Immunology 2004 113 15–22
doi:10.1111/j.1365-2567.2004.01964.x
REVIEW ARTICLE
Improving M cell mediated transport across mucosal barriers: do certain bacteria
hold the keys?
ANGELA L. MAN, MARIA ELENA PRIETO-GARCIA & CLAUDIO NICOLETTI Laboratory of Gut Immunology,
Programme of Gastrointestinal Health and Function, Institute of Food Research, Norwich, UK
SUMMARY
Specialized microfold (M) cells of the follicle-associated epithelium (FAE) of the mucosalassociated lymphoid tissue (MALT) in gut and the respiratory system play an important role in
the genesis of both mucosal and systemic immune responses by delivering antigenic substrate to
the underlying lymphoid tissue where immune responses start. Although it has been shown that
dendritic cells (DC) also have the ability to sample antigens directly from the gut lumen, M cells
certainly remain the most important antigen-sampling cell to be investigated in order to devise
novel methods to improve mucosal delivery of biologically active compounds. Recently, novel
information on the interactions between bacteria and FAE have come to light that unveil further
the complex cross-talk taking place at mucosal interfaces between bacteria, epithelial cells and
the immune system and which are central to the formation and function of M cells. In particular,
it has been shown that M cell mediated transport of antigen across the FAE is improved rapidly
by exposure to certain bacteria, thus opening the way to identify new means to achieve a more
effective mucosal delivery. Here, these novel findings and their potential in mucosal immunity
are analysed and discussed, and new approaches to improve antigen delivery to the mucosal
immune system are also proposed.
Keywords antigen delivery, M cells, mucosal-associated lymphoid tissue (MALT),
mucosal immunity
hydrolytic enzymes.7 These features, along with the closely
packed carpet of microvilli on absorptive cells, prevent
contact and binding of macromolecules and potential
pathogens to the epithelium. However, the mucosal epithelium also has to provide channels through which antigens and microorganisms are delivered to the intestinal
immune system in order to induce immune responses. Indeed, antigenic penetration of epithelial barriers is the first
step required to mount effective mucosal and systemic immune responses and M cells play a key role in this by
regulating access of microorganisms and antigens to areas
of the MALT equipped to generate protective responses.8,9
The importance of finding the best way to induce robust
mucosal responses is highlighted by the accepted view that
the systemic immune system is not adequate to fight the
vast majority of pathogens to which we are continuously
exposed. It is believed that over 95% of human pathogenic
microorganisms target host cells after crossing epithelial
barriers and in many cases the main protective effector
function against these mucosal infections is the production
INTRODUCTION
The epithelia overlying the mucosal surfaces of the respiratory and gastrointestinal tract are exposed throughout
their life to a limitless variety of antigens; their main task
is to provide an efficient barrier against pathogens and
macromolecules.1,2 This is achieved by several means. First,
the epithelium consists of columnar cells joined by tight
junctions that allow passage of water and ions but provide
a mechanical barrier to macromolecules. Secondly, mucosal
surfaces are covered by local secretions of mucus, secretory
IgA antibodies and by a thick glycocalx.3–6 The latter
represents a highly degradative environment due to
the presence of membrane-bound and layer-entrapped
Received 26 May 2004; revised 7 July 2004; accepted 14 July
2004
Correspondence: Dr Claudio Nicoletti, Laboratory of Gut
Immunology, Institute of Food Research, Colney, Norwich
NR4 7UA, UK. E-mail: claudio.nicoletti@bbsrc.ac.uk
2004 Blackwell Publishing Ltd
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A. L. Man et al.
of pathogen-specific local secretory IgA antibody response
that can be achieved solely via activation of the mucosal
immune system.10 This notion, along with the fact that in
adult individuals the area of mucosal surfaces reaches the
impressive size of 400 m,2,11 makes it imperative that we
acquire a better understanding of the mechanism involved
in transepithelial transport of antigens in order to design
new strategies to improve oral delivery of vaccines and
therapeutics.
Although villous-M cells located outside the follicleassociated epithelium (FAE) have been observed recently,12
the transport of antigens and microorganisms across the
intestinal mucosa is carried out mainly by the FAE13,14 that
overlies the gut associated lymphoid tissue (GALT)15
(Fig. 1). A number of features distinguish the FAE from the
absorptive epithelium. There are no receptors for polymeric
immunoglobulin, which suggests that these epithelia do not
secrete IgA.16,17 Few mucus-producing, defensin- and lysosyme-producing cells are seen in the follicle-associated
crypts18 and the production of membrane-associated
digestive hydrolases is reduced significantly.19 However, the
most remarkable feature of the FAE is the presence of
highly specialized antigen-sampling membranous or microfold (M) cells.20 M cells are not restricted to the gut but are
also present within the upper respiratory tract, and particular attention is being given to M cells located within the
nasal-associated lymphoid tissue (NALT).21 The importance of the NALT as a delivery route of great potential to
induce strong mucosal immune responses is becoming
increasingly evident and various groups have attempted to
produce appropriate vaccine preparations to target specifically the local M cell population.22,23
M CELL MEDIATED TRANSPORT ACROSS
MUCOSAL BARRIERS
Sampling the gut: M cell versus dendritic cell
It has been shown recently that also dendritic cells (DC)
sample epithelial cell antigens24 and bacteria25 from the gut
lumen (Fig. 2). DC in the gut are located beneath the FAE
of the Peyer’s patch26,27 and all along the whole intestinal
epithelium within the lamina propria.28 In vitro experiments
have shown that DC can open the tight junctions between
intestinal epithelial cells and send their cellular processes
into the lumen where they directly internalize bacteria.25
The expression of tight junction proteins, such as
Claudin-1, Occludin and Zonula Occludens-1, enables DC
to maintain the integrity of the epithelial barrier while
sending their dendrites into the gut lumen. This mechanism
has been demonstrated clearly in vitro, although at this time
its real relevance in vivo remains to be determined fully.
