Departments of Pathology and Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, California 92697-4800
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ABSTRACT |
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The hypothesis that epithelial cells release
preformed antibiotic peptides as components of mucosal innate immunity
has gained experimental support in recent years. In the mammalian small
intestine, Paneth cells secrete granules that are rich in -defensins
and additional antimicrobial peptides into the lumen of the crypt. The
-defensins are homologues of peptides that function as mediators of
nonoxidative microbial cell killing in phagocytic leukocytes, and they
are potent microbicidal agents in in vitro assays. Because certain
mouse
-defensins stimulate cultured epithelial cells to secrete
chloride ion, those peptides appear to be capable of interacting
directly with the apical membranes of neighboring cells and perhaps
influencing crypt physiology. In instances of crypt disruption or
induced Paneth cell deficiency, crypt intermediate cells appear to
compensate by accumulating and secreting Paneth cell antimicrobial
peptides. Challenges for the future will be to understand the
mechanisms of this epithelial plasticity and to show that Paneth cells
contribute directly to innate immunity in the crypt microenvironment.
innate immunity; crypt epithelium; cryptdins
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INTRODUCTION |
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THE EPITHELIAL MONOLAYER that lines the mammalian small intestine is both the route of nutrient absorption and an active barrier between the external environment and the circulation. At first appearances, adaptations of the small bowel to improve nutrient uptake by amplification of absorptive surface area would seem to increase the risk of mucosal colonization by potential pathogens. Nevertheless, the bacterial load of the small bowel remains very low relative to that of the colon.
Many factors contribute to the low bacterial numbers of the small intestine. Examples include the normal physiological activities of the gut such as motility, secretion of digestive and pancreatobiliary juices, mucous secretions of goblet cells, and humoral and cytotoxic immune responses of B and T lymphocytes to specific antigens. In addition, however, epithelial cells are known to actively release gene-encoded, antibiotic peptides that contribute to a biochemical barrier against microbial colonization. In addition to the studies of small intestinal antimicrobial peptides to be reviewed briefly here, several investigators have shown that the mucosa of the airway, skin, gingiva, tongue, cornea, reproductive tract, urogenital tract, and colon also participate in innate immunity (6).
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ANTIMICROBIAL PEPTIDES |
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The production of antimicrobial proteins and peptides is widely distributed phylogenetically. Originally described as responses of circulating hemocytes to cuticle injury in insect larvae, antimicrobial peptides are now recognized as effectors of a biochemical barrier against potential pathogens in plants, insects, amphibia, teleosts, and mammals (15). Selective pressure for the synthesis and release of antimicrobial peptides in response to infection, or the constant threat of infection, appears to have been widespread, since peptides with potent in vitro antimicrobial activities have been isolated from all phyla examined, including plants, invertebrates, and species lacking clonal immune mechanisms (14). These preformed peptide antibiotics are highly variable in primary structure, and they may be inducible, synthesized continually and accumulate in cytoplasmic granules for regulated secretion, or they may be released on a constitutive basis.
Gastrointestinal expression of antimicrobial peptides also is evolutionarily conserved. In the gastrointestinal tract of the frog, for example, magainins, the primary antimicrobial peptides of frog skin, are found in stomach glands, and they also are present in glands at the base of epithelial folds in the frog intestine (32). In invertebrates, midgut production of insect defensins becomes activated in the mosquito and Stomoxys calcitrans after consumption of a blood meal (5, 21), and the midgut of Manduca sexta larvae contains antimicrobial peptides in granules (21). These findings suggest that the production and secretion of antimicrobial peptides by mammalian intestinal epithelia is a conserved innate immune mechanism rather than a recent evolutionary development.
