EXCEED THE SPACE PROVIDED. In this project, we seek to discern the mechanism by which GM-CSF regulates alveolar macrophage (AM) FcyR-mediated phagocytosis, a function vital to lung host defense. Based on our published (Immunity 15: 557-567;2001) and preliminary data, we propose the hypothesis that GM-CSF is required in the lung constitutively to stimulate high levels of PU. 1 in AMs and that PU. 1 is required in AMs constitutively to stimulate their terminal differentiation and for expression of FcTRc_, Hck and possibly other downstream targets critical for effective FcyR-mediated phagocytosis. In humans, anti-GM-CSF autoantibodies are strongly associated with development of idiopathic pulmonary alveolar proteinosis (PAP) and defects of AM function. GM-CSF gene targeted (GM-/-) mice develop histologically similar PAP and have severely impaired pulmonary clearance of microbial pathogens. Our preliminary data show that GM-CSF critically regulates AM FcTR-mediated phagocytosis in vivo by stimulating high PU.1 levels in AMs. PU.1 appears necessary for expression of several downstream targets required for effective Fcq,R-mediated phagocytosis including FcRc_ and Hck but not FcT or Syk. Although these results demonstrate the importance of GM-CSF/PU.1, they do not indicate if one or both act constitutively or provide a 'commitment'-type stimulus. Nor do they indicate if Hck is critical or clearly define the mechanism by which PU. 1 regulates FcyR-mediated phagocytosis. To answer these questions, we will utilize existing murine transgenic models wherein GM-CSF expression is normal (GM+/+), absent (GM-/-), or overexpressed only in the lungs constitutively (SPC-GM+/+ GM-/-) or conditionally under external control of orally administered doxycycline (double transgenic GM-CSF expressing (DTxGM) mice)).
In Aim 1, we will determine: 1) the temporal relationship between lung-specific GM-CSF expression in vivo and AM FcTR-mediated phagocytosis (evaluated ex-vivo) and AM terminal differentiation; 2) if GM- CSF, in vivo, provides 'rheostatic' or 'on-off' control of AM FcTR-mediated phagocytosis; and 3) the kinetics of gain and loss of such AM functions.
In Aim 2, we will determine: 1) if retrovirus-mediated PU.1 expression in mAM cells (a novel AM cell line derived from GM-/- mice) reimplanted into the lungs of GM-/- mice prevents PAP and restores normal lung defense; and 2) if retroviral replacement of FcTRIII_ rescues FcTR-mediated phagocytosis in mAM cells in vitro.
In Aim 3, we will use retroviral transduction of mAM ceils to express FcyRIIIc_ and/or individual Src-family kinases (SFKs) to establish which family members can effectively rescue wild type levels of FcyR-mediated phagocytosis in GM-/- AMs. We will also determine if Hck of another SFK enhances FcTR-mediated phagocytosis in AMs by regulating either pseudopod extension, actin polymerization, or particle internalization.
These aims will clarify the mechanism by which GM-CSF regulates AM phagocytosis in vivo and will establish a regulatory link between GM-CSF, PU.1 and Hck/SFKs in AMs. Results of this research are expected to help establish the feasibility of the therapeutic use of GM-CSF in the treatment of lung infections by a wide variety of microbial pathogens. PERFORMANCE SITE ========================================Section End===========================================
| Carey, Brenna; Staudt, Margaret K; Bonaminio, Dana et al. (2007) PU.1 redirects adenovirus to lysosomes in alveolar macrophages, uncoupling internalization from infection. J Immunol 178:2440-7 |
| Uchida, Kanji; Beck, David C; Yamamoto, Takashi et al. (2007) GM-CSF autoantibodies and neutrophil dysfunction in pulmonary alveolar proteinosis. N Engl J Med 356:567-79 |
| Trapnell, Bruce C; Whitsett, Jeffrey A; Nakata, Koh (2003) Pulmonary alveolar proteinosis. N Engl J Med 349:2527-39 |
