INTRACELLULAR-DISTRIBUTION OF LYSOZYME IN RAT ALVEOLAR TYPE-II EPITHELIAL-CELLS

This study investigated the intracellular distribution of lysozyme, a protein that is synthesized and secreted by rat alveolar type II epith elial (ATII) cells and alveolar macrophages, using a polyclonal antibo dy generated against purified rat lysozyme. Lysozyme was immunoprecipi tated with this antibody from Triton X-100 lysates of ATII cells cultu red on a basement membrane derived from Englebreth-Holme-Swarm mouse s arcoma (EHS) and radiolabeled with S-35- methionine. ATII cells cultur ed on EHS basement membrane for several days were fixed and labeled wi th antibodies to surfactant apoprotein A (SP-A) and lgp-120 (a lysosom al glycoprotein), or lysozyme and lgp-120, and studied by confocal mic roscopy. Organelles were identified that stained positively for either anti-lysozyme or anti-lgp-120; a second population of organelles cont ained both markers. Similarly, two populations of SP-A-contaaining org anelles were identified; one contained the lysosomal glycoprotein lgp- 120. In addition, confocal images demonstrated that both SP-A and lyso zyme were secreted by ATII cells, as evidenced by the accumulation of secretory Products within the lumen of the cyst-like aggregates. When the subcellular localization of SP-A and lysozyme was studied by analy tical cell fractionation, two populations of organelles were identifie d that contained SP-A or lysozyme. The lighter population accounted fo r approximately 32% of SP-A and 33% of total intracellular lysozyme an d was recovered in the same region of the gradient as secretory lamell ar bodies. The more dense population co-localized with lysosomes and a ccounted for approximately 67% of both SP-A and lysozyme recovered. We stern blots of cell fractions revealed intact lysozyme in all the cell fractions. The results of these experiments suggest that lysozyme has a similar intracellular distribution as surfactant apoprotein A in AT II cells. Lysozyme is found in fractions containing lamellar bodies wh ere it is packaged for secretion, and in lysosomal fractions where it may undergo degradation.