Depletion of eosinophils in mice through the use - Journal of ...

Depletion of eosinophils in mice through the use of antibodies
specific for C-C chemokine receptor 3 (CCR3)
J. Christopher Grimaldi, Nai-Xuan Yu, Gabrielle Grunig, Brian W. P. Seymour, Françoise Cottrez,
Douglas S. Robinson, Nancy Hosken, Walter G. Ferlin, Xiaoyan Wu, Hortensia Soto, Anne O’Garra,
Maureen C. Howard, and Robert L. Coffman
DNAX Research Institute of Molecular & Cellular Biology, Palo Alto, California
Abstract: We have generated rat monoclonal antibodies
specific for the mouse eotaxin receptor, C-C
chemokine receptor 3 (CCR3). Several anti-CCR3
mAbs proved to be useful for in vivo depletion of
CCR3-expressing cells and immunofluorescent
staining. In vivo CCR3 mAbs of the IgG2b isotype
substantially depleted blood eosinophil levels in
Nippostrongyus brasiliensis-infected mice. Repeated
anti-CCR3 mAb treatment in these mice
significantly reduced tissue eosinophilia in the lung
tissue and bronchoalveolar lavage fluid. Flow cytometry
revealed that mCCR3 was expressed on eosinophils
but not on stem cells, dendritic cells, or cells
from the thymus, lymph node, or spleen of normal
mice. Unlike human Th2 cells, mouse Th2 cells did
not express detectable levels of CCR3 nor did they
give a measurable response to eotaxin. None of the
mAbs were antagonists or agonists of CCR3 calcium
mobilization. To our knowledge, the antibodies
described here are the first mAbs reported to be
specific for mouse eosinophils and to be readily
applicable for the detection, isolation, and in vivo
depletion of eosinophils. J. Leukoc. Biol. 65: 846–
853; 1999.
Key Words: rodent · FACS · Th1 · Th2
INTRODUCTION
Chemokines are pro-inflammatory polypeptides of 6–14 kDa
that mediate trafficking on various populations of leukocytes [1,
2]. There are currently four documented classes of chemokines:
C-X-C, C-C, C-X 3-C, and -C-. All chemokines of the C-X-C and
C-C classes have a four-cysteine motif and share a high degree
of sequence and functional homology. The C-X-C chemokines
are characterized by an amino acid insertion between the first
two cysteines, whereas the C-C chemokines have the first two
cysteines adjacent to each other. The -C- class of chemokines,
of which lymphotactin is the only member [3], lacks the first two
cysteines but is otherwise highly homologous with other C-C
chemokines. Fractalkine and its murine homolog neurotactin
belong to a new class of membrane-bound C-X 3-C chemokines
and differ from other chemokines by having three amino acids
between the first two cysteine residues, a long mucin-like stalk,
and a short transmembrane region [4, 5]. Chemokine receptors
can, likewise, be classified in four groups: C-C chemokine
receptors (CCR), C-X-C chemokine receptors (CXCR), virusencoded
chemokine receptors (cytomegalovirus-encoded US28
and herpes simplex virus-encoded ECRF3), and the promiscuous
chemokine receptor, Duffy.
The C-C chemokine receptor CCR3 has been shown to be the
principal receptor for the C-C chemokine eotaxin, a potent
eosinophil chemoattractant. CCR3 is expressed primarily by
eosinophils in both humans [6] and guinea pigs [7]. However,
Sallusto et al. have reported significant expression and function
of CCR3 on human Th2 cells [8]. Eosinophilic infiltrates are
common in the lungs of asthma patients and have been
implicated as mediators of both chronic and acute phases of the
disease [9, 10]. Depletion of eotaxin by gene disruption
partially blocks eosinophil infiltration to sites of antigen
challenge [11], and it has been suggested that eotaxin signaling
through CCR3 is an important mediator of eosinophil recruitment
to the asthmatic lung [12, 13]. A second eosinophilspecific
chemokine, eotaxin-2, has recently been shown to bind
to CCR3 [14]. To study the distribution and function of CCR3 in
mouse models of eosinophil infiltration and asthma, we have
produced a panel of rat mAbs specific for mouse CCR3. These
antibodies give strong immunofluorescent staining specific for
mouse eosinophils, but not mouse Th2 cells.
MATERIALS AND METHODS
Generation of the mouse CCR3 gene
Polymerase chain reaction (PCR) primers homologous to the 5� and 3� termini
of the published murine CCR3 cDNA (genebank accession no. U29677) [15]
were used to clone the full-length cDNA unidirectionally into the eukaryotic
expression vector pMe18sneo at the EcoR1 and Xba1 sites. The primer
sequences were as follows: 5�CCR3 Eco, TCC-GGA-ATT-CAT-GGC-ATT-CAA-
CAC-AGA-TGA-AAT-CAA-G; and 3�CCR3 Xba, CTA-GTC-TAG-ACT-AAA-
ACA-CCA-CAG-AGA-TTT-CTT-GCT-CC. PCR was performed using a mouse
Abbreviations: PCR, polymerase chain reaction; DMEM, Dulbecco’s modified
Eagle’s medium; FCS, fetal calf serum; RT, reverse transcriptase; HBSS,
Hanks’ balanced salt solution; PMA, phorbol myristate acetate; PBS, phosphatebuffered
saline; ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein
isothiocyanate; IFN-�, interferon-�; IL-4, interleukin-4; BAL, bronchoalveolar
lavage.
