A multi-center clinical trial sponsored by Fujisawa Healthcare, Inc, was planned to compare the efficacy of treating lung transplant patients with tacrolimus and sirolimus versus tacrolimus and azathioprine in reducing the incidence of acute rejection during the first twelve months after lung transplantation. Infection is a secondary endpoint and is assessed throughout the trial (i.e. for 3 years after randomization). Presently the mechanisms by which these agents may modify lung innate immunity have not been identified. Herein, we are proposing to isolate SP-A and AMs from the bronchoalveolar lavage fluid (BALF) of patients participating in this clinical trial to identify differences in the ability of AMs to kill gram positive and gram-negative bacterial pathogens and to identify differences in quantity of SP-A and modifications thereof. These data will be correlated with incidences of infection and rejection in patients participating in the clinical trial. We are also proposing to identify basic mechanisms by which normal but not nitrated SP-A enhances phagocytosis. These goals will be accomplished by completing the set of measurements outlined in the following specific aims: (1) Measure levels of surfactant lipids and SP-A in bronchoalveolar lavage (BAL) samples from patients treated with tacrolimus and sirolimus vs. tacrolimus and azathioprine. Oxidative modification to SP-A (oxidation and nitration) will be assessed by Western blotting, ELISA and mass spectrometry analysis using techniques already established in our laboratory; (2) Quantitate levels of inflammatory cytokines (TNFa, INFgamma, IL-6 and IL-lb), as well as levels of nitrate and nitrite, the stable end products of NO metabolism, and nitrotyrosine in the BAL of these patients; (3) Assess the extent of pathogen killing (Klebsiella pneumoniae, a gram negative bacterium and Staphylococcus aureus, a gram positive bacterium) by AMs isolated from the lungs of these patients in the presence of SP-A and surfactant lipids, and (4) Identify putative mechanisms responsible for decreased ability of oxidized or nitrated SP-A to mediate pathogen killing by AMs. We propose that SP-A binding to AM receptors leads to activation of phospholipase C (PLC) which releases 1,4,5 inositol triphosphate (IP3) resulting in Ca+2 release from the endoplasmic reticulum. SP-A nitration may lead to decreased binding, diminishing or abrogating intracellular Ca+2 changes. Our data may provide mechanistic insight into why some patients may develop clinical infection and acute and chronic rejection and thereby form the rationale basis for choosing between these two immunosuppressive regiments for the management of patients with lung transplantation.
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