Most antimicrobial peptides expressed by mammalian epithelial cells are members of peptide families that mediate nonoxidative microbial cell killing by phagocytes (15). In polymorphonuclear leukocytes, the peptides are stored in the azurophilic granules, and they mediate killing of ingested microorganisms following phagolysosomal fusion. Epithelial peptides, on the other hand, appear to function in the extracellular compartment at the interface with the external environment (26). The colonic, airway, and reproductive epithelial cells appear to release antimicrobial peptides continually or constitutively. In the small intestine, microbicidal peptides accumulate in secretory granules of Paneth cells for apical release by regulated exocytosis. Because epithelial antimicrobial peptides are homologous to peptides in the azurophilic granules of neutrophils, e.g., defensins, and because those peptides are microbicidal in in vitro assays, they are implicated in immunity at the mucosal surface.
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ANTIMICROBIAL PEPTIDES IN PANETH CELLS |
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Paneth cells are located at the base of the crypts of Lieberkühn
in the small intestine of many mammalian species. They are recognized
by the unusually large apical secretory granules that they release into
the crypt lumen. Most small intestinal crypts are populated by Paneth
cells, and, in mice, amplification of -defensin cDNA sequences in
isolated crypts by RT-PCR has shown that every crypt contains Paneth
cells or Paneth cell transcripts (3). Aside from their unusual
morphology, Paneth cells have certain characteristic features. For
example, although they originate from the same crypt stem cells that
generate all intestinal epithelial cell lineages, they differentiate
during downward migration from the proliferative zone (2). In contrast,
the other epithelial cell populations differentiate terminally as they
ascend the crypts and migrate along the villi toward their eventual
exfoliation. Also, Paneth cells have a 20 day average lifespan, unlike
villous enterocytes that apoptose and exfoliate into the lumen 2-5
days after emergence from the stem cell zone. Histologically, normal Paneth cells develop under germ-free conditions and from murine or
human fetal intestinal xenografts in nude mice (20), demonstrating that
Paneth cell ontogeny does not require luminal bacteria or dietary constituents.
Insights into Paneth cell function have been provided by analyses of
their gene products. For example, secretion of lysozyme and homologues
of phagocytic antimicrobial peptides implicates Paneth cells in enteric
host defense. Specifically, Paneth cell lysozyme, secretory
phospholipase A2
(sPLA2), and -defensins have
well-established antimicrobial activities when assayed in vitro (16).
In mice, these gene products appear during postnatal crypt ontogeny,
coincident with Paneth cell differentiation, and they are useful
markers of the lineage. The secretion of this array of peptide
antibiotics by the Paneth cell, the defensins in particular, is
consistent with an innate immune role.
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DEFENSINS |
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Two known defensin peptide subfamilies have been well described, the
- and
-defensins. Both subfamilies comprise cationic, 3- to 4-kDa
peptides that contain six cysteine residues in three disulfide bonds.
Although their tridisulfide arrays differ in their Cys-Cys pairings,
- and
-defensins have remarkably similar folded conformations
(29). The mammalian
-defensins are major constituents of the primary
granules in phagocytic leukocytes of myeloid origin, and they were one
of the first antimicrobial peptide families to be recognized (6). The
-defensins occur in more tissues than the
-defensins, but they
have not been detected in Paneth cells to my knowledge. They are
expressed in bovine bone marrow and in the mucosa of the colon, airway,
tongue, kidney, skin, and gingiva in humans and in other species.
Inhibition of human
-defensin-1 antibacterial activity by the high
ionic strength of airway surface fluid of cystic fibrosis patients has
been implicated in microbial pathogenesis associated with the disease
(4); nevertheless, the mechanisms involved remain unresolved. Several years subsequent to their discovery in neutrophils,
-defensins were
identified in mouse and human Paneth cells (16).
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ENTERIC ![]() |
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-Defensins are abundant constituents of mouse and human Paneth cell
granules. Human Paneth cells code for two
-defensin peptides, HD-5
and HD-6, and mice express numerous Paneth cell
-defensin isoforms,
termed "cryptdins" for crypt defensin. Six cryptdins have
been purified to homogeneity (16). Immunohistochemical studies of small
intestinal sections have shown that antibodies to mouse cryptdin-1 and
recombinant HD-5 react exclusively with Paneth cells of the respective
species (17, 25). Immunogold detection experiments show that the
antigen is distributed uniformly in Paneth cell granules (17). In mice,
individual isoforms differ in relative abundance in that whole organ
recoveries of cryptdins-1, -2, -5, and -6 are equivalent but levels of
cryptdins-3 and -4 are much lower. The primary structures of
cryptdins-4 and -5 diverge markedly from each other and from the
majority of cryptdins that are variably substituted variants of
cryptdin-1 (16). Cryptdins-2 and -3, for example, differ in sequence
only at position 10 (Thr vs. Lys, respectively). Cryptdin-4 is the most
cathodal enteric defensin and the first
-defensin to contain a
chain-length variation between the fourth and fifth cysteine residues.
