2B4 (CD244) Is Expressed and Functional on Human
Ariel Munitz,* Ido Bachelet,* Shira Fraenkel,* Gil Katz,†Ofer Mandelboim,†
Hans-Uwe Simon,‡Lorenzo Moretta,§¶Marco Colonna,?and Francesca Levi-Schaffer2*
Eosinophils are present in parasitic, allergic, various immunological, and malignant disorders as well as in a variety of idiopathic
hypereosinophilic syndromes. However, their exact role in some of these conditions remains elusive. They can be activated both
in vivo and in vitro by various agonists, such as Igs, lipid mediators, and cytokines. By phenotyping the surface of the eosinophils,
it may be possible to better define their function(s) in different pathophysiological settings. In the present work we screened
eosinophils with a panel of Abs recognizing CD2 subfamily receptors usually present on a number of hemopoietic cells. We have
demonstrated that human peripheral blood eosinophils, but not basophils or neutrophils, express NTB-A. In addition eosinophils
express 2B4, CD84, CD58, and CD48, but not signaling lymphocytic activation molecule or CD2, on their surface (FACS).
Cross-linking of 2B4 on eosinophils elicited a significant release of eosinophil peroxidase (30 min), IFN-?, and IL-4 (18 h).
Moreover, activation of eosinophils via 2B4 induced eosinophil-mediated cytotoxicity toward two malignant cell lines, i.e., mouse
mastocytoma P815 and EBV-infected 721.221 B cell lines. Cross-linking of 2B4 on the surface of eosinophils or pervenadate
treatment elicited ERK and tyrosine phosphorylation, respectively. Furthermore, we showed that eosinophils express slam-asso-
ciated protein. The demonstration that human eosinophils express a functional 2B4 receptor indicates a broader role for these cells
in health and disease. The Journal of Immunology, 2005, 174: 110–118.
phils are found mainly in mucosal surfaces such as the gastroin-
testinal tract (1). However, blood eosinophilia is detected together
with tissue eosinophilia in allergic, some parasitic, immunological,
and malignant diseases as well as in a variety of idiopathic hy-
pereosinophilic syndromes (2–5). Even though eosinophils have
been attributed an effector role in allergic inflammation, such as
the tissue damage activity in asthma, there is still no consensus on
the exact activity of eosinophils in this condition and others.
Eosinophils express a variety of receptors for different agonists
in their pathophysiological environment that can activate them. For
example, they express receptors for IgA, IgG, cytokines, chemo-
kines, and complement components (4–6). Activation of eosino-
phils results in the secretion of specific, crystalloid granule pro-
teins such as major basic protein (MBP),3eosinophil peroxidase
osinophils are bone marrow-derived, blood-circulating
granulocytes with a typical bi-lobuled nucleus and cyto-
plasmic granules. Under physiologic conditions, eosino-
(EPO), eosinophil-derived neurotoxin, and eosinophil cationic pro-
tein. In addition, eosinophils synthesize and release lipid media-
tors, such as platelet-activating factor, leukotriene C4, PGE2, as
well as proinflammatory and immunoregulatory cytokines and
Receptor definition and surface phenotyping of the eosinophils
are possible tools to better understand their functions in different
pathophysiological settings. This approach has led to the identifi-
cation of NK cell-regulating molecules that resulted in a marked
progress of understanding the biology, development, and function
of these cells (7).
A wide range of surface activatory and/or inhibitory molecules,
such as leukocyte Ig-like receptor/Ig-like transcript, Siglecs,
CD28, and CD86 (8–12), that had been previously thought to be
expressed mainly by T and NK cells have recently been found to
be expressed by eosinophils. It appears that a complex of activa-
tory and inhibitory networks can regulate the activities of
Our interest has focused on the receptors belonging to the CD2
subfamily of the Ig superfamily. They include CD2, CD48 (Blast-1
and BCM-1), CD58 (LFA-3), CD84 (Ly9B), CD150 (signaling
lymphocytic activation molecule (SLAM)), CD229 (Ly9), 2B4
(CD244), BCM-like membrane protein, SF2001 (CD2F-10),
NTB-A (SF2000 and Ly108), and CS1 (CD1-like receptor-acti-
vating cytotoxic effects) (13, 14).
Of this family, 2B4 is expressed on virtually all NK cells, T
cells, a large subset of CD8?T cells, and a small percentage of
CD4?T cells, monocytes, and basophils (15). Cross-linking of 2B4
on NK cells triggers their activation (13), and interaction of
2B4 with its high affinity ligand CD48 (16) results in lysis of tumor
cells and of CD48-transfected cells (15). Cross-linking of 2B4 on
NK cells involves multiple distinct pathways (17) and requires the
unique molecule slam-associated protein (SAP) (17–19). Interest-
ingly, mutations or absence in the SAP gene result in a severe
disease, X-linked lymphoproliferative disease (20–22)
*Department of Pharmacology, School of Pharmacy, Faculty of Medicine, and†Lau-
tenberg Center for General and Tumor Immunology, Faculty of Medicine, Hebrew
University of Jerusalem, Jerusalem Israel;‡Department of Pharmacology, University
of Bern, Bern, Switzerland;§Istituto Giannina Gaslini, Genova, Italy;¶Department of
Experimental Medicine, University of Genova, Genova, Italy;?Department of Pathol-
ogy and Immunology, Washington University School of Medicine, St. Louis, MO
Received for publication July 12, 2004. Accepted for publication September 29, 2004.
