Although M cells frequently have distinctive morphologic phenotypes, the only unequivocal criterion for an M cell is demonstration of transcytosis by an epithelial cell within the FAE.
8 The present study shows selective transcytosis of latex particles by M cells in the rabbit conjunctiva and for the first time demonstrates subsequent rapid transport of the transcytosed material to local lymph nodes.
The ability of the guinea pig conjunctiva to take up latex beads was originally explored with TEM by Latkovic and Nilsson.
18 19 After a 24-hour in vivo exposure during which the beads were replenished every 8 hours, the investigators reported beads were present in the epithelial layer throughout the conjunctiva but did not describe any in the underlying substantia propria. This earlier study was designed simply to demonstrate the phagocytic capacity of conjunctival epithelium, and no attempt was made to distinguish between phagocytosis and transcytosis. Furthermore, the level of uptake and differences between regions were not examined in this earlier work. The results of the present study, demonstrating rapid exit of latex beads once they have been transcytosed across the epithelial barrier, make it difficult to compare our findings with the older studies that used repeated exposures over 24 hours.
19
Latex beads have been widely used as a functional marker of M cells in other locations. The original observation that polystyrene latex beads selectively bind to M cells was made in the rabbit Peyer’s patch.
10 Subsequent studies have shown selective binding and bead translocation by M cells in the rabbit nasal mucosa,
11 rat Peyer’s patches,
13 mouse Peyer’s patches,
14 and a human in vitro M cell model.
20 The reason that latex microspheres preferentially associate with and are internalized by M cells is still somewhat unclear but is likely because of their physical properties, as well as the unique apical surface of the M cell.
21 The efficiency of microsphere internalization can be optimized by selecting the appropriate size, charge, and hydrophobicity.
22 23 Interest in the interactions between microspheres and M cells is not simply restricted to identifying cells that translocate particles. The use of antigen-loaded or coated microspheres as a means to stimulate mucosal immunity or to deliver drugs is currently being studied by several groups.
24 25 26
The origin of the “M cell” name came from the initial descriptions of these cells in the human intestine, where their apical membranes were covered with irregular microfolds rather than the more typical homogeneous brush border of microvilli on neighboring enterocytes.
27 Subsequent studies showed that M cells in other locations frequently had microvilli, therefore, the name has evolved to refer to the thin “membranous” rim of cytoplasm that separates the external environment from the underlying lymphoid cells. The presence or the absence of microvilli and their relative length cannot, therefore, be used as a reliable marker for the identification of M cells. It is safe to say that, consistent with their unique function, the apical membrane specializations of M cells are generally different from the surrounding cells. M cells in the rabbit Peyer’s patch have shorter microvilli than the neighboring enterocytes.
28 In contrast, M cells in the conjunctiva, like their counterparts in the rabbit tonsils and cecum, have irregular, frequently branching, microvilli that are longer than those found on surrounding cells.
29 30 31 32
Although M cells are classically found in the simple columnar epithelium of the intestines, their presence in the stratified epithelium of the conjunctiva is similar to their occurrence in the stratified squamous epithelium of the rabbit’s palatine tonsil.
29 As in the rabbit tonsil, cecum, appendix, and sacculus rotondus, M cells in the conjunctiva are predominately on the lateral flanks of the follicle.
17 21 31 33
Although we consistently saw surface binding of latex beads to a distinctive subpopulation of FAE after in vitro incubation at 4°C, we only saw rare examples of beads binding to the FAE surface after in vivo exposure. This suggests the beads are rapidly internalized soon after they bind. The absence of any additional bead binding once the first wave of beads had been internalized is in agreement with the original report of bead uptake by M cells in the rabbit Peyer’s patch.
10 Similarly, M cells in the mouse Peyer’s patch do not show surface binding at the same time they are transporting beads.
14
The rapid exit of latex beads from the conjunctiva and transport to cervical lymph nodes is similar to what has previously been observed in studies of the rat intestinal Peyer’s patches, where latex beads can be detected in the mesenteric lymph within 5 to 10 minutes of instillation into the intestinal lumen.
34
Vimentin has previously been shown to selectively label M cells in the FAE of Peyer’s patch, appendix, cecum, tonsil, and bronchus of the rabbit but not in other species.
16 17 35 36 Consistent with our evidence for transcytosis by M cells in the rabbit conjunctiva, vimentin positive cells were found in ring-like distributions along the peripheral flanks of the follicles in acetone-fixed whole mounts. In cryostat cross-sections of aldehyde-fixed conjunctiva exposed in vivo to latex beads, vimentin immunolabeling coincided with latex bead distribution in the FAE. Although the functional significance of vimentin expression in M cells has not been established, the presence of this rabbit M cell marker is further evidence that the conjunctival FAE has M cells equivalent to those found in other mucosal locations.
Mucosa associated lymphoid tissue (MALT) has been well established in the gastrointestinal, respiratory, and genital tract, and now there is growing evidence for the presence of MALT at the normal ocular surface.
2 3 37 Ocular MALT has been more controversial because of an absence of evidence of the specialized antigen sampling cells in this tissue.
38 39 A key question has been which animal species would be the best model to address this issue. Although conjunctival lymphoid follicles are present in humans and a wide range of mammals,
1 2 3 they are not found in mice and rats.
2 The inability to use these two common laboratory species led us to examine guinea pigs
9 and rabbits as models more closely resembling the human conjunctiva. Guinea pigs offer the advantage that they have been widely used in modeling ocular allergies. This will allow future studies to investigate the impact of allergic reactions and pharmacological treatments on conjunctival M cells, as well as testing whether M-cell-directed immunization can trigger IgA-mediated immune responses that can ameliorate IgE-mediated allergic reactions. Rabbits, on the other hand, have a better characterized immune system, and antibodies are available against CD markers on inflammatory cells, which will allow for characterization of these cell types in the mucosal immune response. Furthermore, because it is possible to make class-specific rabbit hybridomas,
40 future studies will be able to address whether IgA promotes antigen uptake by conjunctival M cells as previously described for M cells in the mouse intestine.
41 Another advantage of the rabbit is the availability of vimentin immunostaining as an M cell marker, which allowed for an additional confirmatory test for these cells in the conjunctiva.
Despite their beneficial role in initiating the mucosal immune response, M cells in other mucosal locations have also repeatedly been shown to be an entry point for opportunistic bacterial and viral pathogens.
4 5 6 It will be essential for future studies to address whether conjunctival M cells can be a similar entry point for pathogens. Furthermore, an intriguing hypothesis linking conjunctival and tear-duct-associated lymphoid tissue in the pathogenesis of dry eye has been proposed.
42 The identification of animal models with functional conjunctival M cells opens the possibility of testing this hypothesis by targeting antigens to these cells to determine whether the inappropriate stimulation of the underlying lymphoid tissue can trigger onset or exacerbation of dry eye.
The mechanism by which antigens are taken up by the conjunctiva has been elusive until a recently published study showed that the M cells selectively bind and transcytose a sialyllactose-binding lectin in the guinea pig conjunctiva.
9 The present study provides further evidence for a functional ocular MALT by demonstrating that the rabbit lymphoepithelium contains M cells with the ability to translocate particulates and, for the first time, demonstrates subsequent tracer transport to local lymph nodes. The rabbit is, therefore, a useful model to study the role of mucosal immunity in the maintenance of the delicate epithelial barrier of the ocular surface.