Furthermore, cryptdin-4 gene is unique because it is expressed
differentially along the length of the small bowel, since
cryptdin-4 mRNA and peptide are not detected in duodenum but occur at
highest levels in distal ileum. The mechanisms regulating this
pattern of expression are not known.
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Most of the defensin peptide in mature phagocytic cells appears to be
fully processed via a pathway that involves two primary cleavage steps
(7). The human -defensin precursors are cleaved sequentially over a
4- to 24-h period to yield intermediates of 75 and 56 amino
acids, and agents that neutralize the acidic subcellular compartment
diminish the conversion rate. The anionic charge of many
-defensin
precursor prosegments has been suggested to have a role in neutralizing
the basic charge of the functional peptide. Also, addition of the
propeptide to in vitro antimicrobial peptide assays inhibits neutrophil
-defensin activity, suggesting that the prodomain may be
cytoprotective. Deletional mutations in the COOH-terminal region of the
HNP-1 human neutrophil
-defensin prosegment impair pro-HNP-1
processing and targeting to granules, suggesting that residues proximal
to the cleavage site may be involved in the recognition and cleavage
steps. Matrilysin (MMP-7), a metalloproteinase expressed abundantly by
Paneth cells of mouse small bowel (30), has been identified as the
enzyme that processes and activates mouse cryptdin precursors
posttranslationally (C. L. Wilson, A. J. Ouellette, D. P. Satchell, T. Ayabe, Y. S. López-Boado, J. L. Stratman, S. J. Hultgren, L. M. Matrisian, and W. C. Parks, unpublished observation). This
finding is consistent with the processing and activation of members of
the large and diverse procathelicidin antimicrobial peptide family by
neutrophil elastase (24).
Paneth cell -defensins are coded by highly conserved genes that
consist of two exons separated by an intron of ~500 bp, and transcripts of these genes are ~1 kb in length. The 5'
untranslated region, signal peptide, and prosequence are coded by exon
1, and exon 2 encodes the
-defensin peptide and the 3'
untranslated region. Paneth cell
-defensin genes map in proximity to
the myeloid
-defensin and
-defensin genes at 8p21-8pter in
humans and at an homologous locus on proximal chromosome 8 in mice.
Levels of
-defensin mRNA and peptide appear to be approximately the
same in human and mouse Paneth cells, but a series of gene duplication events expanded the gene family in mice, producing over 20 different isoforms. Genetic evidence shows that 17 of these isoforms are expressed in a single crypt from mouse jejunum. However, intestinal levels of deduced cryptdin-7 to cryptdin-19 are judged to be much lower
than those of cryptdins 1-6, based on the low relative cloning frequency of these cDNAs and the fact that cryptdin-7 to cryptdin-19 have not been recovered as abundant peptide components of intestinal extracts.
Peptides recognized originally as Paneth cell -defensins also are
expressed by nonintestinal epithelia. For example, HD-5 transcripts and
peptides have been found in human female reproductive tract epithelium
(19, 27). Cryptdins are present in Leydig cells and Sertoli cells of
mouse testis (9), including an
-defensin mRNA that has not
been detected at the cDNA or peptide level in mouse gut (Ouellette,
unpublished observation). Similarly,
-defensins isolated from rabbit
kidney bear little resemblance to the rabbit myeloid
-defensins,
suggesting that the renal epithelium, a well-established site of
expression and release of
-defensins (4), may express
-defensins as well (1, 31). Collectively, these findings suggest that
-defensins are potential mediators of innate immunity on numerous mucosal surfaces.
