Yuan, Qian; Walker, W. Allan
Innate and adaptive immunity are the two essential elements in the host's immune defense mechanism. Innate immunity is evolutionally highly conserved across species from fruit flies to man, whereas adaptive immunity is a highly sophisticated and specialized defense mechanism that developed late in evolution. The innate immune system is the first line of defense against invading microorganisms. It serves a prominent protective function in all tissues and organs, especially the intestinal tract, the genitourinary tract, the respiratory tract and the skin where there is greater exposure to the external environment and to foreign antigens. Innate immunity can provide for (1) the recognition of microbial organisms as foreign; (2) incapacitation of pathogens and (3) adjuvant magnification of the acquired immune response when such a response is warranted (1). Thus the innate immune system not only provides an immediate non-specific response against invading microorganisms but also serves as a bridge to the adaptive immune system by sending messages that activate immune cells such as T lymphocytes that can then mount an antigen specific adaptive immune response. Adaptive immunity uses T and B lymphocytes to mediate and amplify antigen-specific humoral and cellular responses. However, these responses often take days to weeks to achieve maximal activity and require a somatic gene rearrangement that results in immunologic memory. In general, innate immune defense is based on a complex cellular network including both tissue resident cells and migratory cells attracted to the tissue sites, such as Langerhans cells of the skin, tissue dentritic cells and macrophases, natural killer cells (NK) and γδ T cells of the gut.
In contrast to adaptive immunity, the innate immune system provides a relatively non-specific, but immediate protection against invading pathogens and does not generate immunologic memory (2). The mucosal surface of the intestine constantly encounters foreign antigens and pathogenic microorganisms and is an active site for immune suppression of unnecessary harmful reactions (oral tolerance) and for generation of protective responses (acute, self limited inflammation) that maintain immunologic homeostasis in the host. The innate immune system can recognize conserved pathogen-associated molecular patterns (PAMPs) on microorganisms through the Toll-like receptors (TLRs). The TLRs are crucial for the activation of the dentritic cell, a professional antigen-presenting cell (3). TLR-mediated pathogen recognition on dentritic cells leads to a Th1 type lymphocyte response (4). Thus, innate immunity not only acts as an acute non-specific immune defense mechanism, but also is involved in regulating adaptive immune responses.
In addition to a complex cellular network, a number of other components are also critical to the function of the innate immune system of the gut (Table 1). The mechanical barrier function of the intestinal epithelium actively and continuously provides protection against invasion by pathogenic microorganisms residing in the lumen. Therefore, processes that promote the rapid repair of damaged epithelial cells act to maintain barrier function and to prevent invasion by pathogens that cause harm to the host. In addition, various antimicrobial peptides produced by intestinal epithelial cells result in prompt suppression or death of invading microorganisms. The recent progress made in identifying pattern recognition receptors for bacteria, such as TLRs and the caspase recruitment domain (CARD) 15/nucleotide oligomerization domain (NOD) 2 has provided exciting insight into one of the crucial steps in the innate immune response (e.g., host recognition of harmful bacteria).
Dysfunction of any of the components in the innate immune system can lead to a critical impairment of the host's mucosal immune defenses. For example, mutations of the CARD 15/NOD2 can cause chronic intestinal inflammation. This mutation is thought to play an important role in the pathogenesis of Crohn's disease as a result of an abnormal host response to commensal flora of the gut. Damage of the mucosal epithelia with disruption of the mucosal barrier can lead to enhanced invasion by additional intestinal microorganisms. In animal models, a mutation of the metalloproteinase matrilysin (MMP7) enzyme, which causes impaired function of one of the intestinal antimicrobial peptides, defensin, can result in an increased susceptibility to Salmonella infection and a higher mortality rate (5). In this review, we will provide an update on the functions of some of the crucial elements of the innate mucosal immunity system, including TLRs, CARD 15/NOD2, defensins and the scavenger receptor DMBT1, and we will review recent observations that demonstrate their potential importance in human intestinal diseases.
The Toll-Like Receptor Family
The Toll family of receptors was first described in Drosophila. It was found to be important for dorsal-ventral orientation during development of the Drosophila embryo, and it was also found to provide protection against microbial invasion in the adult (6). Since the fruit fly, Drosophila melanogaster, does not have an adaptive immune system and relies entirely on innate immunity for anti-microbial responses, the Toll family plays a pivotal role in immunity in this organism. A number of human homologs of the Drosophila Toll receptors have been identified in humans and are called TLRs. At present, there have been at least ten mammalian TLRs identified and cloned (6–13). Structurally, all mammalian TLRs are type I transmembrane receptors with extracellular leucine-rich repeats (LRR) and an intracellular signaling domain known as the TIR domain, which have a high degree of homology with Drosophila Toll and with the mammalian IL-1 receptor (IL-1R) (Fig. 1) (14). The intracellular TIR domains of the TLRs initiate a cascade of signals that result in the production of inflammatory mediators such as interleukin 8 (IL-8) and other chemokines (Fig. 2). The innate immune system recognizes conserved PAMPs through TLRs expressed on the cell surface of immune cells and enterocytes (Fig. 1) (15–19). The interaction between TLRs and their ligands transmits signals from the surface of the cell into the cytoplasm resulting in an activation of nuclear factor κB (NF-κB), a transcription factor that activates cytokine production and upregulates costimulatory molecules in phagocytes for antigen presentation. This process ultimately leads to activation of T cells in the adaptive immune system and enterocyte secretion of cytokines. A recent review in the Journal of Pediatric Gastroenterology and Nutrition summarizes TLR interaction with endotoxin (LPS) (20) and will not be discussed in detail here.
