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Crohn's disease: a defensin deficiency syndrome?

Fellermann, Klaus; Wehkamp, Jan; Herrlinger, Klaus R; Stange, Eduard F

European Journal of Gastroenterology & Hepatology: June 2003 - Volume 15 - Issue 6 - p 627-634
Review in depth

This comprehensive review promotes the novel concept that a defensin deficiency, i.e. lack of mucosal peptide antibiotics, may play a pivotal role in the aetiopathogenesis of Crohn's disease. Such an impaired function of this chemical barrier is consistent with the epidemiological relationship of good domestic hygiene with the incidence of inflammatory bowel diseases. The disregulated adaptive immune system, formerly believed to be the major cause in the development of Crohn's disease, may reflect only the primary break of the mucosal defence since the immune response is mostly directed against lumenal bacteria. Recent work has identified five different defensins expressed in colonic mucosa. In contrast to ulcerative colitis, Crohn's disease is characterised by an impaired induction of human beta defensins 2 and 3. This deficient induction may be due to changes in the intracellular transcription by NFκB and the intracellular peptidoglycan receptor NOD2, mutated in Crohn's disease. These findings are consistent with the mucosal attachment of lumenal bacteria in inflammatory bowel diseases and the frequent occurrence of other infectious agents. The hypothesis of an impaired mucosal antibacterial activity is also consistent with the benefit from antibiotic or probiotic treatment in certain inflammatory bowel disease states.

Department of Internal Medicine I, Robert Bosch Krankenhaus, Stuttgart, Germany.

Correspondence to Dr Eduard F. Stange, Department of Internal Medicine I, Robert Bosch Krankenhaus, Auerbachstr. 110, 70376 Stuttgart, Germany. Tel: +49 711 810 13404; fax: +49 711 810 13793; e-mail:

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Despite active research for many decades the aetiology of Crohn's disease (CD) is still enigmatic. Most of the research has focused on a potential dysregulation of specific mucosal immunology. These investigations have elegantly described the mucosal cellular populations and cytokine profiles associated with inflammatory bowel disease (IBD) but have not succeeded in finding the aetiological culprit. The alternative hypothesis of a primary defect in the mucosal barrier also has never been substantiated at the molecular level. In this review we will outline a novel concept of how epidemiological, pathophysiological, genetic, molecular, clinical and pharmacological sets of data may be synthesized into a unifying hypothesis compatible with many features of this disease.

Several years ago we became interested in the mucosal system of antibiotic peptides contributing to the defensive array of substances and structures opposed to the invasion of luminal bacteria and other potential invaders. Most of the work in the field of defensins had focused on the skin as another border of the body exposed to a multitude of bacteria and has resulted in the isolation of various peptides exhibiting potent antibiotic activity towards both Gram-positive and Gram-negative bacteria as well as enveloped viruses and fungi. A very similar system of antibiotic peptides is apparently synthesized and secreted by the intestinal mucosa as part of innate immunity but has been given little attention. The topic of defensins has recently been covered by a concise overview in this journal and is now presented in some detail with respect to IBDs [1].

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Epidemiology: the role of hygiene

There is a clear-cut north to south gradient of IBD incidences worldwide as well as in Europe [2]. In developing countries infectious intestinal diseases are common, whereas idiopathic IBDs, especially CD, are the rare exception. Similarly, the incidence rates in Scandinavia are several fold higher compared to southern Europe but the reasons for this difference are not clear [2]. Since migration in many instances is associated with adaptation to the incidence rates in the immigrant country there is little doubt that environmental factors are involved [3], although a genetic background cannot be excluded. The increase in incidence rates in these countries appears to be associated with the adaptation to ‘Western lifestyle'. The dominant role of hygiene in this cultural–medical evolution is likely but unproven.