Correspondence: J. Christopher Grimaldi, Genentech Inc., 1 DNA Way,
South San Francisco, CA 94080-4990. E-mail: grim@gene.com
Received November 9, 1998; revised February 8, 1999; accepted February
9, 1999.
846 Journal of Leukocyte Biology Volume 65, June 1999 http://www.jleukbio.org

Th2 cDNA library as template to amplify the 1079-bp CCR3 coding region. The
PCR fragment was then cloned into the pMe18sNeo vector. The final construct
pMe18sNeo/mCCR3 was sequenced on both strands to confirm that no
PCR-generated mutations had occurred.
Transfected cell lines
Stable murine CCR3 transfectants were generated in the mouse fibroblast cell
line NIH3T3 and the rat cell myeloma line Y3 using the pMe18sNeo/mCCR3.
NIH3T3 cells were transfected using lipofectamine (GIBCO-BRL, Gaithersberg,
MD) according to the manufacturer’s protocol. Briefly, a 1:20 dilution of
lipofectamine in optiMEM � I medium (GIBCO) was mixed with 15 µg of
pMe18sNeo/mCCR3 for 30 min at room temperature. The solution was then
added to 3 � 10 6 NIH3T3 cells at 37°C for 5 h. After 5 h the medium was
exchanged with Dulbecco’s modified Eagle’s medium (DMEM) containing 10%
fetal calf serum (FCS). G418 (GIBCO-BRL) at a final concentration of 1 mg/mL
was added at 48 h to select for positive clones. The Y3 cell line was transfected
by electroporation using the Gene Pulser (Bio-Rad, Hercules, CA). Briefly 1 �
10 7 cells with 25 µg of pMe18sNeo/mCCR3 were electroporated at 300 mV and
960 µF. The transfected cells were then allowed to expand for 48 h in DMEM
with 10% FCS before selecting in medium containing 1 mg/mL G418. Twenty
single G418-resistant clones were selected and analyzed for the presence of
CCR3 mRNA by reverse transcriptase-PCR.
RT-PCR of stable G418-resistant transfectants
RT-PCR analyses were performed on the clonal G418-resistant transfectants to
determine the presence of CCR3 message. Total RNA was purified from 1 �
10 7 cells using RNAzol B � (TEL-TEST, Inc., Friendswood, TX), per the
manufacturer’s protocol. The isolated total RNA was treated with DNase 1
(Gene Hunter Inc., Nashville, TN) to eliminate genomic DNA contamination.
Oligo(dT)-primed reverse transcription was performed using 5 µg of DNase
1-treated total RNA in 20-µL reactions. PCR was carried out using 5 µL of the
RT product and the above-mentioned CCR3 primers for 25 cycles in a standard
50-µL reaction. The PCR conditions were 5 min 94°C, then 25 cycles at 94°C
30 s, 55°C 45 s, and 72°C 90 s. The RT-PCR products were then analyzed by
agarose gel electrophoresis.
Calcium flux and chemotaxis assays
Calcium flux assays were performed on a Photon Technologies International
fluorometer using the FeliX � fluorescence analysis software version 1.11. Cells
(1 � 10 7 ) were added to 1 mL of media containing 3 µg/mL Indo-1/AM
(Molecular Probes, Eugene, OR) and allowed to incubate with gentle agitation
at room temperature for 45 min. The labeled cells were then centrifuged and
resuspended in 1 mL of Hanks’ balanced salt solution (HBSS) with 1% FCS and
kept at room temperature. For measurements of relative fluorescence, 1 � 10 6
labeled cells were added to 1.9 mL of 37°C HBSS containing 1.6 mM CaCl2 and
10 mM HEPES. Mouse and human eotaxin, hMCP-2, hMCP-3, hMCP-4,
mRANTES, and hEotaxin-2 (Peprotech, Rocky Hill, NJ) were added at final
concentrations ranging from 1 nM to 1 µM. The most eotaxin-responsive
CCR3-transfected Y3 and NIH3T3 cell lines were chosen for further studies
and named Y3/mCCR3 and NIH3T3/mCCR3, respectively. Chemotaxis of
phorbol myristate acetate (PMA)/ionomycin activated 3� polarized Th2
populations was performed using a range of eotaxin concentrations as
previously described [16].
