Lectin cytochemistry and freeze-fracture study of phagocytosis in the rat retina.

Lectin cytochemistry and freeze-fracture intramembrane morphology have been studied in adult rat retinas during various stages of the phagocytic cycle. Animals were perfused with aldehydes at various times during the first 6 hours of light and the retinas were removed. Chopped sections of tissue were stained with one of two lectin-peroxidase conjugates: ricinis communis agglutinin (RCA), which is specific for n-acetylgalactosamine and galactose, or wheat germ agglutinin (WGA), which is specific for n-acetylglucosamine and sialic acid. Other sections were frozen and prepared for freeze fracture. During the first hour of light onset, numerous phagosomes are already present in the retinal pigment epithelial (RPE) cytoplasm. The engulfment stages of phagocytosis whereby the RPE microvilli wrap around and enclose shed rod outer segment (ROS) tips are rarely observed at this time. Newly formed phagosomes are present in the apical RPE cytoplasm while older and more osmiophilic phagosomes are located deeper within the RPE cytoplasm. Between 2 and 4 hours of light onset fewer phagosomes are present in the RPE cytoplasm, but it is common to observe many ROS tips in various stages of microvillous engulfment. By 6 hours phagosomes are rarely present but some engulfed ROS tips continue to be observed. Both RCA and WGA stain RPE microvilli and plasma membranes of photoreceptor outer segments. WGA, in addition, labels some intradisc membranes of intact and also shed outer segments that are being engulfed but does not label disc membranes in phagosomes. At all times, phagosomes are rimmed by both lectin stains, but their internal contents are never stained. Lysosomes and multivesicular and residual bodies are labeled by both lectins. Freeze-fractured retinas show that as the ROS tips are engulfed, particle-free areas are present on disc P-face (cytoplasmic) membranes. Many newly formed phagosomes also display particle-free membrane areas and older phagosome membranes are completely devoid of particles. These observations show that the changes in WGA staining and intramembrane particle distribution on disc membranes occur in retinas during phagocytosis and suggest that some membrane component that is present for the initial phagocytic interaction may be rapidly degraded thereafter.