Interplay of Innate and Adaptive Immunity Through Toll-Like Receptors
Dentritic cells (DC) reside in an immature state in most tissues and organs and are critical in detecting invading pathogens (21). Dentritic cells can be characterized as either immature or mature. Immature DCs have a high endocytotic capacity and low expression of MHC and costimulatory molecules on their surface, and mature DCs have a high endocytotic capacity and an enhanced surface expression of costimulatory molecules. In addition, mature DCs have the capacity to secrete cytokines and chemokines. The function of dentritic cells is not primarily to destroy pathogens but to present pathogen-associated antigens to T cells thereby activating the adaptive immune response (19). Activation and maturation of immature dentritic cells is triggered by microbial pathogens. Upon activation, the DC expresses pathogen peptides with MHC on its surface and traffics from the site of infection to regional lymph nodes where it produces T cell activation. The interaction of TLRs and PAMPs is essential to dentritic cell activation and maturation (3). TLR-mediated recognition of pathogen-associated molecular patterns and induction of co-stimulation of DCs is required to direct T cell responses against pathogen-derived antigens in a cognate interaction between T cells and APC cells. Production of IL-6 by DCs in response to TLR ligation during infection is critical for T cell activation because it allows pathogen-specific T cells to overcome the suppressive effect of CD4+CD25+ regulatory T cells (22). Therefore, TLRs are important in both the innate immune response of pathogen pattern recognition and in the activation of the adaptive immune response. A recent observation has suggested that submucosal immature dendritic cells can extend an appendage through enterocyte tight junctions and sample pathogens within the lumen. This interaction mediated by TLRs results in maturation of the dendritic cell, antigen presentation and release of cytokines, which determines the T-helper cell phenotype (either Th1 or Th2) (Fig. 3).
The development of immune tolerance to innocuous foreign antigens and luminal commensal microorganisms crossing the mucosal surface is a critical protective mechanism of mucosal defense. This response appears to be mediated by TLRs. Naturally occurring mutations have been identified in the TLR4 gene in C3H/HeJ and C57BL/10ScCr mouse strains (23,24). Complete deletion of TLR4 function results in increased susceptibility to Gram-negative infections. The genetic variations or polymorphisms associated with TLR4 are important in the variations in susceptibility to sepsis across and within species (25–27). TLR4 mutation renders C3H/HeJ mice hyperresponsive to oral peanut antigen with impaired immune tolerance in a murine peanut allergy model (28). In human asthma patients, missense mutations in the extracellular domain of the TLR4 result in a blunted airway response to inhaled LPS (29). Polymorphisms in TLR2 may be associated with diminished in vitro responses to bacterial lipoprotein, a known TLR2 ligand (30). These mutations, if identified in patients, might help explain the increased incidence of allergic conditions or certain infectious disease over the last several decades. Additional research on TLRs and similar receptors may help to define susceptibility to clinical disease and design preventive therapy.
Identification of NOD2/CARD15 Molecules
Although genetic factors have long been thought to play a role in the pathogenesis of Crohn's disease, there had been no confirmation of this consideration until the recent identification of NOD2/CARD15, an intracellular receptor for bacterial endotoxin (lipopolysaccaride, LPS). Using a positional cloning strategy, the nucleotide oligomerization domain 2 (NOD2) gene was identified on the human chromosome 16q12 within the area of strongest linkage to Crohn's disease (31). NOD2, recently renamed as caspase activating recruitment domain (CARD) 15, belongs to a family of NOD1/apoptotic protease activating factors (Apaf-1) proteins that regulate apoptosis. Some of them also activate nuclear factor κB (NFκB) (32,33). Structurally, NOD2/CARD15 encodes a cytoplasmic protein with at least three domains: two amino terminal CARDs linked to a nucleotide-binding domain (NBD) and a carboxy terminus component containing leucine rich repeats (LRR) (Fig. 4). This LRR domain can confer responsiveness to bacterial products (e.g., microbial molecular patterns such as LPS, flagellin, etc.) (Table 1).
Functions of NOD2/CARD15
NOD2/CARD15 was initially thought to be involved in the lipopolysaccharide (LPS) response pathway (34). A recent study suggests, however, that bacterial peptidoglycan, a component of both Gram negative and positive bacteria cell walls, is the main pathogen associated molecular pattern that induces NFκB activation through CARD15 (35). NOD2/CARD15 acts as an antibacterial factor in Caco-2 intestinal epithelial cells by preventing invasion of Salmonella typhimurium, whereas mutant forms of CARD15 lose the protective effect (36). Initially, NOD2/CARD15 expression was considered to primarily exist in monocytes (33), but recent data have shown that its expression can be identified in both dentritic cells and intestinal epithelial cells (37,38). In analyses of ileal and colonic tissues from IBD patients, CARD15 expression was found to be localized to Paneth cells within the ileum or in metaplastic Paneth cells within the colon (39,40). NOD2/CARD15 expression is enriched in crypts compared with villi. Cells expressing NOD2/CARD15 also strongly express and secrete TNF-α, a potent stimulus to induced NOD2/CARD15 expression (40). It has also been suggested that NOD2/CARD15 may be involved in the degranulation of Paneth cells, an important innate immune response in the intestinal lumen (41).