Indeed, in vivo evidence that DC extend their cellular
processes between epithelial cells, take-up bacteria and then
migrate back within the tissue to present antigens to naive
Ag
L
E
E
M
E
E
Ly
DC
Figure 1. Scanning electron micrograph of an individual follicle of
a mouse Peyer’s patch. The follicle is embedded among villi and
strategically protrudes into the gut lumen (L) to sample antigens
via M cells or dendritic cells.
Figure 2. Sampling the gut. M cells internalize luminal antigens
and microorganisms (Ag) and deliver them to the underlying
lymphoid tissue. The first contact between immune cells (Ly) and
antigen can take place within M cell cytoplasmic pockets that
represent a docking site for a variety of lymphocyte subsets. In
addition, dendritic cells (DC) also sample antigens directly by
extending their dendrites between epithelial cells (E), process them
and finally present them to naive T cells in the context of MHC
class II molecules.
2004 Blackwell Publishing Ltd, Immunology, 113, 15–22
M cell in transepithelial transport
17
T cells is currently lacking. Experiments carried out
in vivo25 using isolated intestinal loops showed an increase
in the number of DC recruited in the intestinal tissue briefly
after the application of the bacterial stimulus, and subsequently DC dendrites can be seen protruding into the gut
lumen between epithelial cells. However, recent in vivo
experiments have raised questions indirectly as to the true
role of DC as antigen samplers in the gut.29 The authors
showed that in mice the transport across the gut barrier of
orally delivered green-fluorescence protein (GFP)-labelled
Enterobacter cloacae takes place exclusively via FAE-associated M cells in the Peyer’s patches and they concluded
that the internalization by DC occurred only following
M cell mediated transport. In this regard, the discovery of
villous-M cells may provide an alternative explanation for
the occasional uptake of bacteria in areas of the gut located
outside Peyer’s patch.12 These data, taken together, suggest
strongly that M cells are a major player in the sampling of
the gut contents, and as such an appealing target for
mucosal delivery of bioactive compounds.30,31
Features of M cells
M cells display different morphological features compared
with the surrounding enterocytes (Fig. 3). The brush border
is poorly organized with short irregular microvilli, and
the thick glycocalx associated with absorptive cells is
absent.32,33 These adaptations allow easy access of luminal
material to the apical domain of M cells, where it is internalized and then transported to the underlying lymphoid
tissue. The mechanisms by which M cells take up microorganisms and macromolecules vary according to the nature of this material. Large particles and bacteria induce
phagocytosis, which is often associated with ruffling of the
apical plasma membrane of the M cell and rearrangement
of the actin cytoskeleton, which permits active formation of
pseudopodia-like structures.34,35 Viruses and other adherent particles are taken up by endocytosis via clathrin-coated
vesicles,36 whereas non-adherent material is internalized by
fluid phase endocytosis.37,38 In all these cases, internalization is followed quickly by transport of endocytotic
vesicles to the endosomal compartment and then by exocytosis to the basolateral membrane. The biochemical
events involved in the intracellular transport of endocytotic
vesicles in M cells are still largely unknown, but it appears
that this is regulated in the same manner as polarized
transport observed in other epithelial cells.39,40 A characteristic of M cells is that, unlike other intestinal epithelial
cells, they possess deeply invaginated basolateral surfaces
that form intraepithelial pockets harbouring a wide variety
of lymphocyte subsets that migrate to this unique compartment from lymphoid tissue.41,42 Thus, M cell pockets
facilitate the contact between the incoming antigens and the
specialized immune system in an environment separated
from regulatory elements of the mucosal immune system.42
The use of a large panel of lectins to stain FAE cells has
revealed that in some cases M cells display a different glycosylation state compared with neighbouring enterocytes.