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 grants from the Aimwell Charitable Trust (U.K.).
2Address correspondence and reprint requests to Dr. Francesca Levi-Schaffer, De-
partment of Pharmacology, School of Pharmacy, Faculty of Medicine, Hebrew Uni-
versity of Jerusalem, POB 12065, Jerusalem 91120, Israel. E-mail address:
3Abbreviations used in this paper: MBP, major basic protein; EM, enriched medium;
EPO, eosinophil peroxidase; HBA, HBSS supplemented with 0.1% BSA and 0.02%
sodium azide; SAP, slam-associated protein; Siglecs, sialic acid-binding Ig-like lec-
tin; SLAM, signaling lymphocytic activation molecule.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc.0022-1767/05/$02.00
In the present work we have hypothesized that eosinophils could
express cell surface receptors belonging to the CD2 subfamily that
may regulate their function. We have now shown a restricted ex-
pression pattern of the CD2 subfamily receptors on the surface of
eosinophils. Eosinophils can be activated by 2B4 to release EPO,
IFN-?, and IL-4. Engagement of 2B4 on FcR?cells or direct 2B4-
CD48 interactions lead to lysis of tumor cells. In addition, we have
demonstrated for the first time that eosinophils express SAP.
Materials and Methods
Three sources of anti-2B4 mAbs were used: P158 (15), C1.7 (Coulter-
Immunotech), and PP35 (23). Anti-CD2 FITC-conjugated mAbs (MT910)
were obtained from DakoCytomation. Anti-CD58 (1C3), anti-CD84 FITC-
conjugated (2G7), anti-HLA-DR PerCp-Cy5.5-conjugated (G46-6), and
mouse IgG2a isotype control mAbs were purchased from BD Pharmingen.
Anti-NTB-A mAbs (MA127 and ON56) were obtained as previously de-
scribed (24). Anti-CD48 (4H9), goat polyclonal anti-SLAM (recognizing
the N terminus of SLAM from human origin), rabbit polyclonal, anti-SAP
(recognizing aa 1–128 representing the full-length of SAP from human
origin), rabbit polyclonal, anti-pERK, anti-ERK, and anti-?-actin mAbs
were obtained from Santa Cruz Biotechnology. Anti-Fc?RI PE-conjugated
mAbs (AER-37) were obtained from eBioscience. Mouse IgG1, IgG2a, and
PE-conjugated mouse IgG2a Abs were obtained from DakoCytomation,
FITC-conjugated mouse IgG1 Abs was purchased from Ancell. FITC
anti-rabbit and FITC anti-goat Abs were obtained from Pierce. Goat
anti-mouse F(ab)? IgG, PE-conjugated goat anti-mouse F(ab)?, HRP-
conjugated goat anti-rabbit, HRP donkey anti-goat, and HRP-
conjugated goat anti-mouse Abs were obtained from Jackson Immu-
noResearch Laboratories). Sheep F(ab)? anti-mouse Ab was purchased
from ICN Pharmaceuticals (Aurora, OH).
Reagents and chemicals
RPMI 1640 supplemented with L-glutamine, heat-inactivated FCS, and
penicillin-streptomycin solutions were obtained from Biological Industries.
All chemicals used in this study were purchased from Sigma-Aldrich and
were of the best available grade.
Eosinophil and neutrophil purification
Eosinophils were purified from the peripheral blood of mildly atopic in-
dividuals (blood eosinophil levels, 5–10%) as previously described (25).
Written informed consent was obtained from all volunteers according to
the guidelines established by the Hadassah-Hebrew University Human Ex-
perimentation Helsinki Committee. Briefly, venous blood (50–100 ml) was
collected in heparinized syringes and left to sediment in 6% dextran (Am-
ersham Biosciences). Leukocytes were centrifuged on Ficoll-Hypaque
(density, 1.077; 25 min, 700 ? g, 22°C; Amersham Biosciences). Neutro-
phils and contaminating lymphocytes were tagged in the granulocyte-en-
riched pellet with micromagnetic beads bound to anti-CD16 and anti-CD3
Abs (Miltenyi Biotec). Eosinophils were purified by passing the cell sus-
pension through a magnetic column (MACS). They were collected at a
purity of at least 98% by Kimura staining (no CD56?cells were observed
in the contaminating fractions, by FACS analysis; data not shown) and at
a viability of at least 98% by trypan blue staining. For neutrophil isolation,
the magnetic column was washed three times with RPMI 1640 containing
0.05 M EDTA and heat-inactivated FCS (2%, v/v). Neutrophils were col-
lected at a purity of ?96% and a viability of ?98%.
Enrichment of human peripheral blood basophils
Peripheral blood was collected and centrifuged on Ficoll-Hypaque as de-
scribed above. Basophils were purified using the basophil isolation kit
(Miltenyi Biotec) according to the manufacturer’s instructions. Basophils
were collected at a purity of ?80% as determined by FACS analysis
For flow cytometry (FACS) analysis, cells (1 ? 105) were incubated in
15% human serum (to block FcRs) in a final volume of 100 ?l of HBSS
supplemented with 0.1% BSA and 0.02% sodium azide (HBA) for 30 min
on ice. Eosinophils and neutrophils were cultured with different Abs rec-
ognizing CD2 subfamily epitopes, followed by goat anti-mouse FITC Abs
(1/200) when needed. For detection of CD2 subfamily receptors on human
basophils, freshly isolated basophils were incubated for 30 min on ice with
anti-HLA-DR PerCP-Cy5.5-conjugated (1/10), anti-Fc?RI PE-conjugated
Abs (1/5), and an anti-CD2 subfamily receptor recognizing Ab, followed
by goat anti-mouse FITC Abs when needed.