NOD2/CARD15 in Crohn's Disease
More than 60 sequence variants have been identified in the NOD2/CARD15 gene. Three main variants (R702W, G908R, and 1007fs) seem to be associated with susceptibility to Crohn's disease (31,42–46) (Fig. 5). The majority of Crohn's disease associated mutations directly affect the LRR domain, which is a motif common to bacterial resistance R protein in plants and mammals, notably the TLR family which enables recognition of PAMPs. These data suggest that a defect in the host response to peptidoglycan may have some role in Crohn's disease susceptibility. It is possible that mutations in the LRR domain affect its sensing function to intraluminal bacteria leading to aberrant activation of the NFκB/Rel pathways and the production of inflammatory cytokines. In Crohn's disease, mutations of the NOD2/CARD15 gene are most tightly associated with ileal involvement, young age of onset, more extensive granuloma formation and perhaps also with fistulizing and/or fibrostenotic phenotypes (46–48). Inheritance of Crohn's disease occurring as a result of NOD2/CARD15 polymorphisms is manifested somewhere between a gene dosage and an autosomal recessive phenotypic disease expression (49). Heterozygotes have a relatively small increase in risk (of the order of that of smokers) of developing Crohn's disease, whereas homozygotes and compound heterozygotes have a much greater relative risk (49). In a recent study of CARD15 mutations in 55 pediatric patients with Crohn's disease in Saxony, Germany (50), 14 different polymorphic and/or disease-related nucleotide alterations were identified and 65% of the genomic DNA samples harbored at least one of 6 mutations in the CARD15 gene. The cytosine insertion mutation 3020insC was significantly more common in children with Crohn's disease than healthy controls (36% vs. 4%). Patients with at lease one of the 6 CARD 15 disease-associated mutations demonstrated a high risk of inflammation located in the terminal ileum and ascending colon, and in 10 of 19 patients with two mutations, intestinal resection was necessary because of stricturing (50). The genotype-phenotype correlation suggests that it may be possible to predict the outcome in a child with Crohn's disease based on the CARD 15 mutation(s) carried by the patient. Analysis of disease-associated CARD 15 mutations may facilitate more prompt and accurate diagnosis in children with equivocal clinical presentations. Finally, in high risk populations, it may be helpful to identify children carrying the mutations even before the onset of clinical manifestations of disease and especially if new therapeutic interventions become available to prevent the occurrence of Crohn's disease.
ANTIMICROBIAL PEPTIDES (DEFENSINS)
Paneth Cells and Antimicrobial Peptides
Antimicrobial peptides are a group of host defense effector molecules in the innate immune system. They can be divided into general categories: (1) peptides released into internal fluids such as lymph; (2) peptides localized within circulating phagocytic cells and (3) peptides released onto mucosal surfaces (51). These antimicrobial peptides generally function by disrupting the bacterial cell membrane. Interaction of positively charged (cationic) antimicrobial peptides with negatively charged (anionic) elements of the bacterial membrane (including LPS in gram-negative bacteria, polysaccharides such as teichoic acid in gram-positive bacteria and phospholipids such as phosphotidyl-glycerol) results in insertion of these peptides into the membrane and creation of membrane pores. These pores then lead to disruption of energy and ionic gradients across the membrane with subsequent cell lysis (52,53).
Paneth cells are specialized epithelial cells located in clusters at the base of crypts of Lieberkühn in the small intestine and in close proximity to epithelial stem cells (54). Paneth cells are most numerous in the terminal ileum and are recognized by unusually large apical eosinophilic secretory granules. These secretory granules are rich in antibiotic peptides, including lysozyme (55), secretory phospholipase A2 (56,57), α-defensins (58–60), trypsin (54) and angiogenins (61). Paneth cells release secretory granules into the crypt lumen when stimulated by cholinergic agonists, such as pilocarpine and nethanochol (62,63) and bacterial stimuli (60,64). The anatomically close proximity of Paneth cells to epithelial stem cells in the crypts led to an initial hypothesis that Paneth cells protect stem cells by releasing antimicrobial peptides to destroy luminal pathogens which might injure the stem cell. In a recent study, NOD2/CARD15 expression was noted in the Paneth cell in close proximity to its secretory granules (39). This close proximity prompted speculation that NOD2/CARD15 might be involved in degranulation and peptide release by Paneth cells in response to bacterial molecular patterns. Therefore, Paneth cells are critical contributors to innate immune defense in the intestine (65).
Defensin Family of Antimicrobial Peptides
Defensins are a family of evolutionarily related vertebrate antimicrobial peptides (3–4 kDa, positively charged, arginine-rich) with a characteristic β-sheet-rich fold and a framework of six disulfide-linked cysteines forming three disulfide bridges (52). There are two main defensin subfamilies, α- and β-defensins, which differ in the length of peptide segments between the six cysteines and the pairing of the cysteines connected by disulphide bonds (52). Defensins are encoded by single genes as pre-propeptides that are then processed to their active forms by enzymetic cleavage. α-defensins consist of 29 to 35 amino acid residues and are shorter than β-defensins which consist of 38 to 42 residues. Defensins are abundant in cells and tissues involved in innate host defense against microbial infections, including leukocytes, macrophages and intestinal Paneth cells (Table 2). α-defensins are produced and stored as pre-propeptides in mature neutrophils and Paneth cells whereas β-defensins are constitutively expressed in the epithelial compartment and can be induced to higher levels of expression upon infection or inflammation. Therefore, Paneth cell α-defensins and epithelial cell β-defensins exert their antibacterial effect in the intestinal lumen whereas the neutrophil α-defensins serve as an intracellular antimicrobial peptide.
