred to pcDNA-DEST40 via ? phage site-
specific recombination as described by the manufacturer of GATEWAY
cloning technology (Invitrogen Life Technologies). HEK 293T cells were
transfected with the indicated plasmids or, as a control, full-length CD93 in
1240CD93 IS SHED FROM MYELOID CELLS
pCDNA3.1 using LipofectAMINE (Invitrogen Life Technologies) accord-
ing to the manufacturer’s instruction. Supernatants were used as a source
of recombinant proteins. Proteins were purified by nickel chromatography
according to the manufacturer’s protocol (Invitrogen Life Technologies).
Immunofluorescence and confocal microscopy
Human neutrophils (2 ? 105) in 1 ml of HBSS?/0.25% HSA/20 mM
HEPES were plated on sterilized coverslips in a 12-well plate and incu-
bated at 37°C for 1 h. Cells were washed twice for 5 min each time with
PBS and fixed in 3.7% formaldehyde/PBS for 10 min at room temperature.
Cells were washed twice more with PBS and incubated overnight at 4°C
before staining. For permeabilization, cells were incubated with 0.1% Tri-
ton/PBS for 5 min. The immunostaining procedure performed was previ-
ously described (16). Briefly, cells (permeabilized and nonpermeabilized)
were incubated with 2% BSA/2% normal donkey serum in PBS (blocking
solution) for 1 h at room temperature. Next, cells were labeled with either
an anti-CD93 rabbit polyclonal Ab (1157) or a rabbit IgG control Ab at 5
?g/ml in blocking solution (1 h at room temperature), followed by Cy3-
conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laborato-
ries; 1/500 in blocking solution; 1 h at room temperature). Coverslips were
mounted with Vectashield (Vector Laboratories). Neutrophils were visu-
alized using a Zeiss LSM 510 META confocal microscope.
CD93 is shed from the surface of PDBu-stimulated monocytes
To determine whether CD93 was susceptible to ectodomain cleav-
age, human monocytes were treated with increasing concentrations
of the phorbol ester PDBu, a known potent inducer of shedding
(25). A decrease in cell surface CD93 was detected by flow cy-
tometry with increasing concentrations of PDBu, with a maximum
decrease in surface expression at 50 nM PDBu (Fig. 1A). To assess
the kinetics of the PDBu-induced decrease in surface expression,
monocytes were treated with 50 nM PDBu, and surface expression
was assessed at 2, 5, 10, 20, and 60 min. The decrease in cell
surface expression was significant at 20 min after addition of
PDBu (p ? 0.02, by paired Student’s t test) and was also enhanced
at 60 min (80.6% shedding; n ? 3; Fig. 1B). To determine whether
sCD93 was present in medium from PDBu-stimulated monocytes,
medium was analyzed in a sandwich ELISA using anti-CD93
mAbs R139 and R3. The sCD93 was detected at higher levels (6-
to 17-fold) in medium from PDBu-stimulated cells compared with
for 30 min at 37°C and analyzed for CD93 expression by flow cytometry. Depicted is the average mean fluorescence of three experiments with SD relative
to the untreated control. B, Monocytes were treated with 50 nM PDBu for various time periods and analyzed by flow cytometry after staining with
anti-CD93 mAb (R3). Depicted is the average mean fluorescence from three experiments (?SD). ?, p ? 0.02; ??, p ? 0.001 (by paired Student’s t test).
C, Monocytes (1.16 ? 106) in 1 ml were treated with 100 nM PDBu for 20 min. Supernatants were left untreated (?) or were cleared with 5 ?g of
anti-CD93 Ab (R139; f) or isotype control (IgG2b; o), followed by protein G-Sepharose and tested for CD93 by ELISA, as described in Materials and
Methods. Shown is the average of triplicate samples (?SD) from one experiment, representative of two. D, Equal volumes of cell lysates from human
monocytes treated with 50 nM PDBu for increasing amounts of time were separated by 4–20% gradient SDS-PAGE, transferred to PVDF, and probed with
5 ?g/ml polyclonal anti-CD93 cytoplasmic tail Ab 1150. The blot was stripped and reprobed for ?-actin to control for equal protein loading. The thin arrow
points to full-length CD93. Three thick arrows point to CD93 cytoplasmic tail containing cell-associated fragments running at ?25 kDa. E, Monocytes (1 ?
107) in 500 ?l of serum-free medium (HL1) were treated with or without PDBu for 30 min. Supernatants were immunoprecipitated with IgG2b or R139
and resolved by 8% nonreduced SDS-PAGE, and CD93 was detected by Western blot with 1 ?g/ml biotin R3.