For intracellular staining, eosinophils were fixed in 4% paraformalde-
hyde in HBA for 15 min at room temperature, then permeabilized in HBA
containing saponin (0.1%), BSA (1 mg/ml), and human serum (10%) for 30
min on ice. Anti-SAP or irrelevant control Abs (2 ?g/ml) were added to
these fixed permeabilized cells (30 min on ice), then incubated with FITC
anti-rabbit Abs (1/600, 30 min on ice).
After staining, the cells were analyzed on a FACSCalibur system (BD
Biosciences). For each staining, at least 10,000 events were collected, and
data analysis was performed using CellQuest software (BD Biosciences).
Cell culture and activation
Ninety-six well plates (Nunc) were precoated with sheep anti-mouse F(ab)?
in PBS (25 ?g/ml, 2 h, 37°C, 5% CO2). Afterward, plates were washed
three times with PBS and incubated with C1.7 (anti-2B4) or irrelevant
isotype-matched control Ab (1–5 ?g/ml, 2 h at 37°C, 5% CO2) and washed
again three times. Freshly isolated eosinophils were seeded in these pre-
coated wells (2 ? 105/200 ?l) in medium containing RPMI 1640, 200 U/ml
penicillin, 200 ?g/ml streptomycin, and 5% (v/v) heat-inactivated FCS
(enriched medium (EM)) and incubated for 30 min to 18 h (37°C, 5%
CO2). At the end of the incubation, cells were centrifuged (250 ? g, 5 min,
4°C), and supernatants were collected, aliquoted, and stored at ?80°C until
assessed for EPO, IFN-?, and IL-4.
EPO release was determined by a colorimetric assay as previously de-
scribed (26). Briefly, eosinophil culture supernatants (50 ?l) were incu-
bated (10–15 min, 37°C, 5% CO2) with a substrate solution consisting 0.1
mM O-phenylenediamine dihydrochloride in 0.05 M Tris buffer (pH 8.0)
containing 0.1% Triton X-100 (37°C, 5% CO2) and 1 mM hydrogen per-
oxide (Merck). The reaction was stopped by the addition of 100 ?l of 4
mM sulfuric acid (BDH), and absorbance was determined at 492 nm in a
spectrophotometer (PowerWave XS; Bio-Tek Instruments).
Cytokine determination assay
IFN-? and IL-4 in the eosinophil culture supernatants were quantified using
commercially available specific ELISA kits according to the manufactur-
er’s instructions. IFN-? was detected using DuoSet (R&D Systems); IL-4
was detected by Eli-pair (Diaclone). The lower limits for assay sensitivity
are 7 and 1.1 pg/ml for IFN-? and IL-4, respectively.
Redirected cytotoxicity assays
The cytotoxicity of human peripheral blood eosinophils was assessed using
the FcR?mouse mastocytoma P815 cell line (target cells) as previously
described (27) with slight modifications. Briefly, target cells were labeled
with [35S]methionine (Amersham Biosciences) overnight and thereafter
seeded on 96-well, U-shaped plates (Nunc; 5 ? 103/100 ?l; in EM),
washed three times (250 ? g, 5 min, 4°C), and incubated in the presence
or the absence of C1.7 Ab or matching isotype control (1 ?g/ml) for 1 h on
ice. Freshly isolated eosinophils or YTS NK cells as positive controls (ef-
fector cells) were added (100 ?l/well, in EM) at different E:T cell ratios
(50–6.25:1 and 10:1, respectively). After 18-h incubation (37°C, 5% CO2),
the cells were centrifuged (250 ? g, 5 min), and the supernatants were
collected for the cytolytic activity assay. The percentage of specific lysis
was calculated by the following equation: (a ? b)/(c ? b), where a indi-
cates the radioactivity of target cells mixed with effector cells, b is the
radioactivity in the supernatant of the target cells alone, and c is the ra-
dioactivity in the supernatant of target cells alone after lysis with 0.1
Direct cytotoxicity assays
The cytotoxicity of human peripheral blood eosinophils was assessed using
the MHC-I deficient, EBV-transformed B cell line 721.221 (target cells)
that expresses high levels of CD48. To study 2B4-CD48 interactions, target
cells were labeled with [35S]methionine as described above and incubated
in the presence or the absence of anti-CD48 mAb or matching isotype
control (5 ?g/ml) for 1 h on ice. Simultaneously, freshly isolated eosino-
phils or YTS NK cells (effector cells) were incubated in the presence or the
absence of C1.7 or matching isotype control (5 ?g/ml) for 1 h on ice. Both
target and effector cells were washed, resuspended, and cocultured (200
?l/well, in EM) at different E:T cell ratios (50–6.25:1 and 10:1, respec-
tively). After 18-h incubation (37°C, 5% CO2), the cells were centrifuged
(250 ? g, 5 min), and cytolytic activity was assessed as described above.
