Download The role of transepithelial transport by M cells in microbial invasion

209
Journal of Cell Science, Supplement 17, 209-215 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
The role of transepithelial transport by M cells in microbial invasion and
host defense
Marian R. Neutra1 and Jean-Pierre Kraehenbuhl2
department of Pediatrics, Harvard Medical School and Gl Cell Biology Laboratory, Children’s Hospital, 300 Longwood
Avenue, Boston, MA 02115 USA
2Swiss Institute for Experimental Cancer Research and Institute of Biochemistry, University of Lausanne, CH-1066
Epalinges, Switzerland
SUMMARY
Transepithelial transport of antigens by M cells in the
epithelium associated with lymphoid follicles in the
intestine delivers immunogens directly to organized
mucosal lymphoid tissues, the inductive sites for
mucosal immune responses. We have exploited M cell
transport to generate and characterize specific mono­
clonal IgA antibodies that can prevent interaction of
pathogens with epithelial surfaces. The relative protec­
tive capacities of specific monoclonal IgA antibodies
have been tested in vivo by generation of hybridoma
tumors that result in secretion of monoclonal IgA into
the intestine. Using this method, we have established
that secretion of IgA antibodies recognizing a single sur­
face epitope on enteric pathogens can provide protec­
tion against colonization or invasion of the intestinal
mucosa.
INTRODUCTION
organized mucosal lymphoid tissue (Bockman and Cooper,
1973; Neutra et al., 1987; Owen 1977). Antigen process­
ing and presentation at these sites results in IgA-commit­
ted, antigen-specific B lymphocytes that proliferate locally,
then leave the mucosa, migrate via the bloodstream, and
finally ‘home’ to mucosal or glandular sites throughout the
intestine as well as other mucosal and secretory tissues
(Cebra et al., 1976; McDermott and Bienenstock, 1979).
These disseminated cells terminally differentiate to become
subepithelial plasma cells that produce polymeric IgA anti­
bodies (Mestecky and McGhee, 1987; Kraehenbuhl and
Neutra, 1992). Transepithelial transport in the basal to
apical direction is then required for transport of IgA into
glandular and mucosal secretions, and this is mediated by
polymeric immunoglobulin (poly-Ig) receptors in a variety
of epithelial and glandular cells (Mostov et al., 1980; Kuhn
and Kraehenbuhl 1982; Apodaca et al., 1991).
Organized mucosal lymphoid tissues, recognized by the
presense of lymphoid follicles, are present in the oral cavity,
the bronchi, and throughout the small and large intestines
(Owen and Ermak, 1990; Owen and Nemanic, 1978). Single
follicles or small clusters are located along the length of
the gastrointestinal (GI) tract, with increasing frequency in
the colon and rectum (O’Leary and Sweeney, 1986;
Fujimura et al., 1992) and aggregated follicles are found in
the lingual and palatine tonsils, adenoids, appendix and
Peyer’s patches in the small intestine. Whether single or
aggregated, mucosal lymphoid follicles are separated from
The lining of the intestine is a vast monolayer of highly
polarized epithelial cells that provides an effective barrier
to most of the macromolecules, microorganisms and toxins
present in the intestinal lumen. One component of this bar­
rier is the thick and complex coat of glycoproteins and glycolipids on the apical brush borders of intestinal absorptive
cells. The epithelium is nevertheless vulnerable to enteric
pathogens that express surface adhesins, enzymes, and other
specialized mechanisms for colonization of epithelial sur­
faces and invasion of the mucosa. It is thus not surprising
that the intestinal mucosa is heavily populated with cells of
the immune system. Indeed, the intestinal lining contains
more lymphoid cells and produces more antibodies than any
other organ in the body (Mestecky and McGhee, 1987;
Seilles et al., 1985). The vast majority of antibodies pro­
duced at this site are of the IgA isotype, and are exported
into secretions.