CD93 is shed from human monocytes in response to PDBu. A, Human monocytes were stimulated with increasing concentrations of PDBu
1241 The Journal of Immunology
medium from nonstimulated cells. This activity in the ELISA was
depleted when the medium was absorbed with anti-CD93 (R139),
but not with the isotype control Ab (IgG2b), verifying that the
ELISA was specific (Fig. 1C; n ? 2). Full-length CD93 expression
decreased over time after stimulation with PDBu, whereas a grad-
ual increase in smaller cell-associated fragments containing the
CD93 cytoplasmic tail was detected by Western blot (Fig. 1D),
indicative of proteolytic processing of CD93. The presence of a
soluble form of CD93 in tissue culture supernatants was confirmed
by immunoprecipitation of CD93 (using R139) from monocyte
supernatants after treatment with 50 nM PDBu. A 75,000 Mrband
and a 50,000 Mrband reactive with anti-CD93 (R3; Fig. 1, arrows)
were apparent after treatment with PDBu (but not without PDBu;
n ? 2; Fig. 1E).
Soluble CD93 contains both the CRD domain and the EGF
Because both anti-CD93 mAbs R3 and R139 recognized the sol-
uble fragment(s) of CD93 in the culture supernatant from PDBu-
stimulated cells, recombinant proteins encoding either the CRD
domain of CD93 or the CRD domain followed by the EGF repeats
(CRD-EGF5; Fig. 2A) were expressed and used to map the inter-
action site of the Abs to determine the nature of the soluble frag-
ment. The proteins were expressed in HEK293T cells, and super-
natants were tested by Western blot and ELISA. When a sandwich
ELISA was performed using a capture Ab recognizing the V5
epitope tag found on both recombinant fusion proteins, reactivity
with R3 was detected for both fusion proteins, whereas reactivity
with R139 was detected only with the V5-CRD-EGF5fusion pro-
tein, indicating that R3 recognizes the CRD domain and R139
recognizes a region within the EGF repeats (Fig. 2B). Furthermore,
after purification by nickel chromatography, both proteins were
recognized with R3 by immunoblot analysis (Fig. 2B, inset). Also,
the fusion protein encoding the CRD-EGF5displayed reactivity in
an R3/R139 sandwich ELISA, whereas no reactivity by ELISA
was demonstrated for the CRD alone (Fig. 2C). These data dem-
onstrate that R3 and R139, Abs that modulate phagocyte activity
(20, 26), recognize distinct domains within CD93 and suggest that
sCD93 consists of the CD93 N terminus and contains both the
CRD and EGF5repeats, because the mAbs recognize these distinct
extracellular domain fragments generated by expression in HEK293T cells. B, Supernatants from HEK293T cells transfected with CD93 fusion proteins
were tested by sandwich ELISA using the anti-epitope tag V5 Ab to capture (1/1000) and either R3 (f) or R139 (p) to detect. The presence of purified
recombinant protein domains was verified by Western blot with R3 (5 ?g/ml; inset). C, Supernatants from B were tested by sandwich ELISA using the
anti-CD93 mAb R139 to capture (1 ?g/well) and biotinylated anti-CD93 R3 mAb to detect (2 ?g/ml), demonstrating the presence of sCD93 produced
constitutively in transfected HEK293T cells and supporting the immunoreactivity seen in B.
R3 recognizes the CD93-CRD, and R139 recognizes the EGF repeat domain of CD93. A, Schematic of CD93 and recombinant CD93
mal human donors was diluted (1/3 or 1/10) and tested for the presence of
sCD93 by sandwich ELISA with R139 and R3. The average of triplicate
wells (?SD) is shown. B, Soluble CD93 in plasma was depleted by ab-
sorption with 20 ?g of R139 or IgG2b coupled to protein G-Sepharose per
100 ?l of plasma or was competed by addition of 20 ?g of R139 or 20 ?g
of R3 to 115 ?l of plasma before dilution and analysis. The average of
triplicate wells (?SD) is shown.
Human plasma contains sCD93. A, Plasma from three nor-
1242CD93 IS SHED FROM MYELOID CELLS
Soluble CD93 is detected in human plasma/serum
To determine whether sCD93 was produced in vivo, human
plasma was analyzed for the presence of CD93 by ELISA. Plasma
from three healthy donors contained measurable levels of sCD93,
and these levels were similar among the three donors (Fig. 3A).