It is supported by the finding that good domestic hygiene in infancy has been shown to be a risk factor for CD but not for ulcerative colitis (UC), even within a country [4]. Thus, the risk of developing CD is increased 3-fold if a separate toilet is available and 5-fold if there is hot tap water in the household [4]. Similarly, Helicobacter pylori seroprevalence was substantially reduced in CD (odds ratio 0.18) but not in UC. CD was also associated with childhood eczema and frequent use of a swimming pool [5]. In addition, CD occurs more often in members of small families as opposed to those with many children. Since intrafamilial transmission of common pathogens is frequent, the single child is particularly prone to be raised under more hygienic conditions with lower risk of acquiring gut infections [6,7]. Most likely, these various factors associated with the incidence of CD serve as indicators of a rather clean environment, leading to a diminished confrontation with pathogenic or non-pathogenic microorganisms. As a result the intestinal innate immune system is probably not ‘trained’ to confront minor infections without recruiting the full array of specific immune functions which act only at the expense of a relevant inflammation.

Another important aspect in this regard is the apparently frequent association of a recent intestinal infection with the first appearance of CD and the prevalence of superinfection in pre-established IBD [8]. Although these relationships have not been fully understood the interpretation has been made that an infection in some way triggers a relapse of the idiopathic bowel disease by breaking mucosal tolerance. Despite their self-limited character, these infections may initiate a cascade of inflammatory events leading to chronic relapsing disease in genetically susceptible hosts (the ‘hit and run’ hypothesis). Alternatively, the host with IBD may be more likely to contract an intestinal infection because of a defective innate defence system (see below).

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Pathophysiology: the role of luminal bacteria

It has always been an intriguing hypothesis that IBDs are caused by a specific, hitherto unrecognized, infection. For example, Mycobacterium paratuberculosis has been considered by various groups to cause not only Johne's disease in cattle but also CD in humans [9,10]. The debate has been ongoing for many years and is beyond the scope of the present considerations. It should be noted, however, that even very recently, using novel techniques like granuloma isolation with laser capture microdissection, many more Crohn's samples were shown to be positive for mycobacteria than were controls [11]. Thus, although mycobacteria are far from proven to be causative agents, it is apparent that the mucosa in CD frequently harbours unusual and potentially pathogenic bacteria. In some instances listeria have been isolated [12] or specific mucosal adherent Escherichia coli [13]. Interestingly, there is a tremendous increase in the mucosal associated bacterial counts in the neoterminal ileum after ileocaecal resection for CD and this colonization may be related to postoperative relapse [14]. Measles infection in CD is also a very controversial issue but it adds up to the list of transmissible agents recovered from Crohn's mucosa [15]. Taken together, these findings indicate that Crohn's mucosa is often the target of various infections but positive proof that the disease is caused by these agents is missing. Most importantly, the immune response in the gut mucosa is not specific to any of these suspicious agents but rather unspecific to a multitude of organisms.

Since 1939, a series of clinical reports and laboratory investigations have suggested that the intestinal faecal stream may play a significant part in the pathogenesis of CD [16]. This has been corroborated by experiments where small bowel contents have been infused into the distal loop of an ileostomy which triggered an inflammatory response [17]. The human large intestine is known to contain hundreds of culturable bacterial species and morphological and molecular analysis suggests at least an equal number of unculturable species [18]. Also, distal small intestinal contents are contaminated by upwardly mobile colonic flora. Thus, CD is obviously localized mostly in sites with a heavy bacterial load, i.e. in the ileocaecum or large bowel and only rarely in the proximal gut or stomach. Possibly, the colonization of ileal mucosa with an unusual luminal bacterial content after formation of an ileoanal pouch may pose a problem in pouchitis.

However, it has only recently been appreciated that the mucosal immune response in IBD is directed towards a multitude of common luminal bacteria. The most convincing evidence for a break in mucosal tolerance in intestinal inflammation stems from the observation that knockout mice lacking several relevant genes, including interleukins 2 or 10, develop experimental colitis only when raised in contaminated but not in sterile conditions [19]. This fits well with the consistent finding of a break in mucosal tolerance towards various luminal bacteria in IBDs [20,21]. It may be concluded that these diseases are not autoimmune diseases in the strict sense, i.e. reactivity against autologous tissues, but only in a more general sense, i.e. immune response towards commensal bacteria. The permeable mucosal barrier may also explain the development of anti-Saccharomyces cerevisiae antibodies especially in familial CD [22] as well as antibodies to various other microbes, including E. coli.

