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

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