111The Journal of Immunology
Cell lysates were prepared by adding lysis buffer (1 M Tris, 0.5 M EDTA,
150 mM NaCl, 0.02% sodium azide, 0.1% SDS, 1% Triton X-100, and
0.5% sodium deoxycholate) to freshly isolated eosinophils, neutrophils, or
YTS NK cells (5 ? 106), followed by vortex mixing. Cell debris was
removed from the lysates by centrifugation (18,000 ? g, 15 min, 4°C). The
supernatant was precleared using isotype-matched control Ab and then
incubated with anti-2B4 (i.e., C1.7, P158, and PP35; 8 h, 4°C), followed by
protein A/G (20 ?l; 8 h, 4°C; Santa Cruz Biotechnology). The immuno-
precipitates were washed four times with lysis buffer, eluted from the
Sepharose beads by boiling for 15 min in sample buffer (0.1% bromophe-
nol blue, 1.5% 2-ME, 10% SDS, and 0.5 M Tris, pH 6.8), and analyzed by
Cell lysates were prepared from freshly isolated eosinophils, neutrophils,
or YTS NK cells (5 ? 106) in lysis buffer as described above. Samples
were analyzed on 12.5% SDS-PAGE under reducing conditions. The gels
were electrotransferred to polyvinylidene difluoride membranes (Bio-Rad).
The membranes were incubated for 1 h at room temperature with rabbit
anti-SAP Abs (1/500). After the membranes were washed, proteins were
detected by secondary immunopure goat anti-rabbit Abs conjugated with
HRP (1/5000), followed by ECL detection (Amersham Biosciences).
In assays determining phosphorylated forms of ERK, freshly isolated
eosinophils (5 ? 106/400 ?l, in EM) were incubated with C1.7 or isotype-
matched control Abs (1 ?g/ml) for 30 min on ice. Cells were washed twice
(250 ? g, 4°C, in EM) and cross-linked with sheep anti-mouse F(ab)? (25
?g/ml) for 30 min on ice. Then cells were again washed twice (250 ? g,
4°C, in EM) and incubated for the indicated times (37°C, 5% CO2). For
positive control, eosinophils were incubated (5 ? 106/well, in EM) in 12-
well plate (Nunc) with PMA (50 ng/ml) for 4 min (37°C, 5% CO2). After
stimulation, cells were lysed and blotted as described above.
RNA isolation and RT-PCR amplification
Total mRNA was extracted from freshly isolated eosinophils, neutrophils
(10 ?106), and YTS NK cells (8 ? 106) using the RNeasy Mini kit (Qia-
gen). The first-strand cDNA synthesis reaction was catalyzed by Super-
Script II RNA reverse transcriptase (Invitrogen Life Technologies) and
oligo(dt)12–18primer, according to the manufacturer’s instructions. PCR
amplifications were conducted using the following primer sequences 5?-
GCCATGGACGCAGTGGCT-3? and 5?-TGGGGCTTTCAGGCAGACA
TC-3? for SAP (28), generating a 400-bp fragment. The MasterCycler (Ep-
pendorf) was used for the PCR amplifications with the following settings:
35 cycles at 94°C for 30 s, 57°C for 1 min, and 72°C for 1 min. Before the
first cycle, denaturation at 94°C for 3 min was performed. At the end of all
cycles, a primer extension period of 10 min at 72°C was included. The
primer sequences for ?-actin, which was used as a control to test the ef-
ficiency of cDNA synthesis, were 5?-GTACAGGGATAGCACAGCCT-3?
and 5?-TCACCAACTGGGACGACATG-3? generating a fragment of
200bp. Amplified products were electrophoresed on 1.5% agarose gel
stained with ethidium bromide and were photographed under UV light.
Statistical significance was calculated using parametric analysis (ANOVA,
followed by Tukey-Kramer post hoc test). Values were considered signif-
icant at p ? 0.05.
Expression of CD2 subfamily receptors on human peripheral
blood eosinophils, neutrophils, and basophils
We used a panel of Abs to examine the cell surface expression of
CD2 subfamily receptors on freshly isolated human peripheral
blood eosinophils, neutrophils, and basophils. Representative flow
cytometer dot-plots are shown in Fig. 1A. Eosinophils were found
to express several CD2 subfamily receptors, including NTB-A,
2B4, CD84, CD58, and CD48 (n ? 15). Interestingly, the expres-
sion of NTB-A was confined only to eosinophils, because ba-
sophils (n ? 4) and neutrophils (n ? 15) did not express this
receptor. In addition, eosinophils as well as basophils, but not neu-
trophils, expressed 2B4 (n ? 4 and 15, respectively). CD84, CD58,
and CD48 were also expressed by basophils and neutrophils (n ?
4 and 15, respectively). Because 2B4 and NTB-A display a dif-
ferential expression pattern among the three cell types, we next
screened these cells with different mAbs recognizing 2B4 and
NTB-A to discern whether this pattern is Ab dependent. All three
mAbs recognizing 2B4, i.e., P158, PP35, and C1.7 (13, 15, 23),
could similarly identify 2B4 on the surface of the eosinophils (Fig.
1B) and basophils, but not on neutrophils (data not shown). In
addition, the two mAbs recognizing NTB-A, i.e., ON56 and
MA127 (24), displayed the same expression outline on eosinophils
(Fig. 1B), neutrophils, and basophils (data not shown).