Transepithelial transport plays two crucial roles in the
mucosal immune response (Fig. 1). First, samples of anti­
gens and microorganisms must be transported from the
intestinal lumen across the epithelium in order to be
processed in lymphoid tissues and elicit a mucosal immune
response (Neutra and Kraehenbuhl, 1992; Kraehenbuhl and
Neutra, 1992). This appears to be the special role of M
cells, a unique epithelial cell type located exclusively in the
lymphoid follicle-associated epithelia over sites containing
Key words: mucosal immunity, intestinal epithelium, oral
vaccines
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M. R. Neutra and J.-P. Kraehenbuhl
E N V IR O N M E N T
IN D U C TIV E S ITE
ORGANIZED MUCOSAL
LYMPHOID TISSUE
E F F E C T O R SITE
DIFFUSE MUCOSAL
LYMPHOID TISSUE
Fig. 1. The role of transepithelial transport in induction and secretion o f IgA antibodies. Luminal antigens are separated from cells o f the
mucosal immune system by the intestinal epithelial barrier. Antigens are transported into the organized mucosa-associated lymphoid
tissue o f the intestine by M cells in follicle-associated epithelia. Antigen-sensitized, IgA-committed B cells leave the mucosa and migrate
to distant glandular and mucosal sites where they terminally differentiate into polymeric IgA-producing plasma cells. Epithelial and
glandular cells transport polymeric IgA by receptor-mediated transcytosis, releasing slgA into secretions where it can interact with
antigens and pathogens.
the lumen by a highly specialized epithelium containing
antigen-transporting M cells (Fig. 2).
TRANSPORT OF ANTIGENS BY M CELLS
Since transepithelial transport by M cells seems to be
required for initiation of a secretory immune response,
increasing the efficiency of this transport is an important
component of mucosal immunization strategies (Neutra and
Kraehenbuhl, 1992). The M cell apical surface lacks the
closely packed microvilli and thick enzyme-rich coat of
absorptive enterocytes, and displays many broad
microdomains from which endocytosis occurs (Neutra et
al., 1987). M cell apical membranes nevertheless contain
abundant glycoconjugates that can serve as binding sites for
cationic macromolecules and possibly for lectin-like micro­
bial surface molecules (Neutra et al., 1987; Bye et al., 1984;
Owen and Bhalla, 1983). Microorganisms, particles and
lectins that adhere either selectively or nonselectively to M
cell apical membranes are endocytosed and transcytosed
with high efficiency (Neutra et al., 1987; Pappo and Ermak
1989). This is consistent with the observation that materi­
als that can adhere to mucosal surfaces tend to evoke strong
secretory immune responses (DeAizpurua and Russell­
Jones, 1988; Mayrhofer, 1984). Certain pathogenic viruses
and bacteria adhere selectively to M cells and are efficiently
transported, but niether the microbial surface molecules that
mediate adherence nor the M cell surface molecules that
serve as receptors have been identified. It is probable that
microorganisms, which may be considered multivalent par­
ticulate ligands, could find their receptors particularly
accessible on M cells. It was recently demonstrated that
Fig. 2. Diagram o f an M cell. The M cell basolateral surface is
modified to form an intra-epithelial pocket into which
lymphocytes (L) and macrophages (MAC) migrate. Antigens,
microorganisms and particles that adhere to the M cell apical
membrane are efficiently endocytosed and transported into the
pocket, and hence to the underlying mucosal lymphoid tissue.
immunoglobulins in the lumen adhere selectively to the
apical membranes of M cells, and IgA-antigen complexes
were found to adhere to M cells much more efficiently than
antigen alone (Weltzin et al., 1989). This raises the possi­
bility that slgA could in some cases promote re-uptake of
antigens and microorganisms by M cells, perhaps to boost
the secretory immune response to pathogens that have not
been effectively cleared from the lumen. Studies of this
transport system are hampered by the fact that M cells are
Transepithelial transport by M cells
relatively rare occupants of the intestinal lining, however,
and the M cell phenotype has not been replicated in cul­
ture.
M cells take up macromolecules, particles and microor­
ganisms by adsorptive endocytosis via clathrin-coated pits
and vesicles (Neutra et al., 1987), fluid-phase endocytosis
in either coated (Neutra et al., 1987) or uncoated vesicles
(Bockman and Cooper, 1973; Owen, 1977), and phagocy­
tosis involving extension of cellular processes and reorga­
nization of submembrane actin assemblies (Winner et al.,
1991). Bacteria that adhere to M cells can form broad areas
of close interaction: during uptake of Vibrio cholerae, for
example, the bacterial outer membrane and the M cell apical
membrane are separated by a uniform 10-20 nm space.
Endocytosed material is delivered into apical endosomal
tubules and vesicles (Neutra et al., 1987) some of which
contain the late endosome or lysosome membrane marker
lgpl20 and (in some species) MHC class II antigen (Allan
et al., 1993). M cell endosomes are acidified (Allan et al.,
1993), but it is not known whether they contain proteases
and whether endocytosed materials are partially degraded
during transepithelial transport.