α-defensins are abundant constituents of mouse and human Paneth cell granules. Human Paneth cells have two α-defensin peptides, human defensin 5 and 6, whereas mice express numerous Paneth cell α-defensin isoforms termed “cryptdins” (66). Human defensin 5 is stored in Paneth cell granules in its precursor form. The process of activation to its mature form likely occurs in the crypt lumen upon granule secretion. Trypsin released from Paneth cells after microbial or inflammatory stimuli acts as a critical prodefensin convertase that mediates the conversion of precursor human defensin 5 into its mature form (67). Defensin gene expression is developmentally regulated and exhibits species-specific tissue distributions. For example, human α-defensins are found in neutrophils and Paneth cells but not in macrophages, whereas rabbit α-defensins are expressed in both neutrophils and macrophages and mouse α-defensins are present in Paneth cells and testes (1). Human α-defensin (HD) 5 and 6 are expressed in the developing fetus as early as the 13th week of gestation (detected by RT-PCR) with HD-5 present in both the small intestine and colon and HD-6 present only in the small intestine (68). By week 17 of gestation, both HD-5 and HD-6 expression is limited to the small intestine. By week 24, enteric defensin expression reaches levels detectable by Northern blot mRNA analysis. In the late second trimester of gestation, both the number of Paneth cells and the level of enteric α-defensin mRNA are significantly lower than in the adult. In contrast, mouse α-defensins of the small intestine (cryptdins) are present at low levels before birth but rapidly increase during weaning (69–71).
Anitmicrobial Functions of Defensins
Paneth cell α-defensins have anti-microbicidal activity against a variety of microbes. Recombinant Paneth cell human defensin 5 is active against Listeria monocytogenes, Escherichia coli, Salmonella typhimurium, and Candida albicans (72). A developmental profile of the expression of human enteric defensins 5 and 6 demonstrated that enteric defensin mRNAs were expressed at extremely low levels during fetal life compared with term newborns and adults (68). This low level expression may contribute to the immaturity of intestinal innate defense and predispose to the development of necrotizing enterocolitis (NEC). A study of small intestinal samples from infants with NEC demonstrated that human defensin 5 and 6 mRNA was increased threefold in the NEC infant as compared with age matched controls suggesting a possible involvement of defensins in the pathogenesis of NEC (73). Enhanced expression of human defensins 5 and 6 has also been found in colonic specimens from patients with inflammatory bowel disease suggesting a role of human enteric defensins in controlling microbial invasion of the colon in IBD. The expression of enteric defensins may also be increased by inflammation or inflammatory cytokines (74).
In animal studies, mice with a homozygous disruption of the metalloproteinase matrilysin (MMP7) enzyme failed to hydrolyse intestinal prodefensins to defensins and were more susceptible to infection with Salmonella typhimurium. Mice with the mutation required an eightfold lower oral innoculum for a 50% mortality (75). It also took a longer time for these mice to clear infection with an enteropathogenic strain of Escherichia coli (75). Transgenic expression of human defensin 5 in mice at physiologic levels protects them against lethal intestinal infection with Salmonella tryphymurium (76). Taken together, these animal studies provide crucial evidence for the protective role of defensins in enteric infections.
Increasing evidence has also underscored the role of defensins in the activation and recruitment of cells important in the adaptive immune response. Human α-defensins are chemotactic for monocytes, dendritic cells and T lymphocytes at concentrations 10- and 100-fold below those required to produce direct bactericidal damage (77). Human β-defensins have chemotactic activity for immature dentritic cells and memory T cells through the chemokine receptor CCR6 (78). Human α-defensins enhance the expression of TNF-α and IL-1β in activated human monocytes and reduce the expression of VCAM-1 in human endothelial cells activated by TNF-α (79). Collectively, these data indicate that human defensins are not only critical in innate host immune defense, but also are an active participant in regulating adaptive immunity.
SCAVENGER RECEPTOR DMBT1
DMBT1 is a Pattern Recognition Receptor
Scavenger receptors are a group of functionally related but structurally unrelated molecules expressed by myeloid cells (macrophages and dentritic cells) and certain endothelial cells. They play an important role in the uptake and clearance of effete components such as modified host molecules and apoptotic cells (80). They recognize bacterial molecular patterns such as gram-positive bacteria (lipoteichoic acid) and bacterial CpG DNA and thus serve as pattern recognition receptors similar to TLRs and CARD 15/NOD2 (80).
The gene for DMBT 1 (human deleted in malignant brain tumor) encodes a large cysteine rich B protein scavenger receptor (81). It was initially thought to be a possible tumor suppressor gene because of the high frequency of its homozygous deletion and the lack of expression in a variety of cancers. Identification of two mucosal defense-related molecules, gp-340 and salivary agglutinin (alternatively spliced products of DMBT1) suggests that DMBT1 is likely to be another pattern recognition receptor in the innate immune system (82–84).