In mice, for example, small intestine Peyer’s patch M cells
2004 Blackwell Publishing Ltd, Immunology, 113, 15–22
Figure 3. Electron micrograph showing an M cell (M) enclosed
between two adjacent enterocytes (E) in the FAE. The apical region
is characterized by the presence of more irregular and scattered
microvilli compared to those of enterocytes. This adaptation allows
easy access to the M cell apical surface of material in the gut
lumen (L). Lymphocytes (Ly) move into M cell pockets from the
underlying lymphoid tissue (LT) through openings in the basal
lamina (bl). Arrowheads indicate tight junctions.
predominantly express (1–2)-fucose that can be detected by
Uleus europeaus (UEA-1) lectin.43,44 The pattern is different
in caecal patches, where M cells are stained by the same
lectin but also express other terminal saccharides.45,46 In
humans, the glycosylation pattern is different from that of
other species and M cells preferentially display the sialyl
Lewis A antigen.47 Surface glycoconjugates play an
important role in microbial–M cell interactions and the
wide variability of these molecules on the M cell surface has
been interpreted as an effective way to generate a broad
repertoire of M cell surface binding molecules to recognize
bacteria-borne lectins.48 The presence of M cell-specific
glycoconjugates on M cell surface has an immediate relevance in terms of vaccine targeting. Indeed, M cell-specific
glycoconjugates have been used with some success as targets for oral and nasal delivery of antigens and particulate
carrier.49,50
An important aspect of M cell biology that has been
overlooked is its potential in participating in the genesis of
mucosal immune responses. Interestingly, although the role
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A. L. Man et al.
of M cells in processing and presenting antigens has not
been investigated thoroughly, it was reported that M cells
are provided with enzymatic components, including cathepsin E typical of antigen-presenting cells.51 Furthermore,
although the presence of major histocompatibilty complex
(MHC) Class II molecules remains controversial it, was
reported by some authors that M cells expressed Ia molecules on the basolateral plasma membrane and organelles,
such as prelysosome, lysosome and endosome and the
expression of which was enhanced by pretreatment with
interferon (IFN)-c.52 The notion that M cells are not only
simple conduits for antigen transport and may have a more
active role in the early phase of mucosal immune responses
is also suggested by their ability to produce immunoregulatory cytokines, such as interleukin (IL)-1.53
M cells and bacteria
The capacity of M cells for antigen sampling can also
facilitate invasion by potentially harmful intestinal pathogens, a process mediated by bacterial invasin.54 The most
dramatic examples of specific targeting of pathogens to M
cells are provided by studies performed on Salmonella,55,56
Shigella57 and Yersinia.58 In most of these cases the infection of M cells results in the disruption of the cells and the
ensuing alteration of the integrity of the epithelial barrier.
However, more recent studies have revealed that penetration of M cells by bacteria does not always end in catastrophe for the M cells and gut barrier. We observed
that short-term exposure to a non-intestinal bacterium,
Streptococcus pneumoniae R36a, induced rapid and significant changes of the morphology35 and function59,60 of the
FAE. The most notable was an apparent increase in the
number of fully operational M cells that led to an
up-regulation of microsphere transport across the FAE59
(Fig. 4) and to the systemic circulation.60 More recently the
Figure 4. Confocal laser scanning micrograph of the FAE of
Peyer’s patch. A significant increase in the number of microparticles bound, internalized and translocated across the FAE is
observed following short-term exposure to S. pneumoniae (a) compared to control tissue (b). The binding and translocation is highly
specific for the FAE as microparticles can not be seen in contact
with or internalized by villous epithelial cells (V) (L ¼ gut lumen).
ability of S. pneumoniae to up-regulate rapidly M cell
mediated transport of microparticles has been confirmed
by others.61 In this case, the authors reported that the
up-regulation of antigen transport was not accompanied by
a real increase in the number of M cells but rather by the
acquisition, by predetermined M cells already inhabiting
the FAE of both morphological and functional features of
fully mature and differentiated M cells. Furthermore, it is
worth highlighting the fact that both groups observed that
these events were not accompanied by any detectable
alteration of the integrity of the epithelial barrier. These
data demonstrate that this experimental model is an
excellent tool to address basic but still unsolved issues of
M cell biology and function. First, it will help to shed light
on the still controversial issue of origin of this cell type
within the FAE. Secondly, and more important from an
immunological point of view, it provides the first experimental evidence that M cell mediated antigen transport to
the mucosal immune system can be improved in vivo; a
finding that has a direct bearing on mucosal delivery of
vaccines and therapeutics.
M cell formation and function
The issue of M cell origin within the FAE provides a fascinating puzzle for not only cell biologists; indeed, due to
their high efficiency in sampling the gut lumen this is an
enticing objective for scientists seeking to design more
effective methods of mucosal immunization and drug
delivery. As such, the dynamics of M cell formation within
specialized gut epithelia has been the subject of debate since
their discovery in 1974.20 Intestinal epithelial cell maturation and differentiation is a well-known phenomenon
which is completed in a topographically well-organized
migration.62–64 The epithelium of each follicle derives from
surrounding crypts, each of each is a clonal unit65,66 characterized by two distinct axes of migration and differentiation. Cells located on the villous side of the crypt
differentiate into absorptive enterocytes, goblet and
enteroendocrine cells. Cells on the FAE side of the crypt
move onto the dome, acquiring features of absorptive
enterocytes and specialized M cells.13 The final differentiation of intestinal epithelial cells occurs as they migrate in
vertical bands to the apical extrusion zone of the villous and
FAE. Within this scenario two different theories on M cell
genesis in the FAE have been formulated. First, it was
postulated that M cells may originate in the crypts as a
distinct cell lineage from stem cells via an independent
differentiation programme; alternatively, it was thought
that they may be formed by the conversion of FAE–
enterocytes upon interaction with the local lymphoid
microenvironment.