The most surprising finding in this regard is the demonstration by Swidsinski et al. that the mucosa in IBDs is heavily contaminated by adherent and sometimes invading bacteria entering from the lumen [23]. In contrast, normal mucosa is virtually sterile when washed a few times in saline. These findings are difficult to reconcile with an immunological dysregulation as the sole basis of intestinal inflammation in these diseases. Rather, there may be a primary defect in the chemical barrier of intestinal defensins which protects the normal mucosa extremely efficiently against adherent or entering microbes. Thus, a thorough understanding of these functionally relevant peptides is paramount to understanding the true pathogenesis of IBD. Indirectly, a change in the expression or function of this chemical defence may indeed explain the changes in bacterial flora in IBDs reviewed by Linskens et al. [24].

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Defensins: genetics, expression and regulation

The question why frogs have an undisturbed wound healing gave rise to the intensive search for antimicrobials in vertebrates and invertebrates. Several potential peptides and proteins have been identified so far (for review of antimicrobial peptides see [25]) including defensins, lysozyme, bactericidal permeability increasing protein, chemokines and many others (an overview of known human antimcrobial peptides is listed in Table 1). Probably the most important peptide family of endogenous antibiotics is the still growing number of defensins [1,26,27]. They comprise a class of cationic antimicrobial peptides with a molecular weight of 3–5 kDa conserved throughout phylogeny. All defensins have been mapped to chromosome 8 in humans [28–30] as well as in mice and the genomic organization suggests one ancestral gene which has been duplicated during evolution [29,31]. The common key feature is three intramolecular disulphide bonds between cysteine residues. Their position allows differentiation between α- and β-defensins.

Table 1

Table 1

Six α-defensins and four β-defensins have been identified in humans so far. The α-defensins comprise human neutrophil peptide 1–4, abundant in granulocytes, and human defensins 5 and 6 synthesized in Paneth cells. The β-defensins are of epithelial origin and abundant in skin, intestine and lung (representative stains for HD-5, HBD-1 and HBD-2 in inflamed colonic tissue are given in Fig. 1). The concept of a certain defensin exclusively formed by specialized tissues or cells has to be revised as inflammation induces epithelial expression of human neutrophil peptides [32] and β-defensins in monocytes and lymphocytes [33].

Fig. 1

Fig. 1

Defensins can be divided into constitutive forms, e.g. HBD-1 with its widespread stable distribution [34] and inducible peptides such as HBD-2 [35]. The mechanisms of activation are currently under investigation. A cytokine driven induction e.g. by IL-1β and TNF-α has been shown in addition to a direct response to bacterial components such as lipopolysaccharides (LPSs) and lipoproteins. Possible signalling pathways involve Toll-like receptors, especially TLR2 and 4 eventually leading to nuclear factor κB (NFκB) mediated activation of transcription [36,37]. Promoter analysis revealed that transactivation depends on the proper function of NFκB response elements [38]. In the signalling pathway triggered by Salmonella enteritidis both calcium and inositol triphosphate appear to play a role [39] whereas the Src dependent Raf-MEK1/2-ERK system is involved in mediating the IL-1 induction [40]. NOD2/CARD15, as an intracellular LPS receptor, induces NFκB [41] which, in turn, is known to trigger HBD-2 transcription. Interestingly, this NFκB response is impaired in the NOD2 insertion mutation associated with CD [42,43] suggesting a diminished innate response to bacterial components.

Human defensin 5 is released as a propeptide from Paneth cells and activated by trypsinogen in the lumen of the intestinal crypts [44]. This differs from mice, where the active peptide is cleaved by the matrix metalloproteinase matrilysin [45]. Post-transcriptional processing and the activation of other defensins remain obscure.