Anti-CCR3 mAb production
Two-month-old Lewis rats (Harlan, Indianapolis, IN) were injected intraperitoneally
every 2 weeks for 10 weeks with 2 � 10 8 Y3/mCCR3 cells suspended in
phosphate-buffered saline (PBS) and irradiated with 10,000 rads. On the 12th
week the same immunization was given intravenously and 4 days later the
spleen was removed for fusion. The splenocytes were fused to SP2/0 cells and
hybridomas selected using the ClonaCell-HY kit (Stem Cell Systems Inc.,
Vancouver, Canada). Hybridomas were expanded in 96-well plates and the
supernatants tested by flow cytometry for their ability to stain NIH3T3/mCCR3
cells but not the NIH3T3 parent line. All positive hybridomas were recloned
and rescreened to ensure that each hybridoma was clonal. Anti-CCR3 mAbs
were isotyped using an Ouchterlony assay (ICN, Aurora, OH) as well as by
enzyme-linked immunosorbent assay (ELISA) using the rat monoAB ID/SP kit
(Zymed, South San Francisco, CA) as per the manufacturer’s protocol.
Epitope mapping of anti-CCR3 mAbs
Experiments to determine the anti-CCR3 mAb epitope consisted of two
experimental methods: cross blocking by flow cytometry and peptide-specific
ELISAs. Cross blocking experiments were carried out by saturating Y3/mCCR3
cells with each of the anti-CCR3 mAbs at 100 µg/mL in PBS with 3% BSA on
ice for 30 min and then staining with fluorescein isothiocyanate (FITC)conjugated
6SH2-59 anti-CCR3 mAb at 1 µg/mL for 30 min followed by three
washes in PBS with 3% BSA. The stained cells were analyzed for the ability of
the unlabeled antibody to block 6SH2-59 CCR3-FITC staining.
Peptide ELISAs were performed on each of the anti-CCR3 mAbs by coating
96-well ELISA plates with synthetic peptides corresponding to the aminoterminal
peptide and the three extracellular loops of CCR3 (Research Genetics,
Huntsville, AL). The sequences of the peptides were as follows: aminoterminal,
MAFNTDEIKTVVESFETTPHEYEWAPPCEKVRIKELG; loop 1,
LWNEWGFGHYMC; loop 2, HESQDSFGEFSCSPRYPEGEEDSWKRF-
HALR; and loop 3, AFHRTFLETSCEQSKHLDL. Each well was coated
overnight at 4°C with 100 µL of peptide at 2 mg/mL in PBS. The plates were
then washed with PBS containing 0.05% Tween-20 followed by the addition of
100 µL of anti-CCR3 mAb supernatants at room temperature for 2 h. Plates
were washed and horseradish peroxidase-conjugated rabbit anti-rat polyclonal
antibody (Zymed) was added for 1 h. Plates were washed again and the
colorimetric substrate 2,2’-azino-bis (3 ethylbenzthiazoline-6-sulfonic acid)
was added. Plates were read on a Molecular Devices 96-well plate reader
(Mountain View, CA).
Characterization of the anti-CCR3 mAbs for
agonistic and antagonistic activity
Agonistic and antagonistic activity was assayed by calcium flux analysis.
Y3/mCCR3 cells were labeled and calcium mobilization was assayed as
described. For assaying agonistic activity, 50 µg of purified anti-mCCR3 mAb
was added to labeled cells at 37°C and assayed for calcium mobilization over 5
min. For antagonistic activity, 100 µg/mL of purified anti-CCR3 mAb was
added to labeled cells at 37°C for 5 min followed by the addition of eotaxin at a
final concentration of 100 µM. The calcium mobilization was assayed over 10
min, which included the 5-min mAb incubation step.
Preparation of cells
The Th1 clone, D1.1, and the Th2 clone, CDC35, were stimulated and passaged
with rabbit anti-mouse immunoglobulin, as described previously [17, 18].
CD4 � Th1 and Th2 cells were generated in vitro from DO11.10 T cell receptor
transgenic BALB/c mice as described previously [19]. Briefly, naive CD4 � T
cells were cultured with OVA 323-339, the ovalbumin peptide for which the
transgenic T cells are specific, for either 1 or 3 weekly stimulations (designated
1� or 3� polarized). Successful polarization was confirmed by testing for
interferon-� (IFN-�) and interleukin-4 (IL-4) production by activated Th1 and
Th2 cells, respectively. Resting cells were harvested immediately after the last
stimulation; activated cells were resting cells cultured at 37°C for 6hinPMA
(50 ng/mL) plus ionomycin (500 ng/mL). CD4 � and CD8 � splenic T cells were
isolated with a FACSTAR � plus cell sorter and activated as described above.
Stem cells were purified from bone marrow on the basis of positive staining for
C-kit � and Sca-1 � and negative staining for a mixture of lineage-specific cell
surface markers (Ly6G, Mac1, B220, CD4, CD8, and Ter119). Splenic dendritic
cells were purified by the method of Macatonia et al. [20].