The transcytotic pathway of M cells is dramatically
shortened by invagination of the basolateral membrane to
form a large intra-epithelial ‘pocket’. Apical endosomes of
M cells deliver their content not to lysosomes but rather
directly to the membrane domain lining the pocket, and
endocytosed materials are released by exocytosis into the
pocket as early as 10 minutes after apical endocytosis
(Neutra et al., 1987). Since bacteria and particles are read­
ily released from the membrane into the intra-epithelial
space, it is likely that that initial binding involves multiple,
low-affinity interaction sites and that the milieu of the trans­
port vesicle or intra-epithelial pocket allows rapid dissoci­
ation. Whether apical membrane molecules are replaced by
de novo synthesis or by recycling of membrane
microdomains from the pocket is not known. Antigens that
are transported by M cells may interact first with the anti­
gen-presenting cells and lymphocytes present in the intra­
epithelial pocket formed by M cells (Ermak et al., 1990)
but it is not known whether an immune response is gener­
ated in this sequestered site. In any case, the subepithelial
tissue immediately below the follicle-associated epithelium
contains IgM+ B cells, CD4+ T cells, dendritic cells and
macrophages, and in this cellular network antigens and
microorganisms are likely to be efficiently processed and
presented (Ermak and Owen, 1986).
INTERACTION OF MICRO-ORGANISMS WITH M
CELLS
A common mechanism such as lectin-carbohydrate recog­
nition may allow the M cell to ‘sample’ pathogenic lumi­
nal organisms, either by binding of lectin-like bacterial
adhesins to M cell surface glycoconjugates or, conversely,
binding of bacterial surface oligosaccharides to M cell sur­
face lectins. M cell binding of noninvasive bacteria such as
Vibrio cholerae (Winner et al., 1991; Owen et al., 1986)
results in efficient sampling by the mucosal immune
system, and a strong secretory immune response. In the case
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of cholera, secretion of anti-microbial slgA appears to play
a major role in limiting the duration of mucosal disease and
preventing re-infection (Svennerholm et al., 1984; Jertbom
et al., 1986). A variety of pathogenic bacteria and viruses
that bind to M cells, however, exploit the transport mech­
anism that was intended for mucosal protection by using
this transepithelial pathway as an invasion route. For exam­
ple, transport of Salmonella typhi into Peyer’s patch mucosa
(Kohbata et al., 1986) results in a vigorous anti-Salmonella
mucosal immune response, but this occurs too late to pre­
vent spread of bacteria to the liver and spleen and dissem­
inated systemic disease (Chau et al., 1981; Hohmann et al.,
1978). Similarly, M cell binding and transport of Shigella
flexneri (Wassef et al., 1989) and Yersinia enterocoliitica
(Grutzkau et al., 1990) allows these organisms to gain
access to the lamina propria, where they cause mucosal dis­
ease by basolateral invasion of epithelial cells and infec­
tion of mucosal macrophages (Isberg, 1990; Sansonetti,
1991).
Viral pathogens also exploit the M cell transport system.
This has been amply demonstrated in studies of reovirus
pathogenesis in mice (Wolf et al., 1981). Poliovirus adhered
to human M cells in organ culture (Sicinski et al., 1990),
and the retrovirus HIV-1 adhered to M cells of rabbit and
mouse follicle-associated epithelia in mucosal explants
(Amerongen et al., 1991). Selective adherence to M cell
apical membranes effectively targets these pathogens for
efficient transport into the intestinal mucosa. In the case of
reovirus type 3 and possibly poliovirus^ entry into Peyer’s
patch mucosa is followed by invasion of neuronal target
cells and spread to the central nervous system (Nibert et
al., 1991). If HIV is transported by human M cells, the virus
would be delivered directly to target T cells within and
under the follicle-associated epithelium. Although viral par­
ticles are sampled by the mucosal immune system and
immune responses may be generated, entry of virus into
target cells puts them beyond the reach of anti-viral anti­
bodies. Identification of viral surface components that medi­
ate M cell adherence, and the corresponding M cell recep­
tors, remains an important research priority.