The activity in the ELISA was specific, because it was lost after
absorbance with R139, and it was competed by the addition of
R139 or R3, but not isotype control Abs (Fig. 3B). Soluble CD93
was also detected in human serum at levels similar to those in
plasma (data not shown). The concentration of sCD93 was esti-
mated to be 0.2 nM based on the number of molecules per sample
in a standard curve of U937 cell extracts that expresses ?8.1 ?
104molecules/cell (19). The sCD93 in serum and monocyte cul-
ture medium after PDBu stimulation was not reactive with the
anti-CD93 cytoplasmic tail Ab, as determined by ELISA, consis-
tent with ectodomain proteolysis (data not shown).
CD93 shedding is mediated by a metalloproteinase, but is
independent of TACE (ADAM17)
Because metalloproteinases are responsible for most of the re-
ported ectodomain cleavage (1), 1,10-phenanthroline, a zinc-
chelating compound and inhibitor of metalloproteinases, was
tested for its ability to inhibit PDBu-stimulated shedding of
CD93. Monocytes treated with PDBu in the presence of 1,10-
phenanthroline were completely resistant to PDBu-induced
ectodomain cleavage, as assessed by flow cytometry (Fig. 4, A
500 ?l were treated with 5 mM 1,10-phenanthroline, stimulated with 50 nM PDBu for 1 h, and analyzed for CD93 expression by flow cytometry. Shown
is the average mean fluorescence (?SD) relative to the untreated control from two experiments. B, Representative histogram from one of the experiments
described in A. f, Isotype control. The arrow points to PDBu-stimulated cells. Overlapping histograms are untreated (u) and 1,10-phenanthroline treated
(f). The dotted line represents cells treated with PDBu and 1,10-phenanthroline. C and D, Monocytes (1 ? 107) in 500 ?l were treated as described in
A, and supernatants were collected, cells were washed and lysed in 500 ?l lysis buffer, and 100 ?l of supernatant (C) or 100 ?l of cell lysate (2 ? 106
cell equivalents; D) was detected by ELISA. Shown is the average ? SD from triplicate samples, and data are from one experiment, representative of two.
E, Monocytes (?) or lymphocytes (o) were pretreated with TACE inhibitors before addition of 50 nM PDBu. Cells were incubated for an additional 20
min with PDBu, then analyzed for surface CD93 (?) or L-selectin (u) surface expression by flow cytometry. Treatment with R0 compounds is the average
of two experiments, and TAPI compounds were tested in one complete experiment with the L-selectin control (as shown). TAPI compounds showed similar
results with CD93 in at least three other experiments (not shown).
CD93 is cleaved from activated monocytes by a metalloproteinase independent of TACE (ADAM17). A, Human monocytes (1 ? 106) in
1243 The Journal of Immunology
and B). Furthermore, sCD93 could not be detected in superna-
tants from PDBu-stimulated monocytes in the presence of 1,10-
phenanthroline (Fig. 4C), and CD93 remained cell associated
(Fig. 4D). These data suggest that a metalloproteinase is re-
sponsible for the PDBu-induced cleavage event. TACE
(ADAM17) is the sheddase responsible for the cleavage of nu-
merous diverse transmembrane molecules (6); therefore, TACE
inhibitors were used to determine whether CD93 was cleaved
by TACE after PDBu stimulation. Incubation of monocytes
with four different hydroxamic acid-based TACE inhibitors,
TAPI-1, TAPI-2, R0-31-9790, and R0-32-7315, had no signif-
icant effect on PDBu-stimulated shedding of CD93. PDBu-stim-
ulated cleavage of L-selectin on lymphocytes was inhibited by
all TACE inhibitors, as expected, verifying that the inhibitors
were functional (Fig. 4E).
CD93 is shed from human neutrophils
Because CD93 is also expressed on human neutrophils, albeit at
?5-fold less surface density than monocytes (27) (Fig. 4B vs Fig.
5C), we determined whether similar shedding properties were ev-
ident on this cell type. There was less total cell-associated CD93
detected in PDBu-stimulated human neutrophils compared with
untreated control cells, as determined by ELISA (51.2–61.5%
range of three experiments; Fig. 5A) and Western blot (Fig. 5D).
