Figure 5. The neutrophilic phenotype of OVA-treated iPHIL mice rendered eosinophil-deficient during an initial airway challenge is reversed upon subsequent OVA airway challenges by restoration of peripheral eosinophils.

(A) OVA sensitized mice (O) were administered DT ((i.p.) - 15ng/gram body weight) prior to, and during, an initial airway challenge phase on days 20, 21, and 24–27 ((O) arrows). Mice were assessed at two time points in this protocol: (i) Two days following the last of the initial OVA airway challenges - day 28. (ii) OVA sensitized mice that were previously rendered eosinophil-deficient during the initial airway challenge phase were allowed to “rest” (i.e., DT administration was discontinued) 12 days to allow restoration of the peripheral eosinophils prior to re-challenge with 1% OVA (control mice received saline alone) on day 39. (B) Assessments of total BAL cell counts as well as cell differential analyses were performed on cytospins preparations of recovered airway cells at OVA Day 28 vs. Day 41. *P<0.05. (C) Representative photomicrographs of lung sections from iPHIL mice at OVA Day 28 vs. Day 41 are presented relative to lung sections from saline-treated control Day 41 mice. These lungs sections were stained with PAS (dark purple staining cells) as well as immunohistochemistry using an anti-MBP monoclonal antibody (MBP IHC – red staining cells infiltrating the parenchyma), assessing goblet cell metaplasia/airway epithelial cell mucin accumulation and eosinophil infiltration, respectively. These data demonstrated that while both OVA-Day 28 and OVA-Day 41 lungs displayed goblet cell metaplasia/airway epithelial cell mucin accumulation (PAS), iPHIL mice at OVA Day 41 (i.e., after secondary allergen challenge) developed a prominent pulmonary eosinophilia (MBP IHC) in contrast to the complete absence of eosinophils in iPHIL at OVA Day 28. Scale bar = 100μm.