The most convincing experimental evidence supporting
the idea that M cells are derived from undifferentiated crypt
cells comes from studies on differential expression of both
glycoconjugates67,68 and the specific M cell marker,
vimentin in rabbit.69,70 These studies showed that a subpopulation of crypt cells is predetermined as M cells before
attaining their morphological and functional features. This
2004 Blackwell Publishing Ltd, Immunology, 113, 15–22
M cell in transepithelial transport
was supported further by additional study that showed that
the random distribution of the sites where lymphocytes
invade the FAE did not correlate with the organization of
M cells in radial strips that emerged from the dome associated crypts. These studies led the authors to conclude that
M cell formation is restricted to the crypts and that FAE
associated enterocytes do not have the potential to convert
into M cells.
On the other hand, in addition to previous ultrastructural studies that concluded that M cells derived from fully
differentiated enterocytes,71 an in vitro coculture system
was established in which murine Peyer’s patch-derived
lymphocytes converted human intestinal Caco2 cells into
functional M cells.72 Although it is a little unrealistic to
consider these adenocarcinoma cells as mature and fully
differentiated enterocytes,73,74 this model provides direct
evidence that lympho-epithelial cross-talk plays a central
role in controlling the genesis of specialized epithelial cell
populations. The role of immune cells in the formation of
specialized FAE and M cells was also demonstrated in
several experimental models. In one such case75 it was
reported that knock-out (KO) mice that lacked B cells did
show a reduction in the numbers of PP and negligible
numbers of M cell, thus suggesting that B cells play a role in
M cell formation. Although the exclusive role of B cells in
these events was later questioned,76 the ability of lymphocytes to modulate the expression of genes linked to M cell
phenotype has been demonstrated clearly.77,78 In addition,
in vivo studies have shown that bone marrow transplantation in SCID syngeneic mice induced the formation of M
cells, thus further supporting the notion that M cell genesis
is immunoregulated.79 The most compelling evidence for
the ability of immune cells to convert enterocytes into M
cells has come from in vivo studies that involved the use of
bacteria in the gut. First, oral administration of Salmonella
in mice induced a rapid increase (12 hr) in the number of
both IEL and M cells within the FAE80 and secondly, that
short-term exposures (1–3 hr) to a non-intestinal bacterium, S. pneumoniae R36a induced dramatic alterations of
the FAE that included an apparent increase in the number
of fully operational M cells in the periphery of the FAE.60
Finally, it also appears that that the numbers of the newly
discovered villous-associated M cells population increased
following in vivo challenge with S. typhimurium.12 In the
light of recent findings, however,61 it seems that these
apparently conflicting results on M cell genesis within the
FAE can now be reconciled. Indeed, this work produced
evidence that suggested strongly that the rapid increase in
the number of fully operational M cells following certain
stimuli such as Salmonella or S. pneumoniae can be
explained as short-term differentiation of predetermined
M cells already inhabiting the FAE rather than from the
conversion of enterocytes into M cells. Although the nature
of the stimulus required to induce the differentiation of
predetermined M cells into fully operational ones remains
unknown, hypotheses include the release of bacterial
products and bacteria-induced secretion of cytokines by the
host. The presence of cells with the ability to become fully
functional antigen-sampling cells very rapidly would
2004 Blackwell Publishing Ltd, Immunology, 113, 15–22
19
explain the changes of cell population in the FAE following
bacterial challenge and, more importantly, it confirms that
the antigen-sampling capability of the FAE can be manipulated in vivo. With this in mind it is possible to hypothesize that certain bacteria may help us to design novel
strategies for oral delivery of vaccines and drugs.
FUTURE SCENARIOS IN MUCOSAL DELIVERY
Searching for the Trojan horse for mucosal delivery
One of the major problems in devising strategies to improve
M cell mediated delivery of vaccines and therapeutics to the
MALT has been represented so far by the lack of knowledge on the molecular and cellular events underlying M cell
formation. This complicates further the quest to identify
both bacterial and host molecules involved in the bacteriainduced up-regulation of antigen sampling in the gut.
The observations described in the previous section raise
a fundamental question. Can we exploit the ability of certain bacteria to design novel and more effective strategies
to improve M cell-mediated transport into the MALT?
Although the mechanisms underlying the previously described events are still unknown, we can argue reasonably
that this will be possible. Indeed, several observations carried out in vitro point to the fact that soluble mediators
might be involved. Although it is possible that bacterial
products may play an important role in the final differentiation of pre-M cells into competent sampling M cells, it
seems likely that factors secreted by the host gut epithelium
or immune system are involved. First, it was observed that
in vitro conversion of Caco2 cells into M cells by lymphocytes was not restricted to cells in contact with
PP-lymphocytes or B cell lymphoma Raji.72 This was confirmed further in a coculture system in which Caco2 cells
and Raji cells were separated physically during culture.81
These experimental models did not employ bacteria, thus
implying that soluble mediators secreted by B cells had a
major role in the M cell conversion. Given the nature of the
lymphoma Raji B cells it is conceivable that these cells
constitutively secrete a lympho ⁄cytokine which is normally
secreted in vivo upon interaction with bacteria by either
epithelial or, more probably, immune cells. This prospect
opens a fascinating scenario. The advantage of having at
our disposal a molecule with the ability to up-regulate
mucosal antigen uptake is obvious, and novel methods to
improve both oral (via GALT) and nasal (via NALT)
delivery of vaccines and drugs might be within our grasp.