Furthermore, when cell supernatants were analyzed for the pres-
ence of sCD93, 3.75- to 17-fold (range of three experiments) more
sCD93 was detected in culture medium from PDBu-stimulated
cells compared with unstimulated cells (Fig. 5B), demonstrating
that CD93 on neutrophils is also susceptible to PDBu-induced
shedding. However, in contrast to monocytes, PDBu-stimulated
neutrophils displayed an increase in cell surface CD93 expression
(220 ? 69%; n ? 4; Figs. 5C and 8C). These data indicate that
CD93 is shed from PDBu-stimulated neutrophils and also suggests
that CD93 may be stored in neutrophil granules, which are released
in response to PDBu. In support of this hypothesis, confocal anal-
ysis of neutrophils stained with anti-CD93 (1157) demonstrated
prominent intracellular staining in permeabilized cells (Fig. 5E,
lower right panel), whereas nonpermeabilized cells showed pre-
dominantly plasma membrane-associated CD93 (Fig. 5E, upper
50 nM PDBu for 15 min, and cell-associated CD93 from 6 ? 105cell equivalents (A) and sCD93 in culture supernatant (not diluted; B) was detected by
ELISA. Shown is the average (?SD) of triplicate samples from one experiment, representative of three. C, Neutrophil surface CD93 expression after 15-min
exposure to PDBu was analyzed by flow cytometry using R3. The histogram is from one experiment, representative of three. f, IgM control; black line,
untreated; gray line, PDBu-treated. D, Equal volumes of cell lysates from human neutrophils treated with 100 nM PDBu for increasing amounts of time
were separated by 4–20% gradient SDS-PAGE, transferred to PVDF, and probed with 5 ?g/ml polyclonal anti-CD93 cytoplasmic tail Ab 1150. The blot
was stripped and reprobed for ?-actin to control for equal protein loading. The thin arrow points to full-length CD93. The thick arrow points to CD93
cytoplasmic tail containing cell-associated fragment running at ?25 kDa. E, Confocal images of nonpermeabilized (upper) and permeabilized (lower)
neutrophils labeled with anti-CD93 (right) or control IgG (left).
CD93 is shed from activated neutrophils, but surface expression is up-regulated. Human neutrophils (1.5 ? 107) in 500 ?l were treated with
1244CD93 IS SHED FROM MYELOID CELLS
Glycosylation of CD93 does not alter PDBu-induced cleavage
Previous studies showed that CD93 is heavily O-glycosylated, and
that hypoglycosylation of CD93 resulted in a loss of surface CD93
(19). Glycosylation of CD93 can be regulated in the CD93-ex-
pressing CHO ldlD cell line, because growth in medium in the
absence of galactose and N-acetyl galactosamine (GalNAc) is non-
permisive for glycosylation, whereas in the presence of galactose
and GalNAc, glycosylation is restored. To determine whether the
glycosylation state of CD93 contributed to PDBu-induced shed-
ding, the effect of PDBu on surface CD93 expression of ldlD-
CD93 cells grown in the presence or the absence of galactose and
GalNAc was tested. As shown previously, in the absence of ga-
lactose and GalNAc, CD93 surface expression was markedly de-
creased (average, 60.6% decrease; n ? 3; data not shown). How-
ever, PDBu treatment of cultured glycosylation deficient cells
(without sugars) resulted in similar decreases in relative CD93
expression (51.3 and 44.3% in the presence or the absence of sug-
ars, respectively; n ? 3; Fig. 6) as that in control cells. These data
verify that glycosylation of CD93 affects cell surface expression/
stability, but the PDBu-induced shedding event is independent of
and distinct from the instability induced by hypoglycosylation.
Cross-linking CD93 with immobilized Ab induces shedding
To mimic potential ligation of CD93, monocytes were cultured on
anti-CD93 Abs (or isotype control Abs), and cell-associated CD93
was measured by ELISA. After 60 min of adherence to anti CD93,
cell-associated CD93 was decreased by 75.0 ? 13% on R139 com-
pared with IgG2b (n ? 3), and by 94.1% on R3 compared with
IgM (n ? 1) (Fig. 7A); it remained depressed for at least 18 h in
the presence of cross-linking anti-CD93 mAb (decrease of 85.6 ?
7.6% for R139 and 72.4 ? 19.5% for R3; n ? 3; data not shown).
The decrease in cell-associated CD93 at 60 min was partially in-
hibited with 1,10-phenanthroline (19.2 and 29.8% from two sep-
arate experiments; data not shown) consistent with ectodomain
shedding. Analysis of cell lysates by Western blot demonstrated
generation of cell-associated CD93 cytoplasmic tail-containing
cleavage products in response to Ab cross-linking (Fig. 7B), ver-
ifying that cross-linking stimulated proteolytic cleavage of CD93.
