Abstract
Abstract: :
Purpose: Epithelial M cells overlying mucosal lymphoid follicles play a key role in initiating the production of secretory IgA and have also been found to be the site of entry for opportunistic pathogens such as HIV and Shigella. In the rabbit, but not other species, M cells in the intestinal Peyer’s patches, sacculus rotundus, cecum, appendix, palatine tonsil, and bronchus can be specifically labeled with antibodies against the intermediate filament protein vimentin. In addition, M cells in rabbit Peyer’s patches have been found to preferentially bind and translocate polystyrene microspheres. We hypothesized that if the follicle associated epithelium of the rabbit conjunctiva contains M cells, they should show similar vimentin reactivity and preferentially bind and translocate microspheres. Methods: Following induction of anesthesia, the eyelids were sutured together and 300 µl of 1 x 1010/ml 0.5 µm fluorescent polystyrene microspheres in PBS were deposited into the conjunctival sac for 10–30 min (n = 6). After exenteration of the orbit, the conjunctiva was separated from the adnexal tissues and carefully washed in PBS prior to aldehyde fixation. Vimentin staining was performed on either acetone–fixed or aldehyde–fixed and Triton X–100 permeabilized wholemounts using gold–enhancement of colloidal gold conjugated secondary antibodies (n = 5). The wholemounts were then visualized with a combination of stereomicroscopy, confocal microscopy, and scanning electron microscopy. Additional nodules were cryo–sectioned and examined by wide–field and confocal microscopy. Results: The fornix region of the rabbit conjunctiva has numerous, well–developed lymphoid follicles. When these nodules are stained with anti–vimentin antibodies, rings of vimentin positive cells on the surface of the nodule can be seen by stereomicroscopy. A 10–30 min in vivo exposure to 0.5 µm fluorescent polystyrene microspheres results in selective, heavy labeling of the nodular regions. Within 30 min of exposure, the beads had been translocated across the epithelial layer and were present in the underlying nodule region. Double labeling experiments using confocal microscopy demonstrate microsphere binding and translocation overlaps with vimentin labeling. SEM studies are underway. Conclusions: Positive vimentin immunoreactivity and the ability to bind and translocate polystyrene microspheres is strong evidence for the presence of M cells in the rabbit conjunctiva.
Keywords: conjunctiva • microscopy: light/fluorescence/immunohistochemistry • microscopy: electron microscopy