Such a molecule will probably help to reduce or minimize
the dose and consequently the cost of vaccine or drug
preparation, and in addition it may improve the efficiency
of already existing or experimental antigen delivery system.
For instance, recombinant bacteria have been employed
successfully as living delivery vectors for heterologous
antigens.82,83 The knowledge that some bacteria have
an intrinsic ability to up-regulate M cell mediated transport across mucosal barriers may have a great impact in
the selection of the bacterial vector. Alternatively, if
the enhancer of M cell transport was a cytokine the
20
A. L. Man et al.
construction of a more effective bacterial vector would be
possible. The vector should have the ability to target
specifically M cells (i.e. Salmonella) and coexpress a
heterologous antigen along with the enhancer of mucosal
uptake, thus allowing the latter to exert its biological
activity within the FAE. In addition, the efficiency of other
available technologies such as biodegradable microparticles84,85 may also be improved as the enhancer of mucosal
antigen uptake could be microencapsulated easily along
with the desired vaccine or drug preparation.
CONCLUDING REMARKS
As regulator of antigen transport across epithelial barriers in
the GALT and NALT, M cells play an important role in the
genesis of protective immune responses. The importance of
finding better ways to induce effective immune responses at
the mucosal level is stressed by the notion that the systemic
immune response is not well equipped to tackle the vast
majority of pathogens to which we are exposed throughout
life. In addition, mucosal administration of bioactive compounds can be administrated easily with none of the risks
associated usually with parenteral administration, and their
easier and cheaper manufacture may increase their availability to developing countries. In light of these considerations,
it is highly desirable to have a better understanding of the
cellular and molecular events underlying M cell formation
and function in specialized mucosal epithelia. The recent
discovery that the M cell mediated transport across mucosal
barriers can be significantly up-regulated by interaction with
bacteria without altering their integrity gives us a real
opportunity to design novel strategies, or to improve already
existing ones to achieve more effective oral and nasal delivery
of biologically active compounds.
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ACKNOWLEDGMENTS
We wish to thank I. T. Johnson and M. T. Garcia-Conesa for their
suggestions and critics; E. Bertelli, M. Regoli and H. M. Meynell
for transmission, scanning and confocal microscopy and P. Pople
for his help in computer work. Work in our laboratory is sponsored
by research grants from the Biotechnology and Biological Sciences
Research Council, UK.
REFERENCES
1 Madara JL, Nash S, Moore R, Atisook K. Structure and
function of the intestinal epithelial barrier in health and disease.
Monogr Pathol 1990; 31:306–24.
2 Bjarnason I, MacPherson Hollander D. Intestinal permeability.
Gastroenterology 1995; 108:1566–81.
3 McGhee JR, Kiyono H. New perspective in vaccine development: mucosal immunity to infections. Infect Agents Dis 1993;
2:55–73.
4 Frey A, Giannasca KT, Weltzin R et al. Role of glycocalix in
regulating access to plasma membranes of intestinal epithelial
cell: implications for microbial attachment and oral vaccine
targeting. J Exp Med 1996; 1996:1045–59.
5 Kett K, Baklien K, Bakken A, Kral JG, Fausa O, Brandtzaeg P.
Intestinal B cell isotype response in relation to local bacteria
19
20
21
22
23
24
load: evidence for IgA subclass adaptation. Gastroenterology
1995; 109:819–25.
Maury J, Nicoletti C, Guzzo-Chambraud F, Maroux S. The
filamentous brush border glycocalix, a mucine-like marker of
enterocytes hyper polarization. Eur J Biochem 1995; 228:323–
31.
Semenza G. Anchoring and biosynthesis of stalked brush border membrane proteins: glycosidases and peptidases of enterocytes and renal tubuli. Annu Rev Cell Biol 1990; 2:255–313.
Neutra MR, Frey A, Kraehenbuhl JP, Epithelial M cells.
Gateways for mucosal infection and immunization. Cell 1996;
86:345–8.
Neutra MR, Pringault E, Kraehenbuhl JP. Antigen sampling
across epithelial barriers and induction of mucosal immune
responses. Annu Rev Immunol 1996; 14:275–300.
Holmgren J, Czerkinsky C, Eriksson K, Mharandi A. Mucosal
immunization and adjuvants: a brief overview of recent
advances and challenges. Vaccine 2003; 21 (Suppl. 2):89–95.
Brandtzaeg P, Baekkevold ES, Farstad IN et al. Regional specialization in the mucosal immune system: what happens in the
microcompartments? Immunol Today 1999; 20:141–51.
Jang MH, Kweon MN, Iwatani K et al. Intestinal villous M
cells: an antigen entry site in the mucosal epithelium. Proc Nat
Acad Sci USA 2004; 101:6110–5.