However, sCD93 was not detected in supernatants from these ad-
herent cells, even in response to PDBu. This is probably due to the
8- to 10-fold fewer cells per milliliter in the adhesion assay in this
study compared with the cell suspension assay (Figs. 1, 4, and 8),
resulting in sCD93 levels in supernatants that were below the
range of sensitivity of the ELISA.
Inflammatory stimuli trigger CD93 shedding from monocytes
and alter surface expression on neutrophils
Because the CD93 expression profile (monocytes, neutrophils, and
endothelial cells) and its shedding by cell activation are consistent
with a role for CD93 in leukocyte extravasation/inflammation, we
investigated the influence of inflammatory mediators on CD93
shedding. Human monocytes were treated with the proinflamma-
tory cytokine TNF-? or with the bacterial cell wall component
LPS. After 60 min, TNF-? and LPS stimulated shedding of CD93
from monocytes, as assessed by the presence of sCD93 in culture
medium (Fig. 8A), although the amount of shed CD93 was 10–
25% of that seen with PDBu stimulation. FACS analysis demon-
strated that CD93 surface expression after stimulation with TNF-?
and LPS was not significantly altered (Fig. 8B), consistent with the
lower degree of shed receptor and suggesting a modulated re-
sponse to alternative stimuli.
In contrast to monocyte CD93 surface expression and consistent
with the up-regulation of CD93 expression upon PDBu treatment
of neutrophils, TNF-? as well as the inflammatory mediators, C5a
CD93 glycosylation. CD93-expressing LdlD cells were grown in complete
medium (with sugars; f) or in medium lacking galactose and GalNac
(without sugars; o). Cells were treated with or without 300 nM PDBu
(optimal concentration determined for this cell type) for 30 min and ana-
lyzed for surface CD93 expression by flow cytometry. The average geo-
metric mean fluorescence (GMF) relative to control (no PDBu) of three
experiments (?SD) is shown.
PDBu-induced shedding on CHO cells is independent of
ding of CD93. A, Human monocytes (2 ? 106) were plated on two-well,
Lab-Tek chamber slides coated with 8 ?g/ml HSA or 50 ?g/ml Ab for 1 h
at 37°C. After removal of medium and washing, cells were lysed, and
lysates were subjected to sandwich ELISA with Abs R3 and R139. Shown
is the average of triplicate wells from one experiment, similar to three
experiments with R139 and IgG2b. B, Cell lysates from A were electro-
phoresed, transferred to PVDF, and probed with an anti-CD93 cytoplasmic
tail Ab (1150). Full-length CD93 migrates above the 100-kDa marker (ar-
rowhead), and arrows point to cell-associated proteolytic fragments of
CD93 that retain reactivity with the anti-cytoplasmic tail Ab. The blot was
stripped and reprobed for ?-actin as a loading control.
Cross-linking CD93 with immobilized Ab triggers shed-
1245The Journal of Immunology
and platelet-activating factor (PAF), induced a rapid increase
(within 15 min) in CD93 expression on human neutrophils (Fig.
8C). LPS also induced an increase in CD93 expression on neutro-
phils, but with a slower kinetic (by 30 min). In contrast, these
stimuli triggered a decrease in L-selectin expression, suggesting
that CD93 and L-selectin may have opposing functions in neutro-
phil homing (Fig. 8D).
This study demonstrates that CD93 on human monocytes and neu-
trophils is susceptible to protein ectodomain cleavage. The phorbol
ester, PDBu, a known inducer of ectodomain cleavage, triggered
CD93 cleavage in a time- and dose-dependent manner (Fig. 1).
Soluble CD93 was detected in tissue culture supernatants after
exposure to PDBu, and two anti-CD93-reactive species were iden-
tified by Western blot at 75 and 50 kDa in monocyte supernatants
(Fig. 1E). Soluble CD93 was detected in human plasma from
healthy donors (Fig. 3), consistent with a physiologic role for
sCD93, and the CD93 intracellular domain was detected in cells
after shedding. Cleavage of CD93 appears to be mediated by a
metalloproteinase, because it was inhibited by the metalloprotein-
ase inhibitor 1,10-phenanthroline (Fig. 4). Cross-linking CD93
with two immobilized mAbs recognizing different extracellular do-
mains of CD93 resulted in the release of CD93 (Fig. 7), suggesting
that ligand binding may induce CD93 shedding. However, this
decrease in full-length CD93 expression was only partially inhib-
ited by 1,10-phenanthroline, and the ICD-containing cleavage
fragments were not as readily detected after Ab cross-linking com-
pared with PDBu stimulation, suggesting that cross-linking may
also stimulate other forms of CD93 degradation insensitive to the
metalloprotease inhibitor. Incubation with the proinflammatory cy-
tokine, TNF-?, or the bacterial cell wall component, LPS, also
induced CD93 shedding on monocytes, suggesting that sCD93
may be released at sites of inflammation in vivo (Fig. 8).