2B4 activates eosinophils to release EPO, IFN-?, and IL-4
The expression of CD84, NTB-A, and 2B4 on the surface of eo-
sinophils suggests that their responses may be regulated by these
receptors. Based on the knowledge that 2B4 triggers NK cell ac-
tivation (13, 17), we hypothesized that 2B4 may activate human
eosinophils as well. As a first marker of eosinophils activation,
EPO release was evaluated. From preliminary studies we had al-
ready established that cross-linking of the P158 mAb does not
elicit EPO release. In these experiments the best activation was
achieved by cross-linking of the receptor with the C1.7 mAb.
Thus, our next set of experiments was conducted using the C1.7
anti-2B4 mAb. As shown in Fig. 2A, cross-linking of 2B4 induced
eosinophils to release EPO. EPO release, as measured by OD units,
was significantly higher when 2B4 was cross-linked using the C1.7
mAb (OD, 0.59 ? 0.15) in comparison with isotype-matched
cross-linking (OD, 0.15 ? 0.02; p ? 0.0001) or unstimulated cells
(OD, 0.17 ? 0.03; p ? 0.0001).
Next we determined whether cross-linking of 2B4 on eosino-
phils could cause cytokine release. We selected IFN-? and IL-4 as
typical Th1- and Th2-related cytokines, respectively. As shown in
Fig. 2B, cross-linking of 2B4 on eosinophils caused IFN-? release,
ranging between 53.7 and 250.9 pg/ml, in comparison with iso-
type-matched control cross-linking, which ranged between 0 and
45.2 pg/ml (p ? 0.05), and with unstimulated cells, which ranged
between 0 and 8.5 pg/ml (p ? 0.01). Cross-linking of 2B4 on
eosinophils also caused significant IL-4 release (Fig. 2C; 16.5–
97.3 pg/ml) in comparison with cross-linking of isotype-matched
controls (10.48–20.7 pg/ml; p ? 0.05) and unstimulated cells (0–
19.5 pg/ml; p ? 0.01).
2B4 activation mediates eosinophil cytotoxicity toward P815
and 721.221 cells
The demonstration that 2B4 activates human eosinophils to release
cytokines led us to hypothesize that other functions beside cyto-
kine release could be elicited by 2B4 stimulation. Therefore, eo-
sinophil (effector cell (E)) cytotoxicity was tested in a redirected
lysis assay toward the P815 mastocytoma cell line (target cell (T)).
The C1.7 mAb caused a cytotoxic effect of the eosinophils toward
the P815 cells already at an E:T cell ratio of 12.5:1 (Fig. 3A). This
effect became significant at an E:T cell ratio of 25:1 (18.85 ?
1.31% C1.7 mAb vs 2.75 ? 1.38% isotype-matched control; p ?
0.01; n ? 4). Higher E:T cell ratios (50:1 and 100:1) were either
the same or less effective than an E:T cell ratio of 25:1 (data not
Our next aim was to determine whether eosinophils could dis-
play these cytotoxic features toward malignant cells that express
CD48, a high affinity ligand of 2B4 (15, 16). For this set of ex-
periments we cocultured eosinophils with the EBV-transfected B
cell line 721.221 that expresses high levels of CD48, but no MHC
class I. As shown in Fig. 3B, at the highest E:T cell ratio (40:1)
neither neutralization of 2B4 on the surface of eosinophils nor of
CD48 on the surface of target cells blocked the cytolytic effect of
eosinophils (39.87 ? 2.38%). However, at lower E:T cell ratios
1122B4 ACTIVATES HUMAN EOSINOPHILS
(i.e., 20:1 and 10:1) where eosinophils still caused significant cy-
tolysis of 721.221 cells (34.47 ? 2.88 and 31.96 ? 2.71%, re-
spectively), neutralization of CD48 on the surface of the target
cells or of 2B4 on the surface of the eosinophils significantly de-
creased this cytolytic effect. For example, at an E:T cell ratio of
20:1, blocking 2B4 on eosinophils or CD48 on the target cells
decreased the cytolytic effect to 12.56 ? 1.23 and 18.37 ? 1.93%,
respectively (p ? 0.05; n ? 3). In addition, at an E:T cell ratio of
10:1, blocking 2B4 on eosinophils or CD48 on the target cells
decreased cytolytic effect to 11.21 ? 1.45 and 7.88 ? 2.34%,
eosinophils, neutrophils, or basophils were cultured with specific Abs recognizing CD2 subfamily epitopes on the cell surface or appropriate isotype-
matched controls, followed by goat anti-mouse FITC when needed. For basophil determination, basophils were stained with anti-HLA-DR, PerCP-Cy5.5
conjugated, and anti-Fc?RI, PE-conjugated. HLA-DR?/Fc?RI?cells were electronically gated and assessed for CD2 subfamily epitopes The data shown
are representative results from one of 15 (eosinophils and neutrophils) or four (basophils) different experiments. B, Eosinophils were analyzed by FACS
using different mAbs recognizing 2B4 (PP35, P158, and C1.7) and NTB-A (ON56 and MA127), followed by FITC anti-mouse Abs. Bold and dotted
histogram plots indicate isotype-matched control and anti-2B4 or anti-NTB-A mAbs, respectively. Data are representative of 15 different experiments. The
percentage indicates positively stained cells of the total population, as analyzed by CellQuest.