Expression of CCR3, both protein and mRNA
Immunofluorescent staining of cell suspensions was performed with FITCconjugated
6SH2-59 anti-CCR3 mAb at 1 µg/mL. Total RNA was isolated from
cell lines and populations with RNAzol B (TEL-TEST). Northern blots were
performed with 10 µg total RNA and probed with the full-length coding region
of mCCR3 cDNA using standard methods [21]. For quantitative RT-PCR, 4 µg
RNA was treated with DNase 1 and reverse transcribed with SuperScript
(GIBCO). Threefold dilutions of cDNA, representing 0.1–200 pg RNA, were
PCR amplified for 35–40 cycles using conditions as described above for the
cDNA cloning. The primers used were as follows: 5� primer, ATG-GCA-TTC-
Grimaldi et al. Depletion of mouse eosinophils through the use of antibodies 847

AAC-ACA-GAT-GAA-ATC-AAG; 3� primer, GGA-TAG-CGA-GGA-CTG-CAG-
GAA-AAC. PCR products were separated on agarose gels, stained with SYBR �
green I dye (Molecular Probes) and the CCR3 product quantitated by a Storm �
860 phosphoimager (Molecular Dynamics, Sunnyvale, CA). The results were
expressed as arbitrary units of CCR3 mRNA normalized to the content of HPRT
and �-actin mRNA in each sample.
In vivo depletion with anti-CCR3
Eosinophilia was induced in 2-week-old female BALB/c mice by subcutaneous
injection of 500 Nippostrongylus brasiliensis stage 3 larvae (N. brasiliensis). A
single antibody treatment consisted of 0.5 mg purified anti-CCR3 mAb given
intraperitoneally at day 12 after N. brasiliensis infection. Circulating eosinophils
were counted in heparinized blood taken from the tail vein at 0, 24, 48,
96, and 168 h after mAb administration as previously described [22]. For
repeated mAb treatment, the animals were given intraperitoneal injections of 1
mg anti-CCR3 mAb 48 h before N. brasiliensis infection and then 0.5 mg of
mAb on days 3, 8, and 11 after N. brasiliensis infection. The animals were killed
on day 12 for isolation of bronchioalveolar lavage fluid (BAL), lung tissue
sampling, and eosinophil counting in peripheral blood. BAL cytospins and lung
paraffin sections were stained with May-Grünwald Giemsa. For in vitro
restimulation, lung cell suspensions were prepared as described previously [23]
and stimulated on plates previously coated with 10 µg/mL anti-CD3 (Pharmingen,
San Diego, CA). After 3 days the supernatants were removed and IL-4 and
IL-5 concentrations measured by ELISA [23]. Paraffin sections from repeated
antibody treatment experiments were analyzed for eosinophil depletion by two
independent pathologists. Stained sections were scored in a blinded manner on
a scale of 0–4, with 1 being the minimum and 4 being the maximum eosinophil
infiltration.
RESULTS AND DISCUSSION
Much recent evidence suggests that eotaxin and its receptor,
CCR3, are important mediators of allergic inflammation by
stimulating infiltration of eosinophils and, possibly, Th2 cells
into the lung. In order to study CCR3 expression and function
in well-characterized mouse models of eosinophil infiltration
and airway hyper-reactivity, we have produced monoclonal
antibodies to mouse CCR3.
Mouse CCR3 cDNA and stable transfectant
generation
The strategy chosen for producing mAbs to mouse CCR3 was
immunization of rats with a transfected rat cell line expressing
mouse CCR3. The gene for mouse CCR3 was isolated by PCR
using oligonucleotides based on the published mouse CCR3
sequence [15]. Thirty-four different mouse cDNA libraries were
screened by PCR for the presence of CCR3 message. CCR3
cDNA could be detected in three libraries: N. brasiliensisinfected
lung, RAG-2 -/- spleen, and a polarized Th2 population
of CD4 � T cells (data not shown). The presence of CCR3 cDNA
in the latter two libraries was unexpected because several other
T cell and spleen cDNA libraries were negative. The Th2
library had the strongest signal, so it was used as the template to
generate the full-length mouse CCR3 cDNA. This cDNA was
subcloned into the eukaryotic expression vector pMe18sNeo.
The resultant construct pMe18sNeo/mCCR3 was used to transfect
the rat cell line Y3 and the mouse fibroblast cell line
NIH3T3.
Stable CCR3 transfectants were isolated and screened using
multiple procedures to ensure that the cell lines were clonal
and expressed high levels of native, functional mouse CCR3.
Three separate selection steps were used: (1) single cell cloning
of G418 resistance colonies, (2) detection of CCR3 message by
RT-PCR, and (3) testing for positive signal transduction by
eotaxin-induced calcium flux. Several clonal cell lines expressing
CCR3 from both NIH3T3 and Y3 transfections were
isolated. A single clonal transfectant was selected from both the
NIH3T3 and Y3 transfectants based on the highest calcium flux
signal. The two stable mouse CCR3 cell lines used throughout
this work were named Y3/mCCR3 and NIH3T3/mCCR3. The
Y3/mCCR3 cells were used as the immunogen for mAb
production.
