The Roles Of Tyro 3 Subfamily Of Receptor Tyrosine Kinases In Regulating Hematopoiesis

The Tyro 3 subfamily of RTKs, which is composed of three members (Tyro 3, Axl andMer), is widely expressed in various mammalian tissues, such as the nervous, immune,reproductive, vascular and hematopoietic tissues and plays important roles in regulatingspermatogenesis, immune homeostasis, platelet functions, NK cells differentiation,angiogenesis and vascular remodeling. In this study, we mainly focused on investigatingthe roles of Tyro 3 RTKs in regulating hematopoiesis. The expression analyses of Tyro 3RTKs in marrow cells indicated that megakaryocytes (MKs) coexpressed Tyro 3, Axl andMer, B lymphocyte precursors and erythroid lineage cells only expressed Axl and Mer.Furthermore, we used single, double and triple mouse knock-outs for Tyro 3 RTKsubfamily receptors to investigate their roles in regulating hematopoiesis.We first investigated the roles of Tyro 3 RTK subfamily receptors in regulatingmegakaryocytopoiesis and hemostasis. Thrombocytopenia was observed in mice lackingall the three receptors (Tyro 3^-/- Axl^-/- Mer^-/-, TAM), however, any single (Tyro 3^-/-, T; Axl^-/-,A; Mer^-/-, M) or double (Tyro 3^-/- Axl^-/-, T A; Tyro 3^-/- Mer^-/-, TM; Axl^-/- Mer^-/-, AM)mutants exhibited normal platelet level. Thrombocytopenia in TAM mice was caused bydefective megakaryocytopoiesis. In TAM mice, the numbers of MKs per femur decreasedsignificantly, and the MKs exhibited a lower level of ploidy elevated. PPF assays showedthat a few MKs from TAM mice could develop proplatelets, indicating impaired terminalmaturation of TAM MKs. In accord with this finding, electron microscopy demonstratedthat TAM MKs displayed a poor demarcation membrane system and many vaculoes incytoplasm. AM mice also appeared impaired megakaryopoiesis in bone marrow, but theyhad normal platelet level in peripheral blood. Extrameduallary megakarypoiesis in AMmice might compensate the defect of platelet biogenesis in bone marrow. Double and triplemutant of Tyro 3 RTK subfamily receptors exhibited impaired hemostasis owing to plateletdysfunction. Severe impaired platelet aggregation and ATP release abilities were observedin double and triple mutants. Although Single mutants exhibited normal hemostasis, they had mild platelet dysfunction, indicating Tyro 3 subfamily receptors regulated plateletfunctions cooperatively.In addition, we found that Tyro 3 RTKs regulated B lymphocytopoiesis. TAM micedisplayed severe impaired B lymphocytopoiesis, exhibiting about 50% decrease in thenumber of Fractions D and F of B lymphocytes, and 90% reduction of mature Blymphocytes (Fraction F) compared to control mice. In double mutants, AM mice had asimilar phenotype of B lymphocytopoiesis to TAM mice, however, TA and TM miceshowed no apparent alteration in the number of B lineage subsets. Single mutants, A and M,but not T also displayed abnormal B lymphocytopoiesis. Axl gene mutation mainly affectedPre-B stage, while Mer deletion lead to reduction of mature B cells (Fr. F). These resultssuggested that Tyro 3 RTK subfamily receptors possessed discrete functions in regulatingB lymphocytopoiesis. Further, we analysed the responses to thymus dependent (TD)antigen OVA in different genotype mice. The primary OVA-specific IgM and IgG reponseswere reduced in A, M and TAM mice. Though the secondary IgM response was normal,the secondary IgG response was dramatically reduced in M mice. Axl_-/- mice showed areduction in the secondary IgM response, but mounted similar secondary IgG responsecompared to control mice. Both of the secondary IgM and IgG reponses were low in TAMmice. These observations indicated an important role of Axl or Mer receptors in thegeneration of an antigen-specific immune responses.We also demonstrated an essential function for Tyro 3 subfamily receptors inerythropoiesis. The number of Terl19^high erythroid lineage cells in bone marrow decreasedsignificantly in A or M mice. AM mice showed more severe impairment in erythroiddevelopment with a 50% decrease in the number of Terl19^high late stage erythroblast cells.However, an increased Terl19^low early erythroid cells in AM mice suggested the deletion ofboth Axl and Mer partially blocked erythroid differentiation. TAM mice had a similarphenotype to AM mice in erythropoiesis. Though AM and TAM mice displayed apparentdefects in erythropoiesis in bone marrow, they had no anemia, which may owe to the compensation from extramedullary erythropoiesis.In this tudy, we also investigated the roles of Tyro 3 RTKs in regulating macrophagephagocytosis. Previous studies have reported that Mer plays important roles in macrophagephagocytosis of apoptotic cells, but they did not study the roles of Tyro 3 and Axl in thisprocess. Here, we demonstrated that macrophages expressed Axl and Mer, but not Tyro 3.Phagocytosis analysis showed that Mer elimination nearly abolished macrophagephagocytosis of apoptotic cells, and triple mutation impaired phagocytosis of apoptoticcells more severely. In contrast, deletion of Axl, Tyro 3, or both, did not affectphagocytosis of apoptotic cells by macrophages. These results suggested that Tyro 3subfamily receptors played different roles in the clearance of apoptotic cells. Though Merand Axl were involved in engulfment of apoptotic cells, Mer might play a key role and Axlmay function in synergy with Mer. Tyro 3 contributed little to clearance of apoptotic cells.In addition, we found that Tyro 3 subfamily receptors did not affect the phagocytosis ofyeast by macrophages.