MECHANISM OF IgA PROTECTION
There is considerable indirect evidence that specific secre­
tory IgA in the fluids bathing mucosal surfaces can prevent
contact of antigens and pathogens with epithelial cells, a
phenomenon called ‘immune exclusion’ (Tomasi, 1983;
Killian et al., 1988). The molecular mechanisms that under­
lie this protection, however, are not completely understood
and it is likely that this unique effector molecule can play
multiple roles in the changing mucosal environment of the
intestinal lumen. Secretory IgA is an effector molecule,
which functions outside the body in environments usually
devoid of complement and phagocytic cells. Most IgA anti­
bodies studied to date are not ‘neutralizing’ in the classic
sense as they generally do not opsonize in vitro, do not bind
complement or cause release of complement fragment C5a,
and do not directly cause bacterial lysis (Tomasi, 1983; Kil­
lian et al., 1988; Childers et al., 1989). It has been shown
that specific IgA antibodies injected systemically may be
212
M. R. Neutra and J.-P. Kraehenbuhl
ineffective against systemic microbial challenge even when
the same antibodies can protect against mucosal challenge
when they are present in mucosal secretions (Subbarao and
Murphy, 1992; Michetti et al., 1992). IgA antibodies
secreted in response to mucosal bacteria and viruses are
directed primarily against surface antigens or secreted
toxins. Since slgA is a dimer with 4 antigen binding sites,
it can efficiently crosslink target macromolecules and
micro-organisms in the intestinal lumen, thus inhibiting
motility and facilitating entrapment in mucus and clearance
by peristalsis. In the context of the mucosa, slgA can also
collaborate with bacteriostatic proteins and lytic cells to
enhance microbial killing in novel ways (Killian et al.,
1988; Childers et al., 1989).
Oral or mucosal vaccination has been clearly shown to
be the optimal method for induction of secretory IgA and
effective immune protection against many enteric
pathogens. Thus there is rapidly growing interest in testing
and use of novel oral vaccines in experimental animals and
humans (Subbarao and Murphy, 1992; Levine and Edel­
man, 1990; Mekalanos, 1992). The immune responses gen­
erated by enteric viral and bacterial infections and by
mucosal vaccines usually include other components in
addition to secretory IgA, however, such as production of
systemic IgG antibodies and cell-mediated immunity (Can­
cellieri and Fara, 1985). Thus it has been difficult to judge
the relative importance of slgA in protection, or to deter­
mine whether ‘immune exclusion’ by slgA alone can be
sufficient to prevent mucosal infection. It is technically dif­
ficult to collect, purify and analyse polyclonal slgA from
secretions and even more difficult to determine which anti­
genic determinants and antibody specificities contributed to
protection. For these reasons, several laboratories including
our own have produced monoclonal IgA antibodies and
have used them to address these issues in vivo and in vitro.
PRODUCTION AND USE OF MONOCLONAL IgA
ANTIBODIES
Mucosal (but not systemic) immunization favors the for­
mation of antigen-sensitized lymphoblasts of the IgA iso­
type in organized mucosal lymphoid tissues such as Peyer’s
patches. In our laboratories, we have found that fusion of
cells isolated directly from Peyer’s patches after a series of
mucosal immunizations with viruses, bacteria and protein
antigens is an effective method for obtaining IgA hybrido­
mas (Weltzin et al., 1989; W inner et al., 1991; Michetti et
al., 1992; Apter et al., 1991). The antigen specificities of
the IgA antibodies thus obtained serve to identify the micro­
bial surface molecules that are most immunogenic in the
mucosal system. Most of the monoclonal IgAs that we have
produced and analysed have been directed against micro­
bial surface components, and this is consistent with the pro­
posed function of slgA in immune exclusion. Mucosally
derived IgA hybridomas produce IgA primarily in dimeric
form and these antibodies are recognized by polymeric
immunoglobulin receptors on epithelial cells (Weltzin et al.,
1989). Thus they can be delivered into secretions of mice
in vivo or across epithelial monolayers in vitro via the
normal transepithelial transport system with addition of
ORAL IMMUNIZATION
NEUTRALIZATION
Fig. 3. Protocol for production o f IgA hybridomas and secretion
o f monoclonal slgA in vivo. After oral immunization of adult
mice, Peyer’s patch lymphocytes are recovered and fused with
myeloma cells. The resulting hybridomas are screened for
production of IgA antibodies of the desired specificities. Selected
clones of hybridoma cells are injected subcutaneously on the
backs of syngeneic mice, where they form hybridoma ‘backpack’
tumors that produce circulating dimeric IgA. The monoclonal IgA
is recognized by epithelial polymeric immunoglobulin receptors in
the liver and intestine, and is delivered into bile and intestinal
secretions via receptor-mediated transcytosis. This in vivo model
allows identification o f monoclonal IgA antibodies that can
protect against challenge with the corresponding pathogen.
secretory component (Winner et al., 1991; Subbarao and
Murphy, 1992) and can bind recombinant secretory com­
ponent in solution (Michetti et al., 1991). These methods
provide an unlimited source of protease-protected IgA anti­
bodies of known epitope specificities that can be separately
tested in protection assays.
