Caveolae and caveolin isoforms in rat peritoneal macrophages

Caveolea are special (highly hydrophobic) plasma membrane invaginations with a diameter of 50-100 nm. Their characteristic features are the flask- or omega-shape and the lack of basket-like coat composed of clathrin. Caveolin-an integral membrane protein-is the principal component of caveolae membranes in vivo. Multiple forms of caveolin have been identified: caveolin-1alpha, caveolin-1beta, caveolin-2 and caveolin-3. They differ in their specific properties and tissue distribution. In this paper we summarize the morphological and biochemical data providing strong evidence about the existence and function of caveolae in rat peritoneal macrophages. When studied electron microscopically, the surface of both resident and elicited macrophages exhibited omega- or flask-shaped plasma membrane invaginations. There was a significant difference, however, in the number of these profiles: whereas in resident cells only a small amount of them was found on the cell surface, in elicited cells they were abundantly present on the plasma membrane. Using an antibody against the VIP21/caveolin-1 isoform we showed that these plasma membrane pits were indeed caveolae. The number and the appearance of caveolae were found to be in close correlation with the functional activity of these phagocytotic cells, indicating that the formation of caveolae is a highly regulated process. Using Western blot analysis two different proteins ( approximately 29 and approximately 20 kDa)-both labelled with anti-caveolin antibodies-were identified in resident and elicited macrophages that have been isolated from rat peritoneal cavity. The approximately 20 kDa protein was labelled specifically only by anti-VIP21/caveolin-1, while the approximately 29 kDa protein was labelled by both anti-VIP21/caveolin-1 and anti-caveolin-2 antibodies. The presence of the approximately 29 kDa protein was highly characteristic of resident cells, and only a small amount of approximately 20 kDa protein was detected in these cells. Elicitation has resulted in a significant increase in the amount of approximately 20 kDa protein labeled only with anit-VIP21/caveolin-1. Our morphological (confocal and electron microscopical) studies have shown that in resident cells caveolin was present in the cytoplasm, in smaller vesicles and multivesicular bodies around the Golgi area. Only a very small amount of caveolae was found on the cell surface of these cells. In elicited macrophages, caveolae (labelled with anti-VIP21/caveolin-1 antibody) appeared in large numbers on the cell surface, but caveolin detected by anti-caveolin-2 was also found in small vesicles and multivesicular bodies. These data support the idea that the expression of the approximately 29 kDa (caveolin-related) protein is insufficient for caveolae formation in resident cells, it can function as a modified, macrophage-specific caveolin-2 isoform. Our results strongly suggest that caveolin-1 plays a crucial role in the formation of caveolae: it is the amount of caveolin-1 that regulates the appearance of caveolae on the plasma membrane. Studying the endocytotic processes of resident and elicited macrophages we have found that elicited macrophages bound and internalized significantly larger amounts of fluid phase marker (HRP) and immune complex (peroxidase-antiperoxidase-PAP) than resident cells. Serial section analysis, double labelled immunocytochemistry, and filipin treatment were used to demonstrate that caveolae can pinch off from the plasma membrane and can take part in endocytotic processes as alternative carriers in elicited macrophages.