Function of DMBT1 in Mucosa Innate Immunity
The DMBT 1 gene is expressed in the crypts of the small intestine and the neck region of human gastric mucosa (85–87). Through the cysteine rich B domain, the DMBT1 product binds to a variety of bacteria including Streptococcus mutans, Escherichia coli, Lactobacillus casei, Helicobacter pylori and Prevotella intermedia (88). In the gastrointestinal tract, DMBT1 is secreted from the apical membrane of epithelia onto the mucosal surface where binding to invading pathogens such as S. mutans and H. pylori (83), and host defense components such as surfactant protein (SP)-D (89) and secretory IgA (90) can occur. All these ligands are involved in local inflammation in gastric mucosa (91). Studies on airway epithelia have demonstrated that DMBT1 expression is upregulated by stimulation with IL-8 and IL-6, known inflammatory mediators, suggesting that DMBT 1 plays a role in local epithelial inflammatory responses (92). Furthermore, gp-340, an alternatively spliced form of DMBT1, has stimulatory effects on alveolar macrophage chemokinesis suggesting that it directly regulates tissue macrophage protective function (93). Data suggest that DMBT1 acts as a typical pattern recognition receptor in the innate anti-microbial defense system through its interaction with invading pathogens and other local defense components (94). Because of the extensive expression of DMBT1 transcripts in immune organ systems, including spleen, thymus, alveolar macrophages and mucosal T and B cell lines, it has been suggested that DMBT1 may also be involved in the regulation of the adaptive immune response (82,85,95,96). Detected by immunostaining, enhanced DMBT1 expression has been shown in crypt cells of the small intestine and along the neck region of normal human gastric mucosa where intestinal epithelial stem cells are located, implying a possible role in the physiological renewing process of gastrointestinal epithelia.
Loss or reduction in the expression or inactivation of DMBT1 may be involved in tumor formation in the central nervous system, gastrointestinal tract and lung (81,97–99). In vitro studies have demonstrated inflammation-related upregulation of DMBT1 in airway epithelia and differentiation-related downregulation in gastric epithelia (87). The complex interactions of DMBT1 with microbial pathogens, mucosa defense components and possibly trefoil proteins suggest that DMBT1 may serve as a unique, integral factor promoting the regeneration of mucosal epithelia following damage due to pathogen invasion and/or local inflammation (94). Also, impaired gastrointestinal healing after infection and/or inflammation may result from loss of function of the DMBT1 receptor because of its potential role in epithelial regeneration, a common defense mechanism in the innate host defense against microbial invasion. Therefore, it is possible that loss of DMBT1 could potentially lead to impaired mucosal defense against microbial pathogens and a dysregulated adaptive mucosal immune response.
The innate immune system in the gut is comprised of a complex cellular network, multiple pattern recognition receptors and many biologically active secretory peptides or proteins. It plays a critical role in the first line host immune defense provided by the intestinal mucosa. The innate immune system not only serves as the initial step in the ability of the intestinal mucosa to maintain homeostasis but also serves as an important bridge to the activation and regulation of adaptive immune responses. Dysfunction of the innate immune response of the intestinal mucosa may lead to devastating consequences for the host caused by invasion of microbial pathogens, impaired epithelial regeneration and dysreguation of mucosa inflammatory responses, which are thought to play a role in the pathogenesis of inflammatory bowel disease, NEC, enteric microbial infection and malignances.
1. Huttner KM, Bevins CL. Antimicrobial peptides as mediators of epithelial host defense. Pediatr Res 1999;45:785–94.
2. Medzhitov R, Janeway CJ. Innate immunity. N Engl J Med 2000; 343:338–44.
3. Kaisho T, Akira S. Dendritic-cell function in Toll-like receptor-and MyD88-knockout mice. Trends Immunol 2001;22:78–83.
4. Schnare M, Barton GM, Holt AC, Takeda K, Akira S, Medzhitov R. Toll-like receptors control activation of adaptive immune responses. Nat Immunol 2001;2:947–50.
5. Wilson CL, Ouellette AJ, Satchell DP, Ayabe T, Lopez-Boado YS, Stratman JL, et al. Regulation of intestinal alpha-defensin activation by the metalloproteinase matrilysin in innate host defense. Science 1999;286:113–7.
6. Anderson KV, Jurgens G, Nusslein-Volhard C. Establishment of dorsal-ventral polarity in the Drosphila embryo: genetic studies on the role of the Toll gene product. Cell 1985;42:779–89.
7. Medzhitov R, Preston-Hurlburt P, Janeway CAJ. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 1997;388:394–7.
8. Rock FL, Hardiman G, Timans JC, Kastelein RA, Bazan JF. A family of human receptors structurally related to Drosophila Toll. Proc Natl Acad Sci USA 1998;95:588–93.
9. Chaudhary PM, Ferguson C, Nguyen V, et al. Cloning and characterization of two Toll/interleukin-1 receptor-like TIL3 and TIL4: evidence for a multi-gene receptor family in humans. Blood 1998;91:4020–7.
10. Takeuchi O, Kawai T, Sanjo H, Copeland NG, Gilbert DJ, Jenkins NA, et al. TLR6: a novel member of an expanding Toll-like receptor family. Gene 1999;231:59–65.
11. Chuang TH, Ulevitch RJ. Cloning and characterization of a subfamily of human Toll-like receptors: hTLR7, hTLR8 and hTLR9. Eur Cytokine Netw 2000;11:372–8.
12. Hemmi H, Takeuchi O, Kawai T, et al. A Toll-like receptor recognizes bacterial DNA. Nature 2000;408:740–5.
13. Chuang TH, Ulevitch RJ. Identification of hTLR10: a novel human Toll-like receptor preferentially expressed in immune cells. Biochem Biophys Acta 2001;1518:157–61.
14. O'Neill L. A molecular switch for inflammation and host defense. Biochem Soc Trans 2000;28:557–63.
15. Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol 2001;2: 675–80.