To obtain continuous secretion of monoclonal IgA anti­
bodies into intestinal secretions and bile of mice via the
normal receptor-mediated epithelial transport system,
hybridoma cells are injected subcutaneously on the upper
backs of Balb/c mice, resulting in hybridoma ‘backpack’
tumors that release IgA into the circulation (W inner et al.,
1991). In tissues such as liver and intestine where capil­
laries are permeable, dimeric IgA binds to basolateral
epithelial poly-Ig receptors and is delivered into secretions
as monoclonal slgA (Fig. 3). We have used the backpack
tumor method to analyse the specificity and the mechanisms
of IgA protection against micro-organisms in the intestine,
using as models two enteric bacterial pathogens: Vibrio
cholerae, a non-invasive organism that colonizes the
mucosal surface and secretes cholera toxin, and Salmonella
typhimurium, an invasive pathogen.
IgA PROTECTION AGAINST EPITHELIAL
COLONIZATION
Vibrio cholerae causes severe diarrheal disease by coloniz­
ing the mucosal surface of the small intestine. When vib­
rios enter the luminal microenvironment the toxR regulatory
gene is activated resulting in expression of several virulence
factors including the pilus protein that promotes mucosal
Transepithelial transport by M cells
colonization, and cholera toxin (CT) (Miller et al., 1989).
Binding of CT to its glycolipid receptor GM1 on epithelial
cells initiates a series of intracellular events that result in
massive chloride secretion (Holmgren, 1981; Lencer et al.,
1992). V. cholerae evokes polyclonal secretory IgA anti­
bodies (slgA) directed against both CT and bacterial surface
components including the outer membrane lipopolysaccharide (LPS) (Svennerholm et al., 1984) and these are thought
to be involved in limiting the primary infection and pro­
viding protection against subsequent reinfection (Svenner­
holm et al., 1984; Jertbom et al., 1986). Using the hybridoma
backpack tumor method, we have shown that secretion of
monoclonal slgA directed against a strain-specific carbohy­
drate epitope of the surface LPS is sufficient to prevent diar­
rhea and death in mice after a lethal oral dose of V. cholerae
(Winner et al., 1991). Furthermore, a single oral dose of 5
mg of the same anti-LPS IgA protected suckling mice
against subsequent oral challenge for up to 3 hours, although
protection was lost as the IgA was cleared from the upper
small intestine by peristalsis (Apter et al., 1991).
To test the ability of IgA to block the effects of CT and
prevent diarrheal disease, hybridomas were generated that
produce monoclonal IgA antibodies directed against CT; all
of the IgAs recognized the B subunit and none was directed
against the binding site for GMi, the intestinal cell mem­
brane toxin receptor (Apter et al., 1991). These antibodies
were applied to a well-defined intestinal epithelial cell
monolayer culture system (Lencer et al., 1992; Dharmsathaphom and Madara, 1990) to directly measure protec­
tion against toxin action in vitro, and were tested in vivo by
passive oral administration or the backpack tumor method.
When applied to human T84 colon carcinoma cells grown
on permeable supports, the IgAs blocked CT-induced Clsecretion in a dose-dependent manner and completely inhib­
ited binding of rhodamine-labelled CT to apical cell mem­
branes (Apter et al., 1991). Oral feeding of the monoclonal
IgAs protected against oral CT administration in vivo. Both
in vitro and in vivo, however, high doses of IgA were
required for CT neutralization. In contrast, anti-CTB IgA
failed to protect suckling mice against a lethal oral dose of
live V. cholerae organisms, whether the antibodies were
delivered perorally or secreted intraintestinally in mice bear­
ing backpack tumors (Apter et al., 1991). These results cor­
roborate previous human studies in which volunteers orally
immunized with multiple large doses of purified CT were
not protected against oral challenge with V. cholerae,
whereas volunteers immunized orally with live V. cholerae
organisms were completely protected (Levine et al., 1979,
1983). Since CT is not a component of the bacterial surface,
anti-CT IgA alone is not likely to prevent V. cholerae col­
onization, and secretion of toxin from adherent organisms
on the mucosal surface could deliver high levels of CT
directly into the enterocyte glycocalyx and onto apical mem­
branes. Our results show that IgAs directed against the bac­
terial surface provide most efficient mucosal protection.