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Physiological role and dysfunction of defensins

The antimicrobial spectrum varies from one defensin to another. Maximum activity is achieved in the micromolar range and in a low-salt environment. Antibacterial activity of HD-5 has been characterized extensively with activity against E. coli, Listeria monocytogenes, Salmonella typhimurium and Candida albicans [46]. In comparison, HBD-2 is most active against Gram-negative strains [35] whereas HBD-3 has a more Gram-positive spectrum [47,48]. Current proposals for the mode of action include formation of micropores by defensin multimeres within the phospholipid moiety of bacterial membranes resulting in disruption of the membrane [49,50]. Of special interest is the link to adaptive immunity as defensins act as chemokines attracting effector cells [51–53] and their ability to amplify acquired immune responses [54].

The expression of defensins has already been linked to several diseases. For example, necrotizing colitis is associated with an induction of α-defensins [55]. HBD-2 is bactericidal against Helicobacter pylori in vitro and is induced in H. pylori gastritis resolving after eradication treatment [56–58]. Bacterial pneumonia is accompanied by increased systemic and local HBD-2 peptide levels [59,60]. The functional significance in bacterial infection has recently been shown in HD-5 transgenic mice which are protected from lethal salmonella infection [61]. On the other hand, matrilysin deficient mice fail to process defensins efficiently and exhibit higher bacterial counts [45].

If defensin induction is a physiological process in infectious diseases one might postulate that malfunction of this innate defence gives rise to an increase in frequency and/or severity of infections. Improper function of defensins may occur in two ways, lack of function (inactivation) or lack of induction. A model disease for inactivation may be cystic fibrosis. It was postulated that defensins, expressed in respiratory epithelia and present in airway surface fluid of cystic fibrosis patients, are inactivated in the high-salt environment and may account for recurrent pulmonary infections [62,63]. A lack of induction, especially regarding HBD-2, has been encountered in CD (see below) and atopic dermatitis [64]. Bacterial invasion and superinfection is almost absent in psoriasis. In contrast, neurodermatic skin is often colonized by Gram-positive bacteria and superinfections do often occur. This observation has now been attributed to HBD-2, which is largely induced in psoriatic but not in atopic dermatitis skin lesions. Obviously, current efforts to better understand this complex defence system will help clarify many inflammatory as well as immunological features of various infectious as well as ‘idiopathic’ diseases.

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Defensins and inflammatory bowel diseases

Although a decrease or missing induction of antimicrobial peptides may, in principle, lead to a gradual bacterial invasion which could trigger inflammation and loss of mucosal tolerance, only little is known about this aspect of innate immunity in IBD.

In general, α-defensins appear to be induced in both CD and UC (Table 2). Human neutrophil peptides 1–3 as well as lysozyme are expressed in surface enterocytes of mucosa with active IBD but surprisingly not in controls [32]. HD-5 is stored in a precursor form in normal Paneth cells and is expressed by metaplastic colonic Paneth cells [65]. Notably, both α-defensins HD-5 and HD-6 are induced in the colonic mucosa of IBD patients [65–67]. The induction of HD-6 but not of HD-5 is specific for idiopathic IBD and was not observed in infectious colitis.

Table 2

Table 2

The alterations in β-defensins are more intriguing because there is a conspicuous difference between CD and UC. It has been suggested that HBD-1 is constitutively expressed in the intestinal epithelium [68] and qualitative investigations indeed showed constitutive expression in normal tissue and IBD mucosa [69]. Using qualitative reverse transcriptase–polymerase chain reaction (RT-PCR), constitutive expression was observed in 50–60% of normal subjects and IBD patients. Furthermore, we described HBD-1 on the protein level by immunohistochemistry in the colonic epithelium. To extend our previous qualitative RT-PCR findings we quantified β-defensin expression using real-time PCR in colonic mucosa in a recent study [70]. With the quantitative approach a decrease of HBD-1 was found in inflamed mucosa of both CD and UC, respectively. However, it remains to be shown that such a decrease actually translates into a diminished mucosal antibacterial activity.

