Late Breaking Abstract - Identification and functional testing of a novel receptor for SP-A on eosinophils

Abstract

Background: Surfactant protein A (SP-A) is well known for its protective role in pulmonary immunity. Studies from our lab show that mice deficient in SP-A have delayed clearance of eosinophils in the bronchoalveolar lavage after experimental ovalbumin challenge as compared to WT mice. Here, we wanted to investigate the mechanism by which SP-A induces eosinophil apoptosis. Methods: Purified eosinophils from asthmatic volunteers were incubated with SP-A for 16 hours. Induction of eosinophil apoptosis and cell death was measured by quantifying caspase-3 cleavage using Western blotting and Annexin V/Propidium Iodide (PI) staining using flow cytometry. To identify potential binding partners for SP-A, eosinophil lysates were subjected to SP-A pull-down and tandem mass spectrometry (MS/MS) analysis. The pro-apoptotic potential of the identified candidate SP-A membrane receptor was then tested by using a specific antibody to block protein binding capacity prior to direct incubation with SP-A, and subsequently analyzed by flow cytometry. Results: Direct incubation of eosinophils with SP-A resulted in increased cleavage of caspase-3 compared to vehicle control. The percentage of apoptotic eosinophils (Annexin V+, PI-) was also significantly increased upon SP-A treatment. Tandem mass spectrometry identified one membrane-bound protein, myeloid-associated differentiation marker (MYADM), as a candidate SP-A binding partner. Pre-treatment with an antibody to MYADM diminished SP-A-induced eosinophil apoptosis compared to those treated with SP-A only. Conclusions: Our results have identified a novel binding partner for SP-A on eosinophils, MYADM, through which we believe SP-A promotes eosinophil apoptosis.