IgA PROTECTION AGAINST EPITHELIAL
INVASION
The disease caused by Salmonella typhimurium infection in
213
mice is similar to typhoid fever caused by S. typhi in
humans (Edelman and Levine, 1986). In both cases, organ­
isms are ingested orally, rapidly invade the intestinal epithe­
lium, proliferate in the mucosa, and then spread to the liver
and spleen resulting in a potentially lethal systemic disease
(Hohmann et al., 1978; Edelman and Levine, 1986). Entry
into the mucosa occurs first via transepithelial transport by
M cells (Kohbata et al., 1986) and then by adherence, inva­
sion and damage of absorptive enterocytes (Takeuchi,
1975). Immunization strategies designed to protect against
this disease are aimed at preventing the initial interaction
of bacteria with epithelial cells. Indeed, the presense of anti­
Salmonella IgA in intestinal secretions is known to be
closely correlated with protection in experimental animals
and humans (Chau et al., 1981; Edelman and Levine, 1986).
Using monoclonal IgA antibodies, we have recently demon­
strated that secretory IgA antibodies alone can provide this
protection.
A series of anti-Salmonella IgA hybridomas were gen­
erated from Peyer’s patch cells and a monoclonal poly­
meric IgA (Sal4) was characterized that recognizes a sur­
face-exposed carbohydrate epitope on wild type S.
typhimurium (Michetti et al., 1992). Mice bearing subcu­
taneous Sal4 hybridoma tumors secreted monoclonal slgA
into their gastrointestinal tracts and were protected against
a lethal oral challenge with this bacterium. This protection
was directly dependent on specific recognition by Sal4 IgA,
since mice secreting Sal4 IgA from hybridoma tumors
were not protected against an equally virulent S.
typhimurium mutant that lacks the epitope recognized by
Sal4. In these in vivo studies, secretion of monoclonal IgA
was shown to prevent the initial event in pathogenesis,
uptake of Salmonella by M cells into the Peyer’s patch
mucosa. We also used monolayer cultures of epithelial cell
lines (MDCK and HT29) to test IgA protection against
invasion of epithelial cells by Salmonella. It had been pre­
viously shown that invasion of MDCK cells in vitro cor­
relates closely with invasion of CaCo2 cells and with infec­
tion of the small intestinal mucosa in vivo (Finley and
Falkow, 1990; Finlay et al., 1988). Using these in vitro
models, we have demonstrated that specific IgA antibod­
ies can protect absorptive epithelia against invasive Sal­
monella in the absence of mucus, peristalsis and other pro­
tection mechanisms (Michetti et al., unpublished data).
Thus, IgA alone can prevent epithelial contact and mucosal
invasion.
CONCLUSION
In summary, our studies using monoclonal IgA antibodies
have confirmed that the principal role of slgA in the intes­
tine is to prevent contact of antigens and pathogens with
epithelial surfaces. We have established that slgA of appro­
priate specificity, if present in sufficiently large amounts,
can provide protection of the intestinal mucosa in the
absence of other immune protection mechanisms. Identifi­
cation of protective secretory IgA antibodies provides a
means to identify the microbial antigens that should be
included in effective oral vaccines. Elucidation of the mech­
anisms that govern adherence and transepithelial transport
214
M. R. Neutra and J.-P. Kraehenbuhl
by M cells will allow us to target such vaccines to the induc­
tive sites of the mucosal immune system.
We thank the former members o f our laboratories who con­
ducted much o f the work summarized in this review: Pierre
Michetti, Richard Weltzin, Helen Amerongen, Julie Mack, Scott
Winner and Felice Apter. We are indebted to our collaborators
John Mekalanos, Michael Mahon and James Slauch from the
Department o f Microbiology and Molecular Genetics at Harvard
Medical School. The authors are supported by NIH Research
grants HD17557, DK21505 and AI29378 and NIH Center grant
DK34854 to the Harvard Digestive Diseases Center (M.R.N.);
Swiss National Science Foundation grant 31.246404.89 and Swiss
League against Cancer grant 373.89.2 (J.P.K.).
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