The Y3/mCCR3 cell line produced a robust calcium flux
over a range of eotaxin concentrations from 1 µM down to as
little as 0.5 nM (Fig. 1A). The NIH3T3/mCCR3 cell line also
produced a significant calcium flux that was approximately
20% of that seen with the Y3/mCCR3 cell line; the parent cell
lines were both unresponsive (Fig. 1B). Both human and murine
eotaxin produced similar calcium flux responses on the tranfected
lines. The Y3/mCCR3 transfectant produced a detectable
calcium flux in response to two other human CCR3
ligands, human eotaxin-2 and human MCP-4 (data not shown).
However, neither the Y3/mCCR3 nor NIH/mCCR3 transfectants
produced a calcium flux in response to mouse RANTES,
human MCP-2, or MCP-3, even at concentrations as high as 1.5
µM (data not shown). The above data confirmed that mouse
CCR3 was expressed by the Y3/mCCR3 cell line.
Production and characterization
of anti-mCCR3 mAbs
Spleen cells from an immunized rat were fused to SP2/0 cells.
Supernatants of hybridomas were tested by flow cytometry for
positive staining of mCCR3 on NIH3T3/mCCR3 transfectants
and negative staining on NIH3T3 parent lines. Hybridomas
producing supernatants specific for the NIH3T3/mCCR3 transfectant
and negative for the NIH3T3 parent line (Fig. 2A) were
expanded for further study. In total, 30 different anti-mCCR3
mAbs were produced. All anti-mCCR3 mAbs were further
tested on the Y3/mCCR3 as well as the Y3 parent cell line to
confirm specificity for mCCR3 (Fig. 2B).
Using calcium flux analysis, the anti-mCCR3 mAbs were
tested for agonistic as well as antagonistic activity on the
Y3/mCCR3 cell line. None of the antibodies showed any
agonistic activity as demonstrated by the lack of calcium
mobilization when saturating amounts of anti-mCCR3 mAb
were added to Y3/mCCR3 cells. To test whether any of the
anti-CCR3 mAbs were antagonists, a saturating amount of mAb
was added to Y3/CCR3 cells followed by a limiting amount of
eotaxin (100 nM). None of the anti-CCR3 mAbs caused a
reduction in calcium mobilization stimulated by eotaxin. Therefore,
it was concluded that none of the anti-mCCR3 mAbs were
agonistic or antagonistic.
Anti-mCCR3 mAbs recognize
the amino-terminal peptide
Anti-CCR3 mAbs were analyzed by two separate techniques to
determine their epitope specificity on the mouse CCR3 protein.
First, competitive inhibition using unlabeled purified antimCCR3
mAbs was used to block the staining of a FITC-
848 Journal of Leukocyte Biology Volume 65, June 1999 http://www.jleukbio.org

Fig. 1. Calcium mobilization of mCCR3 transfectants. (A) Calcium mobilization in Y3/mCCR3 cell line with various concentrations of eotaxin from 1 µM to 0.5 nM
as well as the Y3 parent line with 1 µM eotaxin. (B) NIH3T3/mCCR3, Y3/mCCR3, and NIH3T3 parent line all stimulated with 1 µM eotaxin.
conjugated anti-mCCR3 mAb named 6SH2-59. Competitive
inhibition showed that all 30 mAbs could block the staining of
6SH2-59. A representative example of these experiments is
shown in Figure 3. This observation suggested that all 30
mAbs might share a common epitope.
To more specifically define the epitope recognized by the
anti-CCR3 mAbs, peptides were generated corresponding to
the four extracellular portions of mCCR3, generally referred to
as the amino-terminal peptide, loop 1, loop 2, and loop 3 (Fig.
4). An ELISA with these four peptides showed that 25 of the 30
mAbs bound only to the amino-terminal peptide and that the
remaining 5 did not bind to any of the peptides. Given that all
30 mAbs can cross-block the binding to CCR3, and that 25 of
Fig. 2. Flow cytometry of mCCR3-transfected cell lines
using anti-mCCR3 mAb. (A) Flow cytometry of NIH3T3/
mCCR3 (bold) and NIH3T3 parent line with FITC-6SH2-
59. (B) Flow cytometry of Y3/mCCR3 (bold) and Y3
parent line with FITC-6SH2-59.
the 30 mAbs recognized the amino-terminal peptide, it is clear
that the epitope(s) recognized by those 25 mAbs is contained
within the amino-terminal peptide. The mAbs used in subsequent
studies, namely, 6SH2-59, 6SH2-88, and 6S2-19-4, all
recognized the amino-terminal peptide.