Expression of CD2 subfamily receptors on human peripheral blood eosinophils, neutrophils, and basophils. A, Freshly isolated and purified
113 The Journal of Immunology
respectively (p ? 0.01; n ? 3). The cytolytic effect was blocked
completely by these procedures at an E:T cell ratio of 5:1 (1.61 ?
1.1 and 1.88 ? 1.6%; p ? 0.001; n ? 3).
Cross-linking of 2B4 results in ERK phosphorylation
Eosinophil activation involves various pathways (29–31). To in-
vestigate whether cross-linking of 2B4 stimulates the MEK1/ERK
pathway, freshly isolated eosinophils were stimulated by cross-
linking the C1.7 mAb for different time periods (Fig. 4). Cells were
then lysed and analyzed by Western blot for ERK phosphorylation
(Fig. 4, upper panel). Cross-linking of 2B4 causes the phosphor-
ylation of ERK as early as 2 min after Ab ligation. This process is
more evident after 4 min and decreases after 6 min. Cross-linking
of an isotype-matched Ab for 4 min did not cause any ERK phos-
phorylation (IgG1), whereas eosinophils stimulated with PMA dis-
played an intense ERK phosphorylation pattern (Fig. 4). To verify
total amounts of ERK protein, the membrane was stripped and
reprobed with anti-ERK Abs (lower panel). Eosinophils were
found to express similar protein levels.
Eosinophils express SAP
An additional signaling molecule essential for 2B4 activation is
SAP (17–20). Thus, the expression of SAP in freshly isolated eo-
sinophils was examined. As shown in Fig. 5A, freshly isolated
eosinophils as well as YTS NK cells express SAP at the mRNA
level, whereas neutrophils do not (n ? 3). SAP expression was
also analyzed at the protein level using both FACS analysis and
Western blot techniques. Intracellular FACS staining revealed that
eosinophils express similar levels of SAP as YTS NK cells,
whereas their expression in neutrophils is negligible (Fig. 5B; n ?
3). In addition, Western blot analysis (Fig. 5C) demonstrated that
SAP is expressed in YTS NK cells as well as in eosinophils, but
not in neutrophils (n ? 8). Interestingly, eosinophils from one of
eight donors did not express SAP (data not shown).
Pervenadate treatment causes 2B4 tyrosine phosphorylation in
Recruitment of SAP to 2B4 is dependent on phosphorylation of
intracellular tyrosine residues. To determine whether 2B4 in eo-
sinophils undergoes tyrosine phosphorylation, eosinophils were
2B4 mAbs. Tyrosine phosphorylation was assessed by Western
blot. As shown in Fig. 6A, only the PP35 mAb was able to pre-
cipitate 2B4 from the eosinophils, whereas the P158 and the C1.7
EPO, IFN-?, and IL-4. Eosinophils were cultured in un-
treated (EM) wells or in wells precoated with sheep anti-
mouse and either isotype-matched Ig (IgG1) or C1.7
mAb (anti-2B4) for 30 min to 18 h. A, EPO in the cul-
ture supernatants was determined by a colorimetric as-
say (A). The data represent the mean ? SEM of five
different experiments preformed in triplicate. p ? 0.01
compared with EM- and IgG1-treated cells. IFN-? (B)
and IL-4 (C) were determined in the culture superna-
tants by ELISAs. The plots represent data obtained from
four donors. Experiments were performed in triplicate.
2B4 activates eosinophils to release
114 2B4 ACTIVATES HUMAN EOSINOPHILS
mAbs were not successful. Furthermore, treatment of the eosino-
phils and YTS NK cells with pervenadate (Fig. 6B) elicited ty-
rosine phosphorylation of 2B4.
In the present study we have screened the surface of human pe-
ripheral blood eosinophils for the expression of CD2 subfamily
receptors and compared it to those of neutrophils and basophils.
Our data demonstrate that eosinophils express NTB-A, 2B4,
CD84, CD58, and CD48. Interestingly, eosinophils were found to
exclusively express NTB-A compared with neutrophils and ba-
sophils. In addition, eosinophils and basophils, but not neutrophils,
expressed 2B4, indicating that the eosinophils’ cell surface mark-
ers resemble those of the basophils rather than the neutrophils (8).
This finding is particularly interesting because this specific expres-
sion pattern of NTB-A (24) could be used to distinguish among
eosinophils, neutrophils, and basophils. CD84, CD58, and CD48
were all expressed in eosinophils neutrophils, and basophils,
whereas CD2 and SLAM were not expressed on any of these cell
Because only NTB-A and 2B4 displayed a differential expres-
sion pattern among the three cell types, we tested various mAbs
recognizing these epitopes to discern whether our findings were
Ab dependent. All Abs examined demonstrated similar expression
patterns of NTB-A and 2B4.
The expression of CD2 subfamily receptors, either specifically
or nonspecifically on human eosinophils may contribute to our
understanding of the role of eosinophils in different pathophysio-
logical settings. The CD2 subfamily receptors interact with each
other in a homotypic or a heterotypic fashion. For example, in
humans CD58 is a high affinity ligand for CD2, whereas CD48 is
a low affinity ligand for CD2 (32), but a high affinity ligand for
2B4 (15, 33). CD84 and SLAM interact with themselves (34, 35).