Neutra MR, Mantis NJ, Kraehenbuhl JP. Collaboration of
epithelial cells with organized mucosal lymphoid tissues. Nature
Immunol 2001; 2:1004–9.
Allan CH, Trier JS. Structure and permeability differ in
subepithelial villus and Peyer’s patches follicles capillaries.
Gastroenterology 1991; 100:1172–9.
Hein WR. Organization of mucosal lymphoid tissue. Curr Top
Microbiol Immunol 1999; 236:1–15.
Pappo J, Owen RL. Absence of secretory component expression
by epithelial cells overlying rabbit gut-associated lymphoid
tissue. Gastroenterology 1988; 95:1173–7.
Gehrke I, Pabst R. The epithelium overlying rabbit bronchusassociated lymphoid tissue does not express the secretory component of immunoglobulin. Cell Tissue Res 1990; 259:397–9.
Giannnasca PJ, Giannasca KT, Falk P, Gordon JI, Neutra MR.
Regional differences in glycoconjugates of intestinal M cells in
mice: potential targets for mucosal vaccines. Am J Physiol 1994;
227:1108–21.
Owen RL, Bhalla DK. Cytochemical analysis of alkaline
phosphatase and esterase activities and of lectin-binding and
anionic sites in rat and mouse Peyer’s patch M cells. Am J Anal
1983; 168:199–212.
Owen RL, Jones AL. Epithelial cell specialization within human
Peyer’s patch: an ultrastructural study of intestinal lymphoid
follicle. Gastroenterology 1974; 66:189–203.
Fujimura Y. Evidence of M cells as portals of entry for antigens
in the nasopharyngeal lymphoid tissue of humans. Virchows
Arch 2000; 436:560–6.
Zuercher AW, Coffin SE, Thurnheer MC, Fundova P, Cebra JJ.
Nasal-associated lymphoid tissue is a mucosal inductive site
for virus-specific humoral and cellular immune responses.
J Immunol 2002; 168:1796–803.
Hou Y, Hu WG, Hirano T, Gu XX. A new intra-NALT route
elicits mucosal and systemic immunity against Moraxella
catarrhalis in a mouse challenge model. Vaccine 2002; 20:2375–
81.
Huang FP, Platt N, Wykes M et al. A discrete subpopulation of
dentritic cells transports apoptotic intestinal epithelial cells to
T cell areas of mesenteric lymph nodes. J Exp Med 2000;
191:435–43.
2004 Blackwell Publishing Ltd, Immunology, 113, 15–22
M cell in transepithelial transport
25 Rescigno M, Urbano M, Valzasina B et al. Dentritic cells
express tight junction proteins and penetrate gut epithelial
monolayers to sample bacteria. Nat Immunol 2001; 2:361–8.
26 Ruedl C, Hubele S. Maturation of Peyer’s patches dentritic cells
in vitro upon stimulation via cytokines or CD40 triggering. Eur
J Immunol 1997; 27:1325–30.
27 Kelsall BL, Strober W. Distinct populations of dentritic cells are
present in subepithelial dome and T cell regions of the murine
Peyer’s patch. J Exp Med 1996; 183:237–47.
28 Iwasaki A, Kelsall BL. Localization of distinct Peyer’s patch
dentritic cell subsets and their recruitment by chemokines
macrophage inflammatory protein (MIP)-3a, MIP-3b, and secondary lymphoid organ chemokine. J Exp Med 2000; 191:1381–
94.
29 Macpherson AJ, Uhr T. Induction of protective IgA by intestinal dentritic cells carrying commensal bacteria. Science 2004;
303:1662–5.
30 Wang X, Hone DM, Haddad A, Shata MT, Pascual DW. M
cell DNA vaccination for CTL immunity to HIV. J Immunol
2003; 171:4717–25.
31 Clark MA, Jepson MA, Hirst BH. Exploiting M cells for drug
and vaccine delivery. Adv Drug Del Rev 2001; 50:81–106.
32 Bockman DE. Functional histology of appendix. Arch Histol
Cytol 1983; 46:271–92.
33 Bye WA, Allan CH, Trier JS. Structure, organization and distribution of M cells in Peyer’s patches. Gastroenterology 1984;
86:789–801.
34 Fujimura Y. Functional morphology of microfold cells (M) in
Peyer’s patches. Phagocytosis and transport of BCG by M
cells into rabbit Peyer’s patches. Gastroenterol Jpn 1986;
21:325–30.
35 Borghesi C, Bertelli E, Regoli M, Nicoletti C. Modifications of
the FAE by short term exposure to a non-intestinal bacterium.
J Pathol 1996; 180:326–32.
36 Neutra MR, Phillips TL, Mayer EL, Fishkind DJ. Transport of
membrane-bound macromolecules by M cells in the FAE of
rabbit Peyer’s patches. Cell Tissue Res 1987; 247:536–46.
37 Owen RL. Sequential uptake of horseradish peroxidase by
lymphoid follicle epithelium of Peyer’s patches in normal
unobstructed mouse intestine: an ultrastructural study.
Gastroenterology 1977; 72:440–51.
