A new procedure for assaying the phagocytosis of isolated rat rod outer segments (ROS) by cultured rat pigment epithelial (PE) cells has been developed. Utilizing an ROS antiserum and the technique of indirect immunofluorescence, ROS which are attached to the external surfaces of the PE cells can be distinguished from those which have already been ingested by the cells. With this assay procedure, large numbers of PE cells can be visualized for study. Most importantly, the procedure makes it possible to separate and to quantitate both ROS attachment and ingestion. This makes it feasible to study the effects of a variety of metabolites on the recognition and attachment phases of phagocytosis, as well as on the ingestion phase. / The Royal College of Surgeons (RCS) strain of rat has been extensively studied in recent years because it has an hereditary retinal dystrophy in which there is a defect in the phagocytosis of shed ROS material by the PE cells. Numerous in vivo and in vitro studies have been unable to localize the step in the phagocytic process which is affected by the genetic mutation. Using cultured, dystrophic PE cells, and the assay procedure described above, it was possible to show that the recognition and attachment phases of phagocytosis are normal in these dystrophic cells. However, the ingestion phase is deficient. After 4 hours of incubation, normal rat PE cells ingested about 80% of the ROS which had attached to the cells. In contrast, after this incubation period, less than 20% of the ROS which had bound to the dystrophic rat PE cells had been ingested. / Since actin is the main component of the microfilaments which surround an attached particle during its ingestion by a phagocytic cell, it seemed likely that actin might be the defective component in the ingestion process for dystrophic PE cells. Utilizing actin antibodies and the technique of indirect immunofluorescence, normal PE cells were shown to have an extensive network of actin filaments. These filament arrangements did not change when the PE cells were challenged with isolated ROS. This was true whether the ROS were externally bound, in the process of being ingested, or completely internalized. However, a localized concentration of actin was seen to take place at many sites of ROS attachment and ingestion. The arrangement of actin filaments in the dystrophic PE cells appeared normal. Additionally, actin was seen to accumulate at the few sites of ROS ingestion. Thus it appears that actin, a contractile protein important for the ingestion phase of phagocytosis, functions normally in the dystrophic rat PE cell. However, the ingestion phase of phagocytosis becomes activated at very few sites of ROS attachment. / The failure of ingestion to take place at all but a few sites of ROS attachment in the dystrophic PE cells raises the possibility that these cells are deficient in those plasma membrane receptor sites which normally mediate ROS ingestion. However, the phagocytic defect in these PE cells may be due to a failure of actin to accumulate and/or form the microfilaments necessary for pseudopod extension. If this is found to be true, then the defect may be in one of the proteins which regulates the intracellular pool of actin, or in the signal necessary to cause actin accumulation and filament formation at sites of ROS attachment. / Source: Dissertation Abstracts International, Volume: 42-01, Section: B, page: 0034. / Thesis (Ph.D.)--The Florida State University, 1981.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_74405 |
| Contributors | CHAITIN, MICHAEL HOWARD., Florida State University |
| Source Sets | Florida State University |
| Detected Language | English |
| Type | Text |
| Format | 206 p. |
| Rights | On campus use only. |
| Relation | Dissertation Abstracts International |
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