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

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