38 Gebert A. The role of M cells in the protection of mucosal
membrane. Histochem Cell Biol 1997; 108:455–70.
39 Druben DG, Nelson WJ. Origin of cells polarity. Cell 1996;
84:335–44.
40 Wolf JL, Bye WA. The membranous (M) epithelial cells and the
mucosal immune system. Annu Rev Med 1984; 35:95–112.
41 Regoli M, Bertelli E, Borghesi C, Nicoleti C. Three (3D)reconstruction of M cells in rabbit Peyer’s patches: definition of
intraepithelial compartment of the follicle-associated epithelium. Anat Rec 1995; 243:19–26.
42 Neutra MR. Current concepts in mucosal immunity. V. Role of
M cells in transepithelial transport of antigens and pathogens to
the intestinal immune system. Am J Physiol 1998; 274:G785–91.
43 Clark MA, Jepson MA, Simmons NL, Booth TA, Hirst BH.
Differential expression of lectin binding sites defines mouse
intestinal M cells. J Histochem Cytochem 1993; 41:1679–87.
44 Falk P, Roth KA, Gordon JI. Lecins are sensitive tools for
defining the differentiation programs of epithelial cell lineages in
the developing and adult mouse gastrointestinal tract. Am
J Physiol 1994; 266:G987–G1003.
45 Gebert A, Hach G. Differential binding of lectins to M cells and
enterocytes in the rabbit caecum. Gastroenterology 1993;
105:1350–61.
2004 Blackwell Publishing Ltd, Immunology, 113, 15–22
21
46 Clark MA, Jepson MA, Simmons NL, Hirst BH. Differential
surface characteristics of M cells from mouse intestinal Peyer’s
patch and caecal patches. Histochem J 1994; 26:271–80.
47 Giannasca PJ, Giannasca KT, Leichtner AM, Neutra MR.
Human intestinal M cells display the sialyl Lewis A antigen.
Infect Immun 1999; 67:946–53.
48 Neutra MR, Giannasca PJ, Giannasca KT. M cells and microbial
pathogens. In: Blaser M, Smith PD, Ravodin JI, eds. Infections of
the GI Tract. New York: Raven Press, 1995:163–78.
49 Clark MA, Blair H, Liang L, Brey RN, Brayden D, Hirst BH.
Targeting polymerised liposomoe vaccine carriers to intestinal
M cells. Vaccine 2002; 20:208–17.
50 Jepson MA, Clark MA, Hirst BH. M cell targeting by lectins: a
strategy from mucosal vaccination and drug delivery. Adv Drug
Del Rev 2004; 56:511–25.
51 Finzi G, Cornaggia M, Capella C et al. Cathepsin E in follicleassociated epithelium of intestine and tonsils: localization to M
cells and possible role in antigen processing. Histochemistry
1993; 99:201–11.
52 Allan CH, Mendrick DL, Trier JS. Rat intestinal M cells
contain acidic endosomal–lysosomal compartments and express
class II major histocompatibility complex determinants.
Gastroenterology 1993; 104:698–08.
53 Pappo J, Mahlman RT. Follicle epithelial M cells are a source
of IL-1 in Peyer’s patches. Immunology 1993; 78:505–7.
54 Granucci F, Ricciardi–Castagnoli P. Interactions of bacterial
pathogens with dentritic cells during invasion of mucosal
surfaces. Curr Options Microbiol 2003; 6:72–6.
55 Kohobata S, Yokobata H, Yabuuchi E. Cytopathogenic effect
of Salmonella typhi GIFU 10 007 on cells of murine ileal Peyer’s
patches in ligated ileal loops: an ultrastructural study. Microbiol
Immunol 1986; 30:1225–37.
56 Jones BD, Ghori N, Falkow S. Salmonella typhimurium initiates
murine infection by penetrating and destroying the specialized
epithelial M cells of the Peyer’s patches. J Exp Med 1994;
180:15–23.
57 Perdomo JL, Cavaillon JM, Huerre M, Ohayon H, Gounon P,
Sansonetti PJ. Acute inflammation causes epithelial invasion
and mucosal destruction in experimental shigellosis. J Exp Med
1994; 180:1307–19.
58 Grutzkau A, Hanski C, Hahn H, Riecken EO. Involvement of
M cells in the bacterial invation of Peyer’s patch: a common
mechanism shared by Yersinia enterocolitica and other enteroinvasive bacteria. Gut 1990; 3:1011–5.
59 Meynell HM, Thomas NW, James PS, Holland J, Taussig MJ,
Nicoletti C. Up-regulation of microsphere transport across the
follicle-associated epithelium of Peyer’s patch by exposure to
S. pneumoniae R36a. FASEB J 1999; 13:611–9.
60 Borghesi C, Taussig MJ, Nicoletti C. Rapid appearance of M
cells after microbial challenge is restricted at the periphery of the
follicle-associated epithelium of Peyer’s patch. Lab Invest 1999;
79:1391–01.
61 Gebert A, Steinmetz I, Fassbender S, Wendlandt KH. Antigen
transport in Peyer’s patches: increased uptake by constant
numbers of M cells. Am J Pathol 2004; 64:65–72.