Although CD93 was originally described as a C1q receptor that
mediated enhanced phagocytosis, recent studies demonstrate that
CD93 does not bind to C1q (12) and that CD93 is not required for
the C1q-mediated enhanced phagocytic activity (10). McGreal et
al. (28) have postulated that CD93, similar to platelet-endothelial
cell adhesion molecule 1, is involved in leukocyte homing based
on a shared tissue distribution pattern with platelet-endothelial cell
adhesion molecule 1 and because a CD93 extracellular domain Fc
fusion protein bound to activated endothelium, suggesting that
there is a CD93 ligand expressed on the endothelium (12). There-
fore, generation of sCD93 induced by proinflammatory mediators
(e.g., TNF-? or LPS) or rapid alterations in CD93 expression on
activated neutrophils may alter the homing properties of mono-
cytes and/or neutrophils at sites of inflammation. Interestingly,
TNF-?, LPS, PAF, and C5a all induced a rapid and nearly com-
plete loss of surface L-selectin expression on neutrophils (Fig. 8D)
while inducing an elevated level of CD93 (Fig. 8C), suggesting
that these two molecules may have opposing functions in neutro-
phil migration. Furthermore, the enzyme(s) responsible for cleav-
age of CD93 differs from the L-selectin sheddase (TACE/
ADAM17; Fig. 4D). Shedding independent of TACE/ADAM17
was also reported for CD44 (8), an adhesion molecule with some
similarities in structure and function to CD93 (29).
107) in 500 ?l of culture medium were treated with or without with 5 ng/ml TNF-? or 100 ng/ml LPS for 60 min at 37°C. Supernatants were analyzed
for sCD93 by ELISA. Shown is the mean of triplicate wells (?SD) from one experiment representative of two. ?, p ? 0.03; ??, p ? 0.003 (by paired
Student’s t test). B, Surface expression of CD93 from monocytes treated as described in A was measured by flow cytometry. Shown is the mean of two
separate experiments (?SD) relative to the control (untreated) sample. C and D, Human neutrophils (1 ? 106) were incubated at 37°C for various time
periods without stimulus (f) or with 5 ng/ml TNF-? (Œ), 100 nM C5a (?), 1 ?M PAF (X), 50 nM PDBu (?), or 100 ng/ml LPS (?) in HBSS?/0.25%,
HSA and 20 mM HEPES. Surface expression of CD93 (C) and L-selectin (D) was measured by flow cytometry. A single experiment with multiple time
points is shown. Similar results for the 15 min point were obtained in three separate experiments.
Inflammatory mediators induce CD93 shedding on monocytes and alterations in CD93 expression on neutrophils. A, Human monocytes (1 ?
1246CD93 IS SHED FROM MYELOID CELLS
One consequence of ectodomain cleavage is the subsequent reg- Download full-text
ulated cleavage of the transmembrane domain, resulting in the lib-
eration of the ICD into the cytoplasm. Although some ICD frag-
ments are susceptible to proteosome-mediated degradation, others
have been shown to act as transcription factors or to act in complex
with transcription factors to alter gene expression. Interestingly,
the cytoplasmic domain of CD93 contains a highly charged jux-
tamembrane domain, predicted to contain a nuclear localization
signal (30), suggesting that the CD93-ICD may be involved in
gene regulation. Furthermore, CD93-ICD fragments are readily
detected in human monocyte cell lysates after PDBu treatment or
Ab cross-linking (Figs. 1D and 7B), implying a relative lack of
susceptibility to proteosome-mediated degradation.
The soluble fragment of the CD93 extracellular domain may
have biological activity, because numerous ectodomain cleavage
products are biologically active (e.g., TNF-?, TGF-?, TGF-?, and
members of the EGF family). Interestingly, Botto et al. (10)
showed that CD93-deficient mice were defective in the clearance
of apoptotic cells from the inflamed peritoneum; however, the
mechanism remains elusive. Perhaps sCD93 is a bridging mole-
cule, responsible for linking the apoptotic cell to the phagocyte,
similar to C1q, protein S, thrombospondin, milk fat globule-EGF
factor 8 protein, and others (31), and is the component responsible
for enhanced clearance of apoptotic cells under inflammatory con-
ditions, when sCD93 would be expected to be present.
