Phenotypic and functional characterization of mouse basophils in vitro and in vivo [electronic resource]

Basophils are rare hematopoietic cells that are found, under certain pathological conditions, throughout the tissues of the body: in the skin in atopic or contact dermatitis; in the lungs in allergic asthma or infection; in the liver, spleen and lungs during Nippostrongylus brasiliensis (N.b.) infection. The three projects described herein identified a new aspect of basophil phenotype, helped to clarify a pathway of basophil development, and developed a new mouse model for analyzing mast cell and basophil functions in vivo. In the first study, we showed that when tested at baseline (i.e., without any form of stimulation) mouse basophils express high levels of E-cadherin transcript and protein. E-cadherin protein was detected at the highest levels in bone marrow basophils, but also was present on blood and spleen basophils. In cultures of mouse bone marrow cells, E-cadherin expression on basophils increased over time, peaking at ~days 3-5, and then decreased by day 11 of culture to levels below those in freshly isolated bone marrow basophils. No baseline expression of E-cadherin protein was detected in human blood or bone marrow basophils. Thus, we have identified the expression of E-cadherin, first identified in epithelial cells, by mouse but not human basophils. In the second study, we found that Runx1P1N/P1N mice, which are deficient in the transcription factor distal promoter-derived Runx1 (P1-Runx1), have a marked reduction in the numbers of basophils in the bone marrow, spleen and blood. In contrast, Runx1P1N/P1N mice have normal numbers of mast cells as well as the other granulocytes, i.e., neutrophils and eosinophils. Runx1P1N/P1N mice fail to develop a basophil-dependent immune response, but responded normally when tested for IgE- and mast cell-dependent reactions. These results demonstrate that Runx1P1N/P1N mice exhibit markedly impaired development of basophils, but not mast cells. However, we found that infection with the nematode parasite Strongyloides venezuelensis, or injections of IL-3, can induce modest expansions of the very small populations of basophils in Runx1P1N/P1N mice. Finally, we found that Runx1P1N/P1N mice have normal numbers of granulocyte progenitor cells that can give rise to all granulocytes, but exhibit a > 95% reduction in basophil progenitors (BaPs). These observations indicate that P1-Runx1 is critical for a stage of basophil development between SN-Flk2+/- cells that have granulocyte progenitor potential and BaPs. In the third project, we generated C57BL/6-Cpa3-Cre; Mcl-1fl/fl mice and showed that they are severely deficient in mast cells and also have a marked deficiency in basophils, whereas the numbers of the many other hematopoietic cell populations examined exhibit little or no changes. Moreover, Cpa3-Cre; Mcl-1fl/fl mice exhibit marked reductions in the tissue swelling and leukocyte infiltration associated with either mast cell- and IgE-dependent passive cutaneous anaphylaxis or a basophil- and IgE-dependent model of chronic allergic inflammation of the skin, and they can't develop IgE-dependent passive systemic anaphylaxis. Our findings support the conclusion that the intracellular anti-apoptotic factor, Myeloid cell leukemia-1 (Mcl-1) is required for normal mast cell and basophil development/survival in vivo in mice, and also show that Cpa3-Cre; Mcl-1fl/fl mice represent a useful model for analyses of roles of mast cells and basophils in health and disease. Taken together, these studies further our understanding and encourage future studies of basophil distribution and function at steady state and during pathological responses.

Advisor: Galli Stephen J. primary advisor. Thesis advisor
Weissman Irving L. primary advisor. Thesis advisor
Engleman Edgar G. advisor. Thesis advisor
Nadeau Kari advisor. Thesis advisor
Sunwoo John B.