Anti-mCCR3 mAbs specifically bind
to eosinophils
The surface expression of CCR3 was analyzed on a variety of
mouse tissues and cell populations by immunofluorescent
staining with FITC-conjugated 6SH2-59. Thymocytes, whole
splenocytes, splenic T and B cells, dendritic cells, and bone
Grimaldi et al. Depletion of mouse eosinophils through the use of antibodies 849

marrow stem cells were negative for CCR3 staining. In the bone
marrow, 0.5–2% of the cells stained with anti-CCR3. CCR3positive
bone marrow cells were purified by cell sorting,
centrifuged onto glass slides, and stained with Wright-Giemsa
stain. This population was comprised of mature (62%), and
juvenile (34%) eosinophils as well as eosinophilic myelocytes
(4%). Less mature cells, such as promyelocytes and blasts, were
not observed in the CCR3-positive fraction. Analysis of lungs
revealed that few anti-CCR3 staining cells could be seen in
normal BAL (Fig. 5A), however, a 33% population of brightly
stained cells was observed in the BAL of mice infected with N.
brasiliensis (Fig. 5B). CCR3-positive and -negative cells in the
BAL of infected mice were separated by cell sorting and
analyzed histologically with May Grünwald Giemsa stain.
Essentially all of the CCR3 positive cells were eosinophils (Fig.
5D), whereas CCR3-negative BAL cells contained no eosinophils
(data not shown).
Anti-mCCR3 mAbs did not detect Th2 cells
Because the initial CCR3 PCR clone was generated from a Th2
cDNA library, we undertook a detailed analysis of CCR3
Fig. 3. Competitive inhibition using FITC 6SH2-59 vs.
unlabeled anti-CCR3 mAb stained on NIH3T3/mCCR3
transfectants. Histograms in bold represent staining with
FITC 6SH2-59 only. Thin line histograms represent
blocked staining by adding unlabeled CCR3 mAb followed
by FITC 6SH2-59. (A) As a control, FITC 6SH2-59
was blocked by 6SH2-59. (B) FITC 6SH2-59 blocked by
6SH2-88; this experiment was conducted for all 30
anti-mCCR3 mAbs and showed similar blockage of FITC
6SH2-59 staining as seen here.
expression in Th cell populations and clones. First, both Th1
and Th2 cell lines were assayed for their response to eotaxin by
calcium flux assay. Calcium mobilization was not detected on
either the CDC35 (Th2) or D1.1 (Th1) clones using eotaxin at
concentrations between 1 µM and 1 nM. Similarly, activated
3� polarized primary Th2 populations generated from ovalbumin-specific,
TCR-transgenic DO11.10 BALB/c mice[19, 24]
produced no significant chemotaxis in response to eotaxin (data
not shown). Surface expression of CCR3 on these same Th
populations and cell lines was assessed by flow cytometry using
FITC-conjugated 6SH2-59 mAb. No CCR3-positive cells were
detected in any of the Th cell lines or populations tested
whether they were resting or activated. Thus, neither Th1 nor
Th2 CD4 � T cells expressed CCR3, at least in amounts that
could be detected by antibody staining, eotaxin-induced calcium
mobilization, or chemotaxis. It is important to note,
however, that a recent publication describes an anti-human
CCR3 mAb that stained human Th2-like T cells [8].
This recent publication [8] led us to examine carefully the
expression of CCR3 mRNA in mouse T cell populations.
Northern analysis of total RNA from resting and activated 1�
Fig. 4. Graphic presentation of the four synthetic peptides
of mouse CCR3 used in ELISA experiments to
determine which extracelluar portion the anti-mCCR3
mAbs recognized.
850 Journal of Leukocyte Biology Volume 65, June 1999 http://www.jleukbio.org

Fig. 5. Flow cytometry of N. brasiliensis (Nb) -infected and -uninfected mouse
BAL stained with FITC 6SH2-59. (A) Uninfected BALB/c, (B) Nb-infected
BALB/c, (C) May-Grünwald Giemsa-stained, unsorted Nb-infected BAL, (D)
FACS-sorted CCR3-positive Nb-infected BAL. The CCR3-positive staining
cells represents a pure population of eosinophils.
polarized Th1 and Th2 populations, CD4 � and CD8 � T cells,
and Y3 showed no appreciable CCR3 message, however, the
Y3/mCCR3-transfected cell line produced an easily detectable
band (Fig. 6A). In a separate Northern experiment, CCR3
mRNA was not detected in resting or activated 3� polarized
Th1 or Th2 cells. As a more sensitive assay of CCR3 mRNA, a
quantitative RT-PCR analysis was performed with samples of
some of the same mRNA preparations used in the Northern blot
analysis. RNA from activated Th2 cells did indeed contain
CCR3 mRNA, but the amounts were approximately 80-fold less
than in a comparable sample from the Y3/mCCR3-transfected
line (Fig. 6B). If CCR3 were expressed on these two cells in a
ratio similar to their mRNA content, it would be quite
consistent with the differences in fluorescent staining and
functional responses between these two cell populations. In a
separate RT-PCR analysis, using a higher cycle number, both
nonactivated Th2 and activated Th1 populations appeared to
express approximately 50-fold lower levels of CCR3 mRNA
than did activated Th2 cells (Fig. 6C). This result could
represent lower levels of CCR3 mRNA expression by all cells or
the presence of a small number of activated Th2 cells in these
populations. As a control, PCR analysis of parallel samples of
all mRNAs without reverse transcription did not show the
CCR3 band, demonstrating that contaminating genomic DNA
was not being detected in these PCR assays. Whether the low
level of expression of CCR3 mRNA reflects a similar low level
of surface CCR3 expression on these cells is not yet known, but
these data make clear that, in the mouse, CCR3 expression is
not a useful basis for distinguishing Th2 cells from other T
cells.