Hence, our results suggest that eosinophils may interact either with
themselves or with other cell types, such as T cells, via these CD2
subfamily receptors. Indeed, it has been shown that a cross-talk
between eosinophils and T cells may occur in asthma (36–39).
Eosinophils express costimulatory molecules, such as CD28 and
CD86 (12, 37), and are able to present Ag (36, 37). Furthermore,
it has been well established that murine asthma is dependent on
eosinophil-T cell interactions (38, 39). Thus, it is likely that this
cross-talk could be mediated by these CD2 subfamily receptors as
well as by additional receptors.
The unique expression pattern of 2B4, the many studies that
demonstrated its function, and the fact that 2B4 has a well-char-
acterized high affinity ligand (15, 33) led us to investigate its role
in eosinophils. The expression of 2B4 on eosinophils suggests that
their responses after recruitment to the site of inflammation may be
regulated by this receptor. Indeed, we found that 2B4 expression
on eosinophils does not change after their culture with various
cytokines (e.g., IL-5, IL-3, GM-CSF, IL-2, IFN-?, IL-4, IL-13, and
TGF-?) and that nasal polyp tissue eosinophils also expressed this
receptor (A. Munitz, I. Bachelet, R. Elyashar, and F. Levi-Schaffer,
It is well known that 2B4 activates NK cells (13, 17, 40, 41).
Thus, we tested whether eosinophils could also be activated via
2B4. Cross-linking of 2B4 caused eosinophils to release EPO.
EPO is a member of the family of haloperoxidases that catalyze the
peroxidative oxidation of halides and pseudohalides (6). Once
EPO is released, it can elicit several effects, some of which are
protective and others destructive (6).
Cross-linking of 2B4 on NK cells has been shown to trigger
cytokine release, primarily IFN-?, and cross-linking of 2B4 trig-
gered IFN-? release from eosinophils as well. Recent studies have
P815 and 721.221 cells. Eosinophil cytotoxicity was tested against
[35S]methionine labeled P815 or 721.221 cells in a redirected lysis or direct
lysis assay, respectively. A, In the redirected lysis assay, the target cells
P815 (target) were incubated in the presence of medium alone (EM), IgG1
isotype control (Isotype), or anti-2B4 Abs (anti-2B4), and eosinophils (ef-
fectors) were added at different E:T cell ratios. The data are representative
results from one of four different experiments performed in duplicate. p ?
0.001 compared with EM- and IgG1-treated cells. B, In the direct lysis
assay, 721.221 cells (target) were incubated in the presence or the absence
of EM, IgG1 isotype control, anti-CD48, or anti-2B4 Abs, and eosinophils
(effectors) were added at different E:T cell ratios. The data are represen-
tative results from one of three different experiments preformed in dupli-
cate. p ? 0.05 and p ? 0.01 compared with IgG1-treated cells.
2B4 activation mediates eosinophil cytotoxicity toward
sinophils were incubated with anti-2B4 or IgG1 isotype-matched control
Abs, followed by sheep anti-mouse F(ab)? for the indicated time points.
Cell lysates were analyzed by Western blot with anti-phospho-ERK Abs
(upper panel). The same membrane was stripped and reprobed with anti-
ERK Abs (lower panel) to check total protein amounts. The data shown are
representative results from one of three different experiments.
Cross-linking of 2B4 results in ERK phosphorylation. Eo-
115 The Journal of Immunology
shown that eosinophils may have a differential cytokine release
profile that is stimuli dependent. For example, it has been shown
that eosinophils release IL-12, but not IL-4, in response to leuko-
cyte Ig-like receptor-7 activation (9). In addition, CD28 activates
eosinophils to release IL-2 and IFN-?, but not IL-10 (12). There-
fore, we investigated whether 2B4 activation may cause eosino-
phils to also release a Th2-related cytokine, such as IL-4. Our
results show that 2B4 can activate eosinophils to release IL-4 as
well as IFN-?.
The demonstration that 2B4 activation on eosinophils can elicit
both EPO and IL-4 release as well as IFN-? raises the possibility
that the function of 2B4 on eosinophils has a broad immunological
importance and can contribute to eosinophil effector functions in
both Th1- and Th2-like responses. In addition, 2B4 functions on
NK cells as a coreceptor to NKp46, NKp44, and NKp30 (42).
Even though the expression of these activating receptors is con-
fined to NK cells, we cannot rule out the possibility that 2B4 func-
tions as a coreceptor on eosinophils as well, orchestrating a re-
sponse with an as yet undefined activatory receptor(s).
Another aspect of 2B4 activation on NK cells is enhancement of
their cytotoxic effects toward several malignant cells (15, 17, 43).
Eosinophils have been documented to be elevated in peripheral
blood and/or to infiltrate the tissue in some malignant disorders (5,
44, 45). In addition, eosinophils have been shown to display direct
and indirect antitumor effects both in vitro and in vivo (45–49).
