Functional and Structural Characterization of Pulmonary Surfactant Fractions Obtained from Bronchoalveolar Lavages

Abstract

The alveolar surface of the lung is protected by a lipophilic material, the pulmonary surfactant, mainly composed by lipids and a few specific proteins: SP-A, SP-B, SP-C and SP-D. The main function of pulmonary surfactant is to reduce the surface tension of the thin aqueous layer covering the alveolar epithelium, avoiding the collapse of the smaller alveoli during the expiration. To achieve this function, surfactant lipids form an interfacial film at the air-liquid interface of this aqueous lining layer. Nevertheless, pulmonary surfactant is synthetized, packed and secreted by type II alveolar pneumocytes as tightly packed membranes assembled in specialized organelles, the lamellar bodies. Currently, the molecular mechanisms that let pulmonary surfactant be transformed from the bilayers of lamellar bodies into the functional interfacial film remain unclear. This is in part because most of the previous studies were made using surfactants obtained from bronchoalveolar lavages, a complex mixture that likely combines freshly secreted surfactant and surfactant that has been already exposed to respiratory dynamics and can be at least partially deactivated. In this work, we have characterized different fractions obtained by density gradient ultracentrifugation of pulmonary surfactant purified from bronchoalveolar lavages of porcine lungs. The analysis of these fractions by transmission electron microscopy showed a heterogeneous mixture of membranous structures. With regard to composition, each fraction showed important differences in cholesterol and the content of surfactant proteins. All fractions presented a relatively good surface activity when analyzed in the captive bubble surfactometer, except for some minority fraction. These results indicate that surfactant from bronchoalveolar lavage is composed by a heterogeneous mixture of membranes differing in composition and structure. We propose that some of these structures could be associated with different stages of surfactant dynamics, including freshly secreted material, interfacial structures and recycled membranes.