Macrophage-epithelial crosstalk during alveolar epithelial repair following pathogen-induced pulmonary inflammation

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CakarovaLidija_2009_07_10.pdf (3.47 MB)

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2009

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DOI:
http://dx.doi.org/10.22029/jlupub-13447

Abstract

Bacterial invasion of the alveolar air space is followed by the fast, tightly regulated immune response facilitating a successful pathogen clearance. Upon pathogen recognition activated resident alveolar macrophages (AMf;) early release pro-inflammatory cytokines, stimulating neighbouring alveolar cells to produce chemokines which in turn mediate the infiltration of neutrophils, exudate macrophages and lymphocytes. The following inflammatory reaction and the pathogen itself leave a damaged alveolar barrier associated with pulmonary oedema and impaired gas exchange. Consequently, epithelial repair processes are initiated to restore the normal lung homeostasis. During the later phase of infection AMf; have been shown to acquire an anti-inflammatory phenotype thereby enhancing alveolar repair processes. However, the potential of early activated, pro-inflammatory AMf to influence epithelial repair remains largely elusive. Therefore, in the present thesis it was investigated whether activated AMf contribute to alveolar epithelial repair upon LPS challenge in vitro and in vivo, as well as in K. pneumoniae pneumonia, and the molecular interaction pathways involved were analysed. The cross-talk between resident alveolar macrophages and alveolar epithelial cells during alveolar repair was assessed in an in vitro co-culture system and an in vivo model of LPS-induced acute lung injury. Gene expression and protein analysis showed that LPS-activated alveolar macrophages stimulated alveolar epithelial cells (AEC) to express growth factors, particularly GM-CSF upon co-culture. Antibody neutralization experiments revealed epithelial GM-CSF expression to be macrophage TNF-alpha dependent. GM-CSF elicited proliferative signalling in alveolar epithelial cells via autocrine activation of the transcription factor STAT 5 and Cyclin D1 expression. Notably, macrophage TNF-alphab induced epithelial proliferation in wild-type but not in GM-CSF-deficient alveolar epithelial cells as shown by [3H]-thymidine incorporation and cell counting. Matrigel:collagen AEC culture preserving the type II phenotype in vitro supported the concept that the proliferative response to GM-CSF is related to the type II AEC phenotype. Moreover, intra-alveolar TNF-alpha neutralization impaired alveolar epithelial type II cell proliferation in LPS-injured mice in vivo, as investigated by flow cytometric Ki67 and immunofluorescence staining of lung sections. Additionally, GM-CSF-deficient mice displayed reduced AEC II proliferation and sustained alveolar barrier dysfunction upon LPS treatment compared to wild-type and SPC-GM mice (overexpressing GM-CSF in AEC II in a GM-CSF-deficient background). Similarly, K. pneumoniae lung infection confirmed the findings in the LPS-model and resulted in early release of macrophage TNF-alpha and epithelial GM-CSF, as well as subsequent TNF-alpha-dependent AEC II proliferation during alveolar repair events.Collectively, these findings indicate that TNF-alpha released from activated resident alveolar macrophages induces epithelial GM-CSF expression, which in turn initiates alveolar epithelial type II cell proliferation and thus contributes to restore alveolar barrier function.

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