Some anti-mCCR3 mAbs can specifically deplete
eosinophils in vivo
Three anti-CCR3 mAbs were chosen for in vivo studies:
6SH2-59 (IgG2a), 6SH2-88 (IgG2b), and 6S2-19-4(IgG2b).
IgG2b is the most efficient complement-fixing rat IgG subclass
and it had been previously demonstrated that the rat IgG2b
anti-mouse Ly-6G antibody, RB6-8C5, could be used to
specifically deplete eosinophils and neutrophils in vivo [25]. To
test whether anti-CCR3 mAbs of the IgG2b subclass could be
used to specifically deplete eosinophils in a similar manner, N.
brasiliensis-infected mice were injected with 0.5 mg of 6S2-
19-4 or 6SH2-88. For comparison, other groups were injected
with the IgG2a anti-CCR3 mAb 6SH2-59 or the IgG2b
anti-Ly-6G mAb, RB6-8C5. Blood eosinophils were counted
before and after administration of the mAbs. Both of the IgG2b
anti-CCR3 mAbs, 6S2-19-4 and 6SH2-88, depleted the circulating
eosinophils to below the levels of uninfected mice within
24 h and these levels remained low for at least 7 days (Table 1).
In subsequent experiments, as little as 100 µg of either mAb
gave maximal depletion of eosinophils at the 24 h time point. In
contrast, no significant depletion was observed with the IgG2a
anti-CCR3 mAb 6SH2-59. Differential blood smears showed
that the IgG2b anti-CCR3 mAbs depleted eosinophils, but not
neutrophils (data not shown), whereas the RB6-8C5 mAb
depleted both cell types [25].
Repeated anti-mCCR3 mAb treatment can
partially deplete eosinophils in N. brasiliensisinfected
lung tissue and BAL fluid
In the experiments discussed above, relatively little eosinophil
depletion was observed in the lung infiltrates or BAL of mice
given a single injection of anti-CCR3 mAb. To determine
whether depletion in these compartments would be more
effective if begun at the initiation of the infection, mice were
given 0.5 mg of 6S2-19-4 intraperitoneally 2 days before N.
brasiliensis infection and then again on days 3, 8, and 11. Mice
were killed on day 12 and the lungs, BAL fluid, and blood were
collected for analysis. As expected, anti-CCR3 mAb-treated
animals had low circulating eosinophil levels comparable to
uninfected control mice (data not shown). Total eosinophil
numbers in BAL fluid, as assessed by differential cell counts,
were reduced from 3.55 � 10 6 � 1.42 � 10 6 in N. brasiliensisinfected
mice to 1.07 � 10 6 � 0.46 � 10 6 in infected,
anti-CCR3-treated mice (n � 6 in each group). Anti-CCR3
mAb treatment did not appear to deplete any cell type other
than eosinophils in either blood or BAL cells. Paraffin sections
of the lungs taken from animals treated with 6S2-19-4 showed a
similar significant reduction in eosinophils, in this case, to
approximately 50% of that in infected controls (data not shown).
Thus the eosinophil population in the lung tissue and BAL were
partially protected from antibody-mediated killing. One possible
explanation is that the expression of CCR3 on many
Grimaldi et al. Depletion of mouse eosinophils through the use of antibodies 851

Fig. 6. Expression of CCR3 mRNA in T cell populations. Total RNA from the cell populations indicated was analyzed by Northern blot (A) or by RT-PCR (B and C)
for relative levels of CCR3 mRNA. (A) 10 µg of total RNA was run in each lane and then blotted and probed for CCR3. (B) Relative levels of CCR3 mRNA in the
Y3/CCR3 cell line and in an activated Th2 population. (C) Relative levels of CCR3 mRNA in resting and activated Th1 and Th2 populations. The levels of PCR
products in panels B and C are expressed as arbitrary phosphoimager units after normalization to HPRT and �-actin mRNA. The units in the two different RT-PCR
experiments (B and C) should not be compared to each other.
eosinophils in the lung and BAL is low enough to evade
antibody-mediated killing. A second possibility is that eosinophils
arise from CCR3-negative precursors within the lung and
are not exposed to antibody or complement levels sufficient to
mediate killing. Using these anti-mCCR3 mAbs on various
murine models of eosinophil-mediated disease will help to
elucidate the importance of CCR3 in disease as well as the role
eosinophils play in the progression of that disease.