The identification of the susceptibility of CD93 to ectodomain
cleavage under a variety of conditions and the observation that the
soluble fragment of CD93 exists in plasma from normal healthy
donors open numerous avenues to study the functional significance
of CD93 and the mechanism by which CD93 modulates phagocy-
tosis and adhesion. Although not addressed in this study, CD93 is
widely expressed on endothelium, so it will be important to deter-
mine whether endothelial CD93 is susceptible to ectodomain
cleavage and to characterize the resulting changes in surface ex-
pression of CD93 after activation of endothelium. The observa-
tions presented in this study warrant further investigation regard-
ing the biological activities of the soluble fragment(s) of CD93
found in plasma, and likely at foci of inflammation, as well as
investigations to elucidate the biological activity of the CD93 ICD
after ectodomain cleavage.
We are grateful to Ozkan Yazan for excellent technical assistance, and
Meiying Zhou for help with the initial shedding experiments. We thank the
staff of the University of California-Irvine General Clinical Research Cen-
ter for help with obtaining human blood for monocyte purification.
The authors have no financial conflict of interest.
1. Arribas, J., L. Coodly, P. Vollmer, T. K. Kishimoto, S. Rose-John, and
J. Massague. 1996. Diverse cell surface protein ectodomains are shed by a system
sensitive to metalloprotease inhibitors. J. Biol. Chem. 271: 11376.
2. Galkina, E., K. Tanousis, G. Preece, M. Tolaini, D. Kioussis, O. Florey,
D. O. Haskard, T. F. Tedder, and A. Ager. 2003. L-selectin shedding does not
regulate constitutive T cell trafficking but controls the migration pathways of
antigen-activated T lymphocytes. J. Exp. Med. 198: 1323–1335.
3. Blobel, C. P. 2000. Remarkable roles of proteolysis on and beyond the cell sur-
face. Curr. Opin. Cell Biol. 12: 606–612.
4. Hoppe, T., M. Rape, and S. Jentsch. 2001. Membrane-bound transcription fac-
tors: regulated release by RIP or RUP. Curr. Opin. Cell Biol. 13: 344–348.
5. Medina, M., and C. G. Dotti. 2003. RIPped out by presenilin-dependent ?-secre-
tase. Cell. Signal. 15: 829–841.
6. Black, R. A. 2002. Tumor necrosis factor-? converting enzyme. Int. J. Biochem.
Cell Biol. 34: 1–5.
7. Peschon, J. J., J. L. Slack, P. Reddy, K. L. Stocking, S. W. Sunnarborg, D. C. Lee,
W. E. Russell, B. J. Castner, R. S. Johnson, J. N. Fitzner, et al. 1998. An essential
role for ectodomain shedding in mammalian development. Science 282:
8. Shi, M., K. Dennis, J. J. Peschon, R. Chandrasekaran, and K. Mikecz. 2001.
Antibody-induced shedding of CD44 from adherent cells is linked to the assem-
bly of the cytoskeleton. J. Immunol. 167: 123–131.
9. Petrenko, O., A. Beavis, M. Klaine, R. Kittappa, I. Godin, and I. R. Lemischka.
1999. The molecular characterization of the fetal stem cell marker AA4. Immu-
nity 10: 691–700.
10. Norsworthy, P. J., L. Fossati-Jimack, J. Cortes-Hernandez, P. R. Taylor,
A. E. Bygrave, R. D. Thompson, S. Nourshargh, M. J. Walport, and M. Botto.
2004. Murine CD93 (C1qRp) contributes to the removal of apoptotic cells in vivo
but is not required for C1q-mediated enhancement of phagocytosis. J. Immunol.
11. Nepomuceno, R. R., A. H. Henschen-Edman, W. H. Burgess, and A. J. Tenner.
1997. cDNA cloning and primary structure analysis of C1qRP, the human C1q/
MBL/SPA receptor that mediates enhanced phagocytosis in vitro. Immunity 6:
12. McGreal, E. P., N. Ikewaki, H. Akatsu, B. P. Morgan, and P. Gasque. 2002.
Human C1qRp is identical with CD93 and the mNI-11 antigen but does not bind
C1q. J. Immunol. 168: 5222–5232.
13. Guan, E., S. L. Robinson, E. B. Goodman, and A. J. Tenner. 1994. Cell surface
protein identified on phagocytic cells modulates the C1q-mediated enhancement
of phagocytosis. J. Immunol. 152: 4005–4016.