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ANTIMICROBIAL ACTIVITIES OF PANETH CELL
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Mouse and human Paneth cell -defensins are potent antimicrobial
agents with selective activities against several varied microbial cell
targets. A recombinant form of HD-5 is active against several species
of bacteria as well as C. albicans,
and the peptide remains active even after partial proteolysis, an
indication of its potential to function within the environment of the
intestinal lumen (18). In vitro assays of cryptdin antibacterial
activities showed that each of the six peptides was microbicidal,
except for cryptdin-2 (16). In suspension, E. coli ML35 cells were killed rapidly by cryptdins-1 and
cryptdins-3 to -6, and, in each case, <1% survival was observed
after 15 min of incubation. Because cryptdins-2 and -3 differ only at
amino acid position 10 (Thr vs. Lys, respectively), that residue
position appears to modulate killing activity in the context of those
particular primary structures. Trophozoites of Giardia
lamblia are highly sensitive to cryptdins-2 and -3, but
cryptdins-1 and -6 have little effect on survival of this enteric
protozoal pathogen as determined by trypan blue exclusion (16). Comparing the primary structures of these peptides implicates amino acid residue 15 in this activity because giardicidal cryptdins-2 and -3 contain arginine at position 15 but inactive cryptdins-1 and -6 contain a glycine at residue 15. By analogy with the crystal structure
of neutrophil defensin HNP-3, amino acid 15 is predicted to be on the
peptide surface in a conserved turn and possibly important in
interactions with eukaryotic cell envelopes (16).
The mechanisms of Paneth cell defensin antimicrobial activity are not
known, but, by analogy with neutrophilic -defensin homologues, it is
likely that they permeabilize the target cell envelope leading to
dissipation of electrochemical gradients. The human and rabbit
neutrophil
-defensins achieve cell killing by membrane disruption,
but, as peptide-to-membrane interaction studies suggest, the details of
the killing mechanism may be very different from peptide to peptide.
Despite their conserved tridisulfide structure, amphipathicity, and
-sheet backbones, human neutrophil
-defensin HNP-2 is a
noncovalent dimer, but NP-1, an
-defensin from rabbit neutrophils,
is a monomer (29). Functionally, the dimeric HNP-2 peptide forms large,
~20 Å, stable multimeric pores after insertion into model
membranes; in contrast, NP-1 generates short-lived defects in
phospholipid bilayers. The interactions of six rabbit neutrophil
-defensins with large unilamellar vesicles of defined lipid
composition showed that the membrane phospholipids, cardiolipin in
particular, strongly affect their permeability to individual
antimicrobial peptides (10). Given these considerations, it seems
prudent to avoid predicting mechanisms for microbial cell killing by
Paneth cell
-defensins.
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BIOLOGY OF PANETH CELLS AND ![]() |
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Paneth cell secretion can be stimulated by cholinergic agonists that appear to act by activation of G proteins coupled to muscarinic receptors (22). Pilocarpine, bethanachol, and the nonspecific G protein activators NaF and AlCl3 induce massive Paneth cell degranulation (23), and muscarinic antagonists inhibit Paneth cell degranulation. In vitro, carbamylcholine interacts directly with isolated mouse ileal crypts, specifically mobilizing cytosolic intracellular calcium in Paneth cells and not affecting calcium flux in the other epithelial cell populations of the crypt. The coupling of specific G proteins to Paneth cell muscarinic receptors remains to be demonstrated, but these findings are consistent with their involvement in the regulation of Paneth cell secretion.