16. Aderem A, Ulevitch RJ. Toll-like receptors in the induction of the innate immune response. Nature 2000;406:782–7.
17. Janeway CAJ, Medzhitov R. Innate immune recognition. Annu Rev Immunol 2002;20:197–216.
18. Hallman M, Ramet M, Ezekowitz RA. Toll-like receptor as sensors of pathogens. Pediatr Res 2001;50:315–21.
19. Akira S, Hemmi H. Recognition of pathogen-associated molecular patterns by TLR family. Immunol Lett 2003;85:85–95.
20. Haller D, Jobin C. Interaction between resident luminal bacteria and the host: can a healthy relationship turn sour?J Pediatr Gastroenterol Nutr 2004;38:123–36.
21. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998;392:245–52.
22. Pasare C, Medzhitov R. Toll pathway-dependent ade of CD4+CD25+ T cells-mediated suppression by dentritic cells. Science 2003;299:1033–6.
23. Poltorak A, He X, Smirnova I, et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in TLR4 gene. Science 1998;282:2085–8.
24. Qureshi ST, Lariviere L, Leveque G, et al. Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (TLR4). J Exp Med 1999;189:615–25.
25. Golenbock DT, Hampton RY, Qureshi N, Takayama K, Raetz CR. Lipid A-like molecules that antagnize the effets of endotoxins on human monocytes. J Biol Chem 1991;266:19490–8.
26. Lien E, Means TK, Heine H, et al. Toll-like receptor 4 imparts ligand-specific recognition of bacterial lipopolysaccharide. J Clin Invest 2000;105:497–504.
27. Poltorak A, Ricciardi-Castagnoli P, Citterio S, Beutler B. Physical contact between lipopolysaccharide and Toll-like receptor 4 revealed by genetic complementation. Proc Natl Acad Sci USA 2000;97:2163–7.
28. Bashir ME, Andersen P, Fuss IJ, Shi HN, Nagler-Anderson C. An enteric helminth infection protects against an allergic response to dietary antigen. J Immunol 2002;169:3284–92.
29. Arbour NC, Lorenz E, Schutte BC, et al. TLR4 mutations are associated with endotoxin hyporesponsiveness in humans. Nat Genet 2000;25:187–91.
30. Lorenz E, Mira JP, Cornish KL, Arbour NC, Schwartz DA. A novel polymorphism in the Toll-like receptor 2 gene and its potential association with staphylococcal infection. Infec Immun 2000;68:6398–401.
31. Hugot JP, Chamaillard M, Zouali H, et al. Association of NOD2 leucine-rich repeat variants with the susceptibility to Crohn's disease. Nature 2001;411:599–603.
32. Inohara N, Nunez G. The NOD: a signaling module that regulates apoptosis and host defense against pathogens. Oncogene 2001; 20:6473–81.
33. Ogura Y, Inohara N, Benito A, Chen FF, Yamaoka S, Nunez G. Nod2, a Nod1/Apaf-1 family member that is restricted to monocytes and actives NF-kappaB. J Biol Chem 2001;276:4812–8.
34. Inohara N, Ogura Y, Chen FF, Muto A, Nunez G. Human Nod1 confers responsiveness to bacterial lipopolysaccharides. J Biol Chem 2001;276:2551–4.
35. Chamaillard M, Philpott D, Girardin SE, et al. Gene-environment interaction modulated by allelic heterogeneity in inflammatory bowel diseases. Proc Natl Acad Sci USA 2003;100:3455–60.
36. Hisamatsu T, Suzuki M, Reinecker HC, Nadeau WJ, McCormick BA, Podolsky DK. CARD15/NOD2 functions as an antibacterial factor in human intestinal epithelial cells. Gastroenterology 2003; 124:993–1000.
37. Gutierrez O, Pipaon C, Inohara N, et al. Induction of Nod2 in myelomonocytic and intestinal epithelial cells via nuclear factor-kappa B activation. J Biol Chem 2002;277:41701–5.
38. Rosenstiel P, Fantiti M, Brautigam K, et al. TNF-alpha and INF-gamma regulate the expression of the NOD2 (CARD15) gene in human intestinal epithelial cells. Gastroenterology 2003;124: 1001–9.
39. Ogura Y, Lala S, Xin W, et al. Expression of NOD2 in Paneth cells: a possible link to Crohn's ileitis. Gut 2003;52:1591–7.
40. Lala S, Ogura Y, Osborne C, et al. Crohn's disease and the NOD2 gene: a role for Paneth cells. Gastroenterology 2003;125:47–57.
41. Aldhous MC, Nimmo ER, Satsangi J. NOD2/CARD15 and the Paneth cell: another piece in the genetic jigsaw of inflammatory bowel disease. Gut 2003;52:1533–5.
42. Ogura Y, Bonen D, Inohara N, et al. A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease. Nature 2001;411:603–6.
43. Hampe J, Cuthbert A, Croucher P, et al. Association between insertion in NOD2 gene and Crohn's disease in German and British populations. Lancet 2001;357:1925–8.
44. Ahmad T, Armuzzi A, Bunce M, et al. The molecular classification of the clinical manifestations of Crohn's disease. Gastroenterology 2002;122:854–66.
45. Cuthbert AP, Fisher SA, Mirza MM, et al. The contribution of NOD2 gene mutations to the risk and site of disease in inflammatory bowel disease. Gastroenterology 2002;122:867–74.
46. Lesage S, Zouali H, Cezard JP, et al. CARD15/NOD2 mutational analysis and genotype-phenotype correlation in 612 patients with inflammatory bowel disease. Am J Hum Genet 2002;70:845–57.
