In the cultures of lung CD34+ cells (Fig. 2a), we detected 2 ± 1 CFU/well in the control culture, and 8 ± 3 versus 6 ± 2 CFU/well in cultures where either IL-5 or rmEotaxin-2 was added alone, respectively. When the combination of rmIL-5 and rmEotaxin-2 was added to the culture of lung CD34+ cells, no further significant increase in CFU/well was observed (10 ± 1 CFU/well; Fig. 2a). Interestingly, previous studies have shown that BM-derived CD34+ cells form CFU when stimulated with rmIL-5.9 Hence, BM CD34+ cells were cultured in parallel as a control for our system. In the cultures of BM CD34+ cells, we detected 1 ± 1

BM CFU/well in the control cultures (no cytokines INCB024360 added), whereas we found no BM CFU in the cultures where rmEotaxin-2 alone was added. In contrast, the cultures where rmIL-5 was added alone, or together with rmEotaxin-2, had 27 ± 3 and 26 ± 2 CFU/well, respectively (Fig. 2b). The optimal time for BM CFU growth was after 8 days of culture, and in lung after 8–14 days of culture. The cells Acalabrutinib were identified as eosinophils on the basis of morphologically homogeneous appearance. A multiparametric cell cycle analysis was used to assess whether the magnetically enriched CD34+ or Sca-1+ newly produced eosinophil-lineage-committed cells proliferate locally within the airways in response to allergen, by analysis of BrdU staining together with 7-AAD staining (total DNA stain). We found

a significant increase in the number of CD34+ CCR3+ BrdU+ and Sca-1+ CCR3+ BrdU+ proliferating cells (i.e. cells within S phase or G2/M phase) Carnitine palmitoyltransferase II in the allergen-exposed animals when compared with the saline exposed animals. This increase was paralleled with an increase in proliferating cells in both SSChigh and SSClow lung cell populations, representing eosinophils

and their progenitors (Fig. 2c,d). We employed double staining of CCR3 together with MBP to further assess whether the CCR3+ cells were committed to the eosinophil lineage. Almost all of the CCR3+ cells gated on both the SSChigh and SSClow cell population co-expressed MBP (ranging between 75 and 99%) (data not shown). Bone marrow, lung and BAL cells were stained for CD34+ CD45+ IL-5Rα+ to evaluate the amount of CD34+ progenitors (CD34+ CD45+ cells) and the classical eosinophil progenitors (CD34+ CD45+ IL-5Rα+ cells) in our model. No differences were found in BM eosinophil progenitors of allergen-exposed animals compared with saline-exposed animals (data not shown). In contrast, lung and BAL CD34+ CD45+ IL-5Rα+ cells were significantly increased in the allergen-exposed animals compared with the saline-exposed animals (Fig. 3a). To further assess whether the IL-5Rα+ newly produced cells proliferate locally within the airways in response to allergen a multiparametric cell cycle analysis for BrdU+ cells together with 7-AAD staining (total DNA analysis) was used.