21,22 Eotaxins, acting via their receptor, CCR3, may therefore not only represent an important link in the mobilization of eosinophils and their progenitors, but also play a role in haematopoiesis at sites of inflammation (i.e. in situ haematopoiesis). Therefore, we hypothesize that CD34+ CCR3+ cells are increased in the airways after allergen exposure. We further hypothesize that these cells, in addition to the classical
CD34+ IL-5 receptor α subunit-positive (IL-5Rα+) eosinophil progenitor cells, have a proliferative capacity and undergo in situ proliferation in response to allergen. In this study, the importance and potential role for these potential progenitor populations in the lung following allergen provocation were investigated in the mouse using both in vivo models (e.g. allergen Vemurafenib supplier provocation of wild-type see more and IL-5 transgenic mice as well as 5-bromo-2′-deoxyuridine (BrdU) labelling of progenitor cell populations in the lung) and ex vivo culture studies (e.g. semi-solid cultures, evaluating colony formation) to identify and characterize these cells. Moreover, the specific role of these progenitor populations in pulmonary allergen-mediated inflammatory responses was
highlighted in vivo by selective depletion with a rat anti-mouse CCR3 monoclonal antibody. This study was approved by the Animal Ethics Committee in Gothenburg, Sweden. Five- to six-week-old male BALB/c mice purchased from Taconic (Ry, Denmark) were used for all in vivo experiments and the in vitro colony-forming assays. Interleukin-5 transgenic mice (line NJ.1638) were used as part of in vivo migration studies (i.e. administration of eotaxin-2) and for in vitro transmigration assays.23 These mice were kept under animal housing conditions and provided with food and water ad libitum. Mice were immunized twice, at an interval of 5 days, PAK5 by intraperitoneal (i.p.) injections of 0·5 ml alum-precipitated antigen containing 8 μg ovalbumin [OVA; bound to 4 mg Al(OH)3, both from Sigma-Aldrich, St Louis, MO] in PBS. Eight days after the second sensitization,
the mice were quickly and briefly anaesthetized with isofluorane (Baxter, Deerfield, IL) and received an intranasal administration of 100 μg OVA in 25 μl PBS on five consecutive days. In addition, one group received 25 μl PBS on five consecutive days as a control for the OVA exposure. Twenty-four hours after the final OVA exposure, the mice were killed and BM, bronchoalveolar lavage fluid (BALF) cells and lung tissue were collected. The BrdU (Roche Diagnostics Scandinavia AB, Bromma, Sweden) was administered to mice as a means to label newly produced cells, of which a proportion are eosinophil-lineage-committed cells. The BrdU was given at a dose of 1 mg in 250 μl PBS by i.p. injection on two occasions, 8 hr apart on day 3 and day 5 before harvesting of samples. Samples were collected 24 hr after the final OVA exposure using the protocol noted earlier.
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