Therefore, we were interested to determine whether 2B4 can trig-
ger eosinophil cytotoxicity toward malignant cells, as it does in
We could show that activation of eosinophils via 2B4 causes
them to display cytotoxic effects toward the FcR?P815 mouse
mastocytoma cell line as well as toward the CD48?721.221 B cell
lymphoma. It is important to note that the vast majority of receptor
studies involving NK stimulation were performed with NK cells
activated by IL-2 (50). However, in this work, freshly isolated
eosinophils, without any previous in vitro priming were tested and
found to be active. Interestingly, eosinophils were not able to lyse
melanoma cells transfected with CD48 (1106mel) and soluble
CD48 did not activate eosinophil mediator release (data not
shown). Importantly, a recent study by Lee et al. (51) has demon-
strated that 2B4 acts as an inhibitory receptor, rather than an ac-
tivating one, on CD48 ligation. Although the study was conducted
on murine 2B4, it is feasible that human 2B4 can exert versatile
signaling cascades as well (5, 51). Thus, we cannot exclude a
possibility that additional cell-cell interactions regulate 2B4-CD48
interactions. Taken together, our results indicate that eosinophils may
participate in the immune response against tumors in direct cell-cell
and receptor-ligand interactions also via 2B4-CD48 interactions.
Examination of the signal transduction pathway of 2B4-stimu-
lated NK cells reveals that activation of 2B4 entails complex in-
teractions involving LAT, Ras, Raf, ERK, and p38 (52–54). Cross-
linking of 2B4 on eosinophils resulted in ERK phosphorylation as
early as 2 min after the ligation and peaked at 4 min. Nevertheless,
the p38 MAPK pathway was not activated (data not shown). Thus,
the signaling cascade elicited by 2B4 on eosinophils slightly differs
from that in NK cells and may recruit diverse pathways.
CD84, 2B4, NTB-A, Ly-9, and SLAM all display cytoplasmic
tyrosine-based motifs similar to ITIMs (13, 14). In 2B4, phosphor-
ylation of these residues and recruitment of SAP, an Src homology
2 domain-containing molecule, results in NK cell activation. SAP
is an adaptor molecule that recruits the tyrosine kinase Fyn and
probably other Src kinases to SLAM and most likely to related
receptors. The expression of SAP is limited to several cell types
and has been shown to be expressed mainly in T and NK cells (55).
Thus, to determine whether SAP may be functional on human
eosinophils, the expression of this unique molecule was examined.
Freshly isolated eosinophils, but not neutrophils, expressed SAP
at both mRNA and protein levels. These results are in contradic-
tion with the findings of Nakajima et al. (20), who showed that
eosinophils do not express SAP. This could be explained by the
different sources of eosinophils, in their case atopic dermatitis pa-
tients and in ours mildly atopic subjects (M. Colonna, unpublished
observations) or by the different Abs used.
PCR analysis, total RNA was extracted from freshly iso-
lated eosinophils (Eos), neutrophils (Neut), or YTS NK
cells (YTS). First-strand cDNA synthesis was catalyzed,
and PCR amplification was conducted. Amplified prod-
ucts were electrophoresed on agarose gel, stained with
ethidium bromide, and photographed under UV light. B,
For FACS studies, fixed, permeabilized cells were in-
cubated with anti-SAP or control Abs, followed by
FITC-labeled goat anti-rabbit. Data are representative
results form one of three different experiments. C, For
Western blot analysis, isolated eosinophils were lysed
and analyzed on SDS-PAGE gel under reducing condi-
tions. After the transfer procedure, the membrane was
incubated with rabbit anti-SAP Abs, washed, and de-
tected by secondary immunopure goat anti-rabbit Abs
conjugated with HRP, followed by ECL detection. The
numbers on the right of the panels indicate the positions
of the m.w. markers.
Eosinophils express SAP. A, For RT-
1162B4 ACTIVATES HUMAN EOSINOPHILS
Interestingly, immunoprecipitation of 2B4 from the surface of
the eosinophils was only successful when using the PP35 Ab, but
not the P158 or the C1.7 Abs. Indeed, these different Abs have
been shown to activate different functions on NK and T cells (13).
For example, the C1.7 Ab-dependent IFN-? release from NK cells
required IL-2, IL-12, or target cells (K562) and had a low effi-
ciency in precipitating 2B4 (13, 19). In addition, the P158 Ab did
not induce any cytokine production from NK cells and had no
effect on T cell-mediated killing (13, 15). Therefore, our results
could also be explained by the differences among these Abs.
Treatment of eosinophils with pervenadate resulted in tyrosine
phosphorylation of 2B4 even though our studies could not dem-
onstrate a direct interaction between 2B4 and SAP in the eosino-
phils. Thus, the role of SAP signaling in eosinophils remains to be
clarified. Intriguingly, eosinophils express several cell surface re-
ceptors that potentially recruit SAP for their function, including
Siglec 10 and Siglec 8 (10, 11) as well as CD84, 2B4, and NTB-A.
Interestingly, among these receptors Siglec-8 is uniquely ex-
pressed on eosinophils, mast cells, and basophils, and NTB-A is
restricted to eosinophils among the myeloid lineage. Therefore,
SAP may have a greater role in regulating eosinophil functions.
In conclusion, the demonstrations that CD2 subfamily receptors
are expressed and that 2B4 is functional on eosinophils are of
particular interest. Defining the conditions in which eosinophils
can interact with CD48 through 2B4 will make it possible to shed
more light on the factors that regulate eosinophil functions in
health and disease.
We thank Dr. I. Golan for his technical assistance, Madelyn Segev for her
editorial assistance. and all members of F.L.S.’s laboratory.
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118 2B4 ACTIVATES HUMAN EOSINOPHILS