62 Hermiston M, Green RP, Gordon JI. Chimeric-transgenic
mice represent a powerful tool for studying how the proliferation and differentiation programs of intestinal epithelial
cells lineages are regulated. Proc Natl Acad Sci USA 1993;
90:8866–70.
63 Heath JP. Epithelial cell migration in the intestine. Cell Biol Int
1996; 20:139–40.
64 Louvard D, Kedinger M, Hauri HP. The differentiating intestinal epithelial cells: establishment and maintenance of functions
22
65
66
67
68
69
70
71
72
73
74
75
A. L. Man et al.
thought interactions between cellular structures. Annu Rev Cell
Biol 1992; 8:157–95.
Ponder BA, Schmidt GH, Wilkinson MM, Wood MJ, Monk
M, Reid A. Derivation of mouse intestinal crypts from single
progenitor cells. Nature 1985; 313:689–91.
Rubin DC, Ong D, Gordon JI. Cellular differentiation in the
emerging fetal rat small intestinal epithelium: mosaic pattern of
gene expression. Proc Natl Acad Sci USA 1989; 86:1278–82.
Giannasca PJ, Giannasca KT, Falk P, Gordon JI, Neutra MR.
Regional differences in glycoconjugates of intestinal M cells in
mice: potential targets for mucosal vaccine. Am J Physiol 1994;
267:G1108–21.
Gebert A, Posselt W. Glyconjugate expression defines the origin
and differentiation pathway of intestinal M cells. J Histochem
Cytochem 1997; 45:1341–50.
Lelouard H, Sahuquet A, Reggio H, Montcourrier P. Rabbit M
cells and dome enterocytes are distinct cell lineages. J Cell Sci
2001; 114:2077–83.
Gebert A, Fassbender S, Werner K, Weissferdt A. The development of M cells in Peyer’s patches is restricted to specialized
dome-associated crypts. Am J Pathol 1999; 154:1573–82.
Smith MW, Peacock MA. Lymphocyte induced formation of
antigen transporting M cells from fully differentiated mouse
enterocytes. In: Robinson JWL, ed. Mechanisms of Intestinal
Adaptation: Lancaster: MTP, 1982:573–83.
Kerneis S, Bogdanova A, Kraehenbulh JP, Pringault E. Conversion by Peyer’s patch lymphocytes of human enterocytes into
M cells that transport bacteria. Science 1997; 277:949–52.
Delie F, Rubas W. A human colonic cell line sharing similarities
with enterocytes as a model to examine oral absorption:
advantages and limitations of the caco-2 model. Crit Rev Ther
Drug Carrier Syst 1997; 14:221–86.
Owen RL. Mid-life crisis for M cells. Gut 1998; 42:11–2.
Golovkina TV, Shlomchik M, Hannum L, Chervonsky A.
Organogenic role of B lymphocytes in mucosal immunity.
Science 1999; 286:1965–8.
76 Debard N, Sierro F, Browning J, Kraehenbuhl JP. Effect of
mature lymphocytes and lymphotoxin on the development of
the follicle-associated epithelium and M cells in the Peyer’s
patches. Gastroenterology 2001; 120:1173–82.
77 El Bahi S, Caliot E, Bens M et al. Lymphoepithelial interations
trigger specific regulation of gene expression in the M cellcontaining follicle-associated epithelium of Peyer’s patches.
J Immunol 2002; 168:3713–20.
78 Lo D, Tynan W, Dickerson J et al. Cell culture modeling
of specialized tissue: identification of genes expressed specifically by follicle-associated epithelium of Peyer’s patch by
expression profiling of Caco-2 ⁄ Raji co-cultures. Int Immunol
2004; 16:91–9.
79 Sharma R, Schumacher U, Adam E. Lectin histochemistry
revealed the appearance of M cell in Peyer’s patches of SCID
mice after syngeneic normal bone marrow transplantation.
J Histochem Cytochem 1998; 46:143–8.
80 Savidge TC, Smith MW, James PS, Aldred P. Salmonellainduced M-cell formation in germ-free mouse peyer’s patch
tissue. Am J Pathol 1991; 139:177–84.
81 Gullberg E, Leonard M, Karlsson J et al. Expression of specific
markers and particle transport in a new human intestinal M-cell
model. Biochem Biophys Res Commun 2000; 279:808–13.
82 Robinson K, Chamberlain LM, Lopez MC et al. Mucosal and
cellular immune responses elicited by recombinant Lactococcus
lactis strains expressing tetanus tocxin fragment C. Infect
Immun 2004; 72:2753–61.
83 Bowe F, Pickard DJ, Anderson RJ, Londono-Arcila P, Dougan
G. Development of attenuated Salmonella starins that express
heterologous antigens. Meth Mol Med 2003; 87:83–100.
84 Mathiowitz E, Jacob JS, Jong YS et al. Biologically erodable
microspheres as potential oral drug delivery systems. Nature
1997; 386:410–4.
85 Brayden DJ. Oral vaccination in man using antigens in particles:
current status. Eur J Pharm Sci 2001; 14:183–9.
2004 Blackwell Publishing Ltd, Immunology, 113, 15–22