The goal of this project is to define the cellular and molecular mechanisms that contribute to lung injury after marrow transplantation. Our overall hypothesis is that cellular and molecular events involved in the pathogenesis of lung injury are determined by elements of the transplant procedure that activate inflammatory and immune responses and promote pneumonitis. Even in the absence of overt pneumonitis, these mechanisms may prime the lung for an exaggerated response to subsequent lung injury. Our experimental approach primarily will involve investigation of a murine model of marrow transplantation with graft versus host disease (GVHD). The model provides an opportunity to examine the cellular and molecular evolution of lung injury while controlling components of the transplant procedure. Studies in patients with idiopathic (non-infectious) pneumonia will be undertaken to investigate the role of similar mechanisms in the clinical setting. We have elected to focus on the specific hypothesis that GVHD activates proinflammatory cytokine production that results in priming of alveolar macrophages. Subsequent challenge with lipopolysaccaride (LPS) results in exaggerated release of tumor necrosis factor (TNF)-alpha and (either directly or indirectly) increased production of mitogenic and fibrogenic cytokines (i.e transforming growth factor (TGF)-alpha and TGF-beta. These latter events may be potentiated by total body irradiation. Our plans to investigate this hypothesis are outlined in the following specific aims.
Aim A. Determine the components of marrow transplant procedure that promote lung injury in a murine model of marrow transplantation. The incidence and severity of pneumonitis will be compared among mice in which irradiation dose and donor T-cell dose are varied.
Aim B. Analyze the cellular and molecular evolution of pneumonitis in the murine model. The following parameters will be examined in specimens obtained at intervals after marrow transplantation: l. Interferon levels in lavage and serum; 2. LPS and lipopolysaccharide binding protein (LBP) in serum and lavage and CD14 receptor expression in lavage and alveolar macrophages; 3. TNF-alpha immunoreactivity in lavage and serum and steady state mRNA levels in lung tissue; and spontaneous and LPS-triggered release of TNF by cultured alveolar macrophages obtained at intervals after marrow transplantation; 4. TGF-alpha, TGF-beta, procollagen steady state mRNA levels and immunolocalization in lung tissue.
Aim C. Determine whether susceptibility to LPS-induced lung injury is modified by marrow transplantation. Animals will be transplanted with a radiation regimen and T-cell dose lower than that associated with pneumonitis (determined in Aim A). They will then be challenged with LPS and parameters of lung injury and cytokine expression will be analyzed.
Aim D. Analysis of cytokine levels in patients with idiopathic pneumonia. Understanding the components of the transplant procedure that contribute to pneumonitis as well as cellular and molecular mechanisms evoked by these events could constitute a scientific foundation for fixture modification of transplant procedures, interventions aimed at minimizing the risk for lung injury, and direct therapeutic measures for idiopathic pneumonia.