To determine whether the very low level of CCR3 expression
by activated Th2 cells renders them sensitive to anti-CCR3
TABLE 1. Depletion of Eosinophils in N. brasiliensis-Infected Mice
Using Anti-CCR3 IgG2b Rat mAb
mAb treatment
(0 h)
Day 10
(24 h)
Day 11
(48 h)
Day 12
(96 h)
Day 14
(168 h)
Day 17
6S2-19-4 (Anti-mCCR3/IgG2b) 447 13 5 8 11
6S2-88 (Anti-mCCR3/IgG2b) 471 8 20 20 13
6S2-59 (Anti-mCCR3, IgG2a) 484 224 445 308 211
RB6-8C5 (Anti-Ly-6G, IgG2b) 450 20 7 2 60
N. brasiliensis (untreated) 297 302 353 253 177
Eosinophil counts in peripheral blood from N. brasiliensis-infected BALB/c
mice treated with 0.5 mg i.p. of mAb on day 10 after infection (0 h). Counts were
taken at 0, 24, 48, 96, and 168 h after the intraperitoneal injection. N � 9, all
values are eosinophils/ml � 10 4 . Normal BALB/c mice have 30 � 10 4
eosinophils/mL.
killing in vivo, IL-4 and IL-5 levels were determined by
restimulation assays in vitro on the lung cells taken on day 12
from mice treated with anti-CCR3 mAb as described above.
Serum was taken from these same animals and assayed for IgE
levels as a sensitive measure of Th2 help [23]. No significant
difference was observed in either IL-4 and IL-5 production in
vitro or in serum IgE levels between untreated and anti-CCR3treated
mice. This demonstrates that IgG2b anti-CCR3 mAbs
do not cause any reduction in Th2 cytokines or in Th2-mediated
responses and would appear to only reduce eosinophils.
In summary, this study demonstrates that mouse CCR3
functions similarly but not identically to the previously published
human CCR3. Human CCR3 transfectants have been
shown to respond to RANTES, MCP-2, MCP-3, MCP-4,
eotaxin, and eotaxin-2 [14, 26]. This study has shown that
eotaxin can produce a robust calcium mobilization in cells
transfected with mouse CCR3. In addition, eotaxin-2 and
MCP-4 can produce significant calcium mobilization in cells
transfected with mouse CCR3 (data not shown), whereas
RANTES, MCP-2, and MCP-3 showed no effect on mouse
CCR3 transfectants (data not shown). The selective expression
of readily detectable mCCR3 on eosinophils matches well with
previous reports of huCCR3 expression and suggests utility of
these antibodies for isolating and enumerating mouse eosino-
852 Journal of Leukocyte Biology Volume 65, June 1999 http://www.jleukbio.org

phils. However, the surface expression of CCR3 on mouse Th2
cells, if present, was too low for detection by flow cytometry. In
addition, our study shows that mouse Th2 cells are not
responsive to eotaxin either by calcium mobilization assay or
chemotaxis. This contrasts with a recent report of preferential
staining with an anti-huCCR3 mAb as well as the eotaxinmediated
chemotaxis and calcium flux of human Th2 cells [8].
Our observation of low levels of mCCR3 mRNA in activated
Th2 cells, however, is consistent with a more recent report on
CCR3 mRNA expression observed in human Th1 and Th2 cells
[27]. It is quite possible that CCR3 expression on Th2
population is tightly linked to an as yet unknown activation
state and this could explain the differences observed between
different reports. We have also demonstrated that anti-mCCR3
mAbs of the rat IgG2b isotype can also be used to achieve
partial to complete depletion of eosinophils in vivo. Treated
animals were used in restimulation assays of lung cells, which
showed no increase in Th2-induced cytokine levels. In short,
these assays taken together show that CCR3 is not a significant
chemokine receptor for mouse Th2 cells. Recent studies have
shown that other chemokine receptors such as CCR8 may have
a far more significant role on Th2-specific cellular migration
[28]. These antibodies should be useful in future studies aimed
at determining the roles of eosinophils in infectious and
inflammatory diseases.
ACKNOWLEDGMENTS
DNAX Research Institute is supported by Schering-Plough
Corporation. The authors would like to thank Debra Liggett for
synthesis of DNA, Dan Gorman, Allison Helms, and Connie
Huffine for DNA sequencing analysis, Dr. Terri McClanahan
and Karin Bacon for providing cDNA libraries, Dr. Donna
Rennick for histology consultation, Dr. Joe Hedrick for chemotaxis
analysis, Dr. Kenneth Soo for his helpful suggestions, and
Gary Burget and Dr. Maribel Andonian for graphics work.
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