14. Ikewaki, N., and H. Inoko. 1996. Development and characterization of a novel
monoclonal antibody (mNI-11) that induces cell adhesion of the LPS-stimulated
human monocyte-like cell line U937. J. Leukocyte Biol. 59: 697–708.
15. Ikewaki, N., H. Tamauchi, A. Yamada, N. Mori, H. Yamao, H. Inoue, and
H. Inoko. 2000. A unique monoclonal antibody mNI-11 rapidly enhances spread
formation in human umbilical vein endothelial cells. J. Clin. Immunol. 20:
16. Fonseca, M. I., P. M. Carpenter, M. Park, G. Palmarini, E. L. Nelson, and
A. J. Tenner. 2001. C1qR(P), a myeloid cell receptor in blood, is predominantly
expressed on endothelial cells in human tissue. J. Leukocyte Biol. 70: 793–800.
17. Li, Y. S., R. Wasserman, K. Hayakawa, and R. R. Hardy. 1996. Identification of
the earliest B lineage stage in mouse bone marrow. Immunity 5: 527–535.
18. Danet, G. H., J. L. Luongo, G. Butler, M. M. Lu, A. J. Tenner, M. C. Simon, and
D. A. Bonnet. 2002. C1qRp defines a new human stem cell population with
hematopoietic and hepatic potential. Proc. Natl. Acad. Sci. USA 99:
19. Park, M., and A. J. Tenner. 2003. Cell surface expression of C1qRP/CD93 is
stabilized by O-glycosylation. J. Cell Physiol. 196: 512–522.
20. Guan, E. N., W. H. Burgess, S. L. Robinson, E. B. Goodman, K. J. McTigue, and
A. J. Tenner. 1991. Phagocytic cell molecules that bind the collagen-like region
of Clq: involvement in the Clq-mediated enhancement of phagocytosis. J Biol.
Chem. 266: 20345–20355.
21. Webster, S. D., M. Park, M. I. Fonseca, and A. J. Tenner. 2000. Structural and
functional evidence for microglial expression of C1qRP, the C1q receptor that
enhances phagocytosis. J. Leukocyte Biol. 67: 109–116.
22. Lionetti, F. J., S. M. Hunt, and C. R. Valeri. Methods of Cell Separation. Plenum
Press, New York, pp. 141–156.
23. Bobak, D. A., M. M. Frank, and A. J. Tenner. 1986. Characterization of C1q
receptor expression on human phagocytic cells: effects of PDBu and FMLP.
J. Immunol. 136: 4604–4610.
24. Goodman, E. B., D. C. Anderson, and A. J. Tenner. 1995. C1q triggers neutrophil
superoxide production by a unique CD18-dependent mechanism. J. Leukocyte
Biol. 58: 168–176.
25. Guo, L., J. R. Eisenman, R. M. Mahimkar, J. J. Peschon, R. J. Paxton,
R. A. Black, and R. S. Johnson. 2002. A proteomic approach for the identification
of cell-surface proteins shed by metalloproteases. Mol. Cell. Proteomics 1:
26. Nepomuceno, R. R., S. Ruiz, M. Park, and A. J. Tenner. 1999. C1qRPis a heavily
O-glycosylated cell surface protein involved in the regulation of phagocytic ac-
tivity. J. Immunol. 162: 3583–3589.
27. Nepomuceno, R. R., and A. J. Tenner. 1998. C1qRp, the C1q receptor that en-
hances phagocytosis, is detected specifically in human cells of myeloid lineage,
endothelial cells, and platelets. J. Immunol. 160: 1929–1935.
28. Albelda, S. M., W. A. Muller, C. A. Buck, and P. J. Newman. 1991. Molecular
and cellular properties of PECAM-1 (endoCAM/CD31): a novel vascular cell-
cell adhesion molecule. J. Cell Biol. 114: 1059–1068.
29. Bohlson, S. S., M. Zhang, C. E. Ortiz, and A. J. Tenner. 2005. CD93 interacts
with the PDZ domain-containing adaptor protein GIPC: implications in the mod-
ulation of phagocytosis. J. Leukocyte Biol. 77: 80–89.
30. Cokol, M., R. Nair, and B. Rost. 2000. Finding nuclear localization signals.
EMBO Rep. 1: 411–415.
31. Savill, J., I. Dransfield, C. Gregory, and C. Haslett. 2002. A blast from the past:
clearance of apoptotic cells regulates immune responses. Nat. Rev. Immunol. 2:
1247 The Journal of Immunology