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In the intestine, lymphoid nodules, individually or aggregated into Peyer’s patches, are sites where antigen recognition begins and mucosal immune responses are initiated. M cells in the epithelium covering these lymphoid nodules are specially differentiated to take up and transport antigenic macromolecules and microorganisms from the lumen across the epithelial barrier, that otherwise restricts potential systemic pathogens to the lumen and prevents unregulated migration into tissues. M cells, enterocytes, goblet cells, and other epithelial cells lining the intestine and other mucosal surfaces are end stage cells, replaced from stem cells in an unending flow, that allows cells, physiologically infected by luminal microorganisms after emerging from the mouths of crypts, to be continually replaced by fresh recruits. Since M cells were first identified in humans in 1974,1 a major limitation and challenge for investigators has been their inability to grow M cells in culture under controlled conditions where regulation of growth, development, and function could be delineated.
Lack of methods for in vitro investigation of lymphoid follicle epithelium, except in short term organ culture, has required deductive approaches to understanding factors which regulate M cell differentiation. Bye et al found that M cells already display detectable differences in enzymatic, cytoskeletal, and surface characteristics upon emerging from crypt mouths.2Remarkably, cells streaming from mouths of crypts surrounding intestinal lymphoid nodules show enterocyte characteristics when they flow from the sides of crypt walls next to villi, but on the opposite side of the same crypts, M cells originate from stem cells abutting lymphoid follicles. This observation generated the hypothesis that lymphocytes or their intercellular mediators provide stimuli directing differentiation of intestinal epithelial stem cells.
A series of refreshingly direct yet technologically sophisticated in vitro and in vivo studies by Kernéis et al have provided new insights into the roles of intestinal lymphoid cells in the regulation of M cell differentiation and maturation. The hypothesis that lymphocytes or their products direct differentiation of intestinal crypt stem cells was tested by injecting Peyer’s patch lymphoid cells beneath the epithelium of non-patch areas of mouse and rabbit intestine. Mucosal lymphoid nodules were generated with typical follicle associated epithelium containing functional M cells. Thus, the controlling stimuli for generation of intestinal lymphoid follicles seem to be factors produced by lymphoid cells aggregated in particular loci by homing receptors or other factors within intestinal vasculature rather than a genetically defined distribution of stem cells uniquely able to generate M cells.
Kernéis et al also developed an in vitro system for generating M cells from cultured human Caco-2 enterocyte cell lines, using dispersed murine Peyer’s patch B and T lymphocytes lacking detectable macrophages or dendritic cells. When these lymphocytes were introduced into chambers separated by filters from confluent sheets of cultured enterocytes, they migrated into spaces between these cells, forming interepithelial pockets in enterocytes with disordered apical microvilli, characteristic of M cells. Alterations in brush border hydrolases typical of follicle epithelium also occurred in enterocytes without obvious lymphocyte contact, implicating soluble factors as well as direct contact in enterocyte differentiation. Interestingly, dissociated thymocytes did not induce formation of new mucosal lymphoid nodules.
Does this mean that differentiated enterocytes in vivo can undergo a mid-life career change into M cells? Not necessarily. Although each mammalian cell contains genetic code with the potential for expressing all the possibilities of any cell in the body, the lack of terminal differentiation which allows Caco-2 cells to replicate in culture more closely resembles that of crypt stem cells than of mature enterocytes. Identifying factors responsible for initiating and suppressing expression of intestinal stem cell differentiation genes in this system may also give clues to regulation of renewal of enterocytes in inflammatory conditions with increased numbers of interepithelial lymphocytes.
The ability of mouse cells to induce differentiation in human enterocytes suggests that both the inductive lymphocyte factors and corresponding epithelial receptors must be important enough to be conserved across species lines. Interestingly, murine lymphocyte stimulation of cultured human enterocytes induced not only structural features characteristic of M cells but also the ability to take up and transport Vibrio cholerae, which are non-invasive.
These studies point the way to future investigations of specific lymphocyte factors which induce M cell morphological and functional differentiation, information which might be exploited to increase uptake and transport at the time of introduction of mucosal vaccines or to retard and block uptake during critical periods after administration of irradiation or radiomimetic immunosuppresive drugs, when patients are otherwise susceptible to systemic infection by endogenous intestinal organisms.
The epithelium that lines the gut is impermeable to macromolecules and microorganisms, except in Peyer’s patches (PP), where the lymphoid follicle-associated epithelium (FAE) contains M cells that transport antigens and microorganisms. A cultured system that reproduces the main characteristics of FAE and M cells was established by cultivation of PP lymphocytes with the differentiated human intestinal cell line Caco-2. Lymphocytes settled into the epithelial monolayer, inducing reorganization of the brush border and a temperature-dependent transport of particles and Vibrio cholerae. This model system could prove useful for intestinal physiology, vaccine research, and drug delivery studies.