Physiological salt and water secretion from the intestine depends on
the activation of chloride channels in the apical membrane of
epithelial cells lining the crypt, the secretory gland of the intestine. Evidence suggests that mouse Paneth cell -defensins may
participate in that process, since cryptdins-2 and -3 activate chloride
channels when applied apically to monolayers of human T84 intestinal
epithelial cells (13). The chloride secretory effect is reversible,
time and dose dependent, and isoform specific, since cryptdins-2 and -3 can form the channel but cryptdins-1 and cryptdins-4 to -6 cannot under
identical conditions. If cryptdins elicit chloride secretion in vivo,
the process would stimulate fluid movement within the crypt, possibly
flushing the lumen and distributing
-defensins and other crypt
secretions to the villus surface. The variety of biological activities
identified for individual neutrophil
-defensin peptides, i.e.,
chemotaxis for monocytes and T lymphocytes, inhibition of natural
killer (NK) cells in vitro, modulation of intestinal and
renal cell volume, alteration of epithelial monolayer barrier
integrity, and the fact that certain peptides may be cytotoxic to
mammalian cells suggest that Paneth cell
-defensins may also be multifunctional.
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PANETH CELL DYNAMICS IN THE CRYPT |
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Conditions that disrupt normal crypt cell biology appear to recruit new
cells to activate Paneth cell -defensin genes and accumulate the
corresponding peptides. For example, in mice that express attenuated
diphtheria toxin A fragment or SV40 large T antigen transgenes under
the control of a functional mouse cryptdin-2 gene promoter, the crypts
undergo a transient Paneth cell deficiency (8). Up to 8 wk of age,
small intestinal crypts of transgenic mice lack apparent Paneth cells,
and the crypts are occupied by undifferentiated crypt columnar cells.
During this period of Paneth cell deficiency, numbers of intermediate
cells and granule-containing goblet cells increase, and those cells
accumulate electron-dense, secretory granules that contain elevated
levels of cryptdin(s) and sPLA2
(8). Thus Paneth cell deficiency appears to induce a compensatory
response in crypt intermediate cells, altering their genetic repertoire
to produce and secrete Paneth cell antimicrobial peptides. These mice
may be useful for determining the levels of
-defensin expressed by
these activated intermediate cells, for learning whether defensins or
newly activated antimicrobial peptide gene products are made during
Paneth cell deficiency, and for characterizing the responses of the
transgenics to oral challenge with microbial pathogens.
Recent findings suggest that the small intestinal crypt epithelium may communicate with and respond to T lymphocytes. For example, infection of mice with Trichinella spiralis induces intestinal goblet cell hyperplasia that appears to be mediated by T helper cells (11). During the course of infection with this parasite, the number of Paneth cells and goblet cells in crypts increases (M. Kamal, D. Wakelin, and Y. R. Mahida, personal communication). Furthermore, Z. Alnadjim and T. A. Barrett of Northwestern University Medical School have shown that T cell activation results in rapid and dramatic crypt cell apoptosis followed by an approximately threefold increase in the number of crypt cells positive for eosinophilic granules (personal communication). It should be of interest to define the soluble mediators and the signaling pathways that induce the crypt epithelium to modify its genetic programs.
Collectively, the experiments in Paneth cell-deficient transgenic mice, in Trichinella infection, and in the experimental T cell activation models suggest that the crypt epithelium is capable of some plasticity with respect to the repertoire of genes that crypt cell populations may express. Part of that response includes a compensatory increase in production of secretory products, including antimicrobial peptides that normally are expressed at significant levels only by Paneth cells. Studies to identify the mechanisms of that apparent plasticity, to determine its relation to innate immunity in the crypt microenvironment, and perhaps to employ these gene products as markers of immunopathogenesis of gastrointestinal diseases may prove to be informative.
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ACKNOWLEDGEMENTS |
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I thank Drs. Terrence A. Barrett, Charles L. Bevins, Wayne Lencer, Yashwant R. Mahida, Michael E. Selsted, and Carole L. Wilson for useful discussions and for permission to cite their unpublished findings.
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FOOTNOTES |
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* Fourth in a series of invited articles on Mucosal Immunity and Inflammation.
This work was supported by National Institutes of Health Grants DK-44632, DK-33506, and HD-31852.
Address for reprint requests and other correspondence: A. Ouellette, Dept. of Pathology, D-440, Med Sci I, College of Medicine, Univ. of California, Irvine, CA 92697-4800 (E-mail: aouellet{at}uci.edu).
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