47. Hampe J, Grebe J, Nikolaus S, et al. Association of NOD2 (CARD15) genotype with clinical course of Crohn's disease: a cohort study. Lancet 2002;359:1661–5.
48. Radlmayr M, Torok HP, Matin K, Folwaczny C. The c-insertion mutation of the NOD2 gene is associated with fustulizing and fibrostenotic phenotypes in Crohn's disease. Gastroenterology 2002;122:2091–2.
49. McGovern DPB, van Heel DA, Ahmad T, Jewell DP. NOD2 (CARD15), the first susceptibility gene for Crohn's disease. Gut 2001;49:752–4.
50. Sun L, Roesler J, Rosen-Wolff A, et al. CARD15 genotype and phenotype analysis in 55 pediatric patients with Crohn's disease from Saxony, Germany. J Pedia Gastroenterol Nutr 2003;37: 492–7.
51. Bevins CL. Antimicrobial peptides as agents of mucosal immunity. Ciba Found Symp 1994;186:250–60.
52. Ganz T. Defensins: antimicrobial peptides of innate immunity. Nat Rev Immunol 2003;3:710–20.
53. Boman HG, Agerberth B, Boman A. Mechanisms of action on Escherichia coli of cecropin P1 and PR-39, two antibacterial peptides from pig intestine. Infec Immun 1993;61:2978–84.
54. Ganz T. Paneth cells—guardians of the gut cell hatchery. Nat Immunol 2000;1:99–100.
55. Peeters T, Vantrappen G. The Paneth cells: a source of intestinal lysozyme. Gut 1975;16:553–8.
56. Harwig SS, Tan L, Qu XD, Cho Y, Eisenhauer PB, Lehrer RI. Bactericidal properties of murine intestinal phospholipase A2. J Clin Invest 1995;95:603–10.
57. Nevakainen TJ, Gronroos JM, Kallajoki M. Expression of group II phospholipase A2 in the human gastrointestinal tract. Lab Invest 1995;72:201–8.
58. Selsted ME, Miller SI, Henschen AH, Ouellette AJ. Enteric defensins: antibiotic peptide components of intestine host defense. J Cell Biol 1992;118:929–36.
59. Porter E, Liu L, Oren A, Anton P, Ganz T. Localization of human intestinal defensin 5 in Paneth cell granules. Infec Immun 1997; 65:2389–95.
60. Ayabe T, Satchell DP, Wilson CL, Parks WC, Selsted ME, Ouellette AJ. Secretion of microbicidal alpha-defensins by intestinal Paneth cells in response to bacteria. Nat Immunol 2000;1:113–8.
61. Hooper LV, Stappenbeck TS, Hong CV, Gordon JI. Angiogenins: a new class of microbicidal proteins involved in innate immunity. Nat Immunol 2003;4:269–73.
62. Satoh Y, Habara Y, Ono K, Kanno T. Carbamylcholine- and catecholamine-induced intracellular calcium dynamics of epithelial cells in mouse ileal crypts. Gastroenterology 108;108:1345–56.
63. Ayabe T, Wulff H, Darmoul D, Cahalan MD, Chandy KG, Ouellette AJ. Modulation of mouse Paneth cell alpha-defensin secretion by mlKCa I, a Ca2+-activated, intermediate conductance potassium channel. J Biol Chem 2002;277:3793–800.
64. Qu XD, Lloyd KC, Walsh JH, Lehrer RI. Secretion of type II phospholipase A2 and cryptdin by rat small intestinal Paneth cells. Infec Immun 1996;64:5161–5.
65. Porter EM, Bevins CL, Ghosh D, Ganz T. The multifaceted Paneth cell. Cell Mol Life Sci 2002;59:156–70.
66. Ouellette AJ. Mucosal immunity and inflammation IV. Paneth cell antimicrobial peptides and the biology of teh mucosal barrier. Am J Physiol 1999;277:G257-G61.
67. Ghosh D, Porter E, Shen B, et al. Paneth cell trypsin is the processing enzyme for human defensin-5. Nat Immunol 2002;3: 583–90.
68. Mallow EB, Harris A, Salzman N, et al. Human enteric defensins—gene structure and developmental expression. J Biol Chem 1996;271:4038–45.
69. Ouellette AJ, Cordell B. Accumulaion of abundant messenger ribonucleic acids during postnatal development of mouse small intestine. Gastroenterology 1988;94:114–21.
70. Ouellette AJ, Greco RM, James M, Frederick D, Naftilan J, Fallon JT. Developmental regulation of cryptdin, a corticostatin/defensin precursor mRNA in mouse small intestine crypt epithelium. J Cell Biol 1989;108:1687–95.
71. Darmoul D, Brown D, Selsted ME, Ouellette AJ. Cryptdin gene expression in developing mouse small intestine. Am J Physiol 1997;272:G197-G206.
72. Porter E, van Dam E, Valore E, Ganz T. Broad spectrum antimicrobial activity of human intestinal defensin 5. Infec Immun 1997;65:2396–401.
73. Salzman NH, Polin RA, Harris MC, Ruchelli E, Hebra A, Zirin-Butler S, et al. Enteric defensin expression in necrotizing enterocolitis. Pediatr Res 1998;44:20–6.
74. Wehkamp J, Schwind B, Herrlinher KR, et al. Innate immunity and colonic inflammation: enhanced expression of epithelial alpha-defensin. Dig Dis Sci 2002;47:1349–55.
