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

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