In this project, we seek to discern the mechanism by which GM-CSF regulates alveolar macrophage (AM) Fc?R-mediated phagocytosis, a function vital to lung host defense. Based on our published study (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 Fc?R?, Hck and possibly other downstream targets critical for effective Fc?R?-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 Fc?R?-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 Fc?R?-mediated phagocytosis including Fc?R? and Hck but not Fc?R 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 Fc?R-mediated phagocytosis. To answer these questions, we will utilize existing murine transgenic models wherein GM-CSF expression is normal (GM+/+), absent (GM-/-), or over expressed 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 Fc?R-mediated phagocytosis (evaluated ex-vivo) and AM terminal differentiation; 2) if GMCSF, in vivo, provides """"""""rheostatic"""""""" or """"""""on-off"""""""" control of AM Fc?R-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) re-implanted into the lungs of GM-/- mice prevents PAP and restores normal lung defense; and 2) if retroviral replacement of Fc?RIII? rescues Fc?R-mediated phagocytosis in mAM cells in vitro.
In Aim 3, we will use retroviral transduction of mAM ceils to express Fc?RIII? and/or individual Src-family kinases (SFKs) to establish which family members can effectively rescue wild type levels of Fc?R-mediated phagocytosis in GM-/- AMs. We will also determine if Hck of another SFK enhances Fc?R-mediated phagocytosis in AMs by regulating 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.
| 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 |