75. Wilson CL, Ouellette AJ, Satchell DP, et al. Regulation of intestinal alpha-defensin activation by the metalloproteinase metrilysin in innate host defense. Science 1999;286:113–7.
76. Salzman NH, Ghosh D, Huttner KM, Paterson Y, Bevins CL. Protection against enteric salmonellosis in transgenic mice expressing a human intestinal defensin. Nature 2003;422:522–6.
77. Yang D, Chen Q, Chertov O, Oppenheim JJ. Human neutriphil defensins selectively chemoattract naive T and immature dendritic cells. J Leukoc Biol 2000;68:9–14.
78. Yang D, Chertov O, Bykovskaia SN, et al. Beta-defensins: linking innate and adaptive immunity through dentritic and T cell CCR6. Science 1999;286:525–8.
79. Chaly YV, Paleolog EM, Kolesnikova TS, Tikhonov II, Petratchenko EV, Voitenok NN. Neutrophil alpha-defensin human neutrophil peptide modulates cytokine production in human monocytes and adhesion molecule expression in endothelial cells. Eur Cytokine Netw 2000;11:257–66.
80. Peiser L, Mukhopadhyay S, Gordon S. Scavenger receptors in innate immunity. Curr Opin Immunol 2002;14:123–8.
81. Mollenhauer J, Wiemann S, Scheurlen W, et al. DMBT1, a new member of the SRCR superfamily, on chromosome 10q25.3–26.1 is deleted in malignant brain tumours. Nat Genet 1997;17:32–9.
82. Holmskov U, Mollenhauer J, Madsen J, et al. Cloning of gp-340, a putative opsonin receptor for lung surfactant protein D. Proc Natl Acad Sci USA 1999;96:10794–9.
83. Prakobphol A, Xu F, Hoang VM, et al. Salivary agglutinin, which binds StreptococcuS. mutans and Helicobacter pylori, is the lung scavenger receptor cysteine-rich protein gp-340. J Biol Chem 2000;275:39860–6.
84. Ligtenberg TJ, Bikker FJ, Groenink J, et al. Human salivary agglutinin binds to lung surfactant protein-D and is identical with scavenger receptor protein gp-340. Biochem J 2001;359:243–8.
85. Mollenhauer J, Herbertz S, Holmskov U, et al. DMBT1 encodes a protein involved in the immune defense and in epithelial differentiation and is highly unstable in cancer. Cancer Res 2000; 60:1704–10.
86. Takito J, Hikita C, Al-Awqati Q. Hensin, a new collecting duct protein involved in the in vitro plasticity of intercalated cell polarity. J Clin Invest 1996;98:2324–31.
87. Kang W, Nielsen O, Fenger C, et al. The scavenger receptor, cysteine-rich domain-containing molecule gp-340 is differentially regulated in epithelial cell lines by phorbol ester. Clin Exp Immunol 2002;130:449–58.
88. Bikker FJ, Ligtenberg TJ, Nazmi K, et al. Identification of the bacteria-binding peptide domain on salivary agglutinin (gp-340/DMBT1), a member of the scavenger receptor cysteine-rich superfamily. J Biol Chem 2002;277:32109–15.
89. Holmskov U, Lawson P, Teisner B, et al. Isolation and characterization of a new member of the scavenger receptor superfamily, glycoprotein-340 (gp-340), as a lung surfactant protein-D binding molecule. J Biol Chem 1997;272:13743–9.
90. Oho T, Yu H, Yamashita Y, Koga T. Binding of salivary glycoprotein-secretory immunoglobulin A complex to the surface protein antigen of StreptococcuS. mutans. Infec Immun 1998;66: 115–21.
91. Murray E, Khamri W, Walker MM, et al. Expression of surfactant protein D in the human gastric mucosa and during Helicobacter pylori infection. Infec Immun 2002;70:1481–7.
92. Mollenhauer J, Helmke B, Muller H, et al. Sequential changes of the DMBT1 expression and location in normal lung tissue and lung carcinomas. Genes Chromosomes Cancer 2002;35:164–9.
93. Tino MJ, Wright JR. Glycoprotein-340 binds surfactant protein-A (SP-A) and stimulates alveolar macrophage migration in an SPA-independent manner. Am J Respir Cell Mol 1999;20:759–68.
94. Kang W, Reid KB. DMBT1: a regulator of mucosal homeostasis through the linking of mcosal defense and regeneration? [Lett]FEBS 2003;540:21–5.
95. Aruffo A, Bowen MA, Patel DD, et al. CD6-ligand interactions: a paradigm for SRCR domain function?Immunol Today 1997; 18:498–504.
96. Resnick D, Pearson A, Krieger M. The SRCR superfamily: a family reminiscent of the Ig superfamily. Trends Biochem Sci 1994;19:5–8.
97. Somerville RP, Shoshan Y, Eng C, Barnett G, Miller D, Cowell JK. Molecular analysis of two putative tumour suppressor genes, PTEN and DMBT, which have been implicated in glioblastoma multiforme disease progression. Oncogene 1998;17:1755–7.
98. Mori M, Shiraishi T, Tanaka S, et al. Lack of DMBT1 expression in oesophageal, gastric and colon cancers. Br J Cancer 1999;79: 211–3.
99. Takeshita H, Sato M, Shiwaku HO, et al. Expression of the DMBT1 gene is frequently suppressed in human lung cancer. Jpn J Cancer Res 1999;90:903–8.
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