The inducible HBD-2, which was described originally in skin [35], is also expressed in the colon during inflammation [68], particularly in UC [69]. Based on the qualitative RT-PCR in our recent study HBD-2 was detected more often in biopsies from UC than in controls or CD. On the protein level we confirmed these findings by immunohistochemistry with a preferential expression in UC. The real-time PCR and the possibility to measure mRNA expression levels further explored the difference between CD and UC. Using light cycler technique the inducible β-defensin 2 was found almost exclusively in inflamed and much less in non-inflamed UC [70]. In CD the inflamed mucosa exhibited a low level of HBD-2 expression comparable to unspecific colitis. In non-inflamed CD the level of HBD-2 was comparable to non-inflamed controls. Most likely, there is a lack of β-defensin induction in CD contributing to a defective antimicrobial barrier or, alternatively, there is an excessive induction in UC.

The third defensin studied was HBD-3 which was reported by Harder et al. as a novel inducible β-defensin in skin [47]. Another group described HBD-3 based on genomic analysis [48]. In a recent study expression of HBD-3 was a rare event, almost limited to inflamed specimens (Wehkamp et al., submitted). Interestingly, HBD-3 levels in the different patient groups closely correlated with that of HBD-2, although defensins are known to be regulated independently. Although HBD-3 was also slightly induced in inflamed Crohn's mucosa, its expression was preferentially enhanced in inflamed and non-inflamed UC. This was unexpected because cell culture experiments indicated a divergent regulation of both defensins [47,48]. A deficiency in the antimicrobial defence systems of defensins may be a reasonable and plausible explanation for the break of the antibacterial barrier function in IBDs.

In conclusion, the decrease of HBD-1 in both IBDs and the lack of induction of both inducible β-defensins HBD-2 and HBD-3 in CD suggest a deficient mucosal barrier function. This may in part be compensated by the induction of the α-defensins. A lack in the innate defence system of antimicrobial peptides may lead to a permanent but slow bacterial invasion triggering the inflammatory process but further direct studies on antimicrobial peptide activity in IBD mucosa are required to validate this hypothesis.

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Therapy: the role of antibiotics and probiotics

If a deficiency of these endogenous antibiotics was triggering relapse one would expect exogenous antibiotics to be an efficacious treatment option. Therefore the different trials using antibiotics for various indications are discussed.

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Remission induction in Crohn's disease

In active CD metronidazole has been shown to be equivalent to sulfasalazine [71] and significantly better than placebo [72]. Similarly, ciprofloxacin was equivalent to mesalazine in the treatment of mild to moderate flare-ups [73]. The combination of both antibiotics may even be as effective as systemic steroids [74]. The best study to date tested the combination of both antibiotics in addition to budesonide in a placebo controlled fashion. Although the authors were unable to show significant differences between the two groups there was a trend towards efficacy in colonic disease [75]. Thus, although not completely convincing antibiotics may offer a therapeutic option in acute flares of CD at least in cases with mild to moderate disease activity.

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Remission maintenance in Crohn's disease

All reported trials have been performed with active disease. Only one placebo controlled study tested the efficacy of ciprofloxacin as adjunct to standard therapy in moderately active CD with a study duration of 6 months. Ciprofloxacin was significantly superior to placebo in remission maintenance [76]. In the postoperative condition metronidazole was effective in preventing endoscopic recurrence after 3 months [77]. In contrast, a prophylactic probiotic treatment did not prevent recurrence after surgery [78].

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Perianal and fistulizing Crohn's disease

No controlled trials for antibiotics exist for this condition, but uncontrolled trials have shown efficacy for metronidazole [79,80] and the combination therapy of metronidazole and ciprofloxacin [81] with respect to pain relief, reduction of draining fistulas and fistula closure.

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Remission induction in ulcerative colitis

Less promising results have been obtained in the treatment of UC. There is no trial testing the efficacy of antibiotics alone. Only an early study on short-term oral tobramycin added to steroids showed a significant benefit in the treatment of acute colitis [82]. In contrast, in addition to steroids neither short-term oral ciprofloxacin in mild to moderate disease [83] nor the intravenous application in severe disease resulted in better outcome [84]. Similarly, metronidazole [85] as well as the combination of metronidazole and tobramycin [86] as an adjunct to steroids failed to improve remission rate in severe UC.

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Remission maintenance in ulcerative colitis

Only one controlled trial using ciprofloxacin as a long-term ‘add on’ to steroids and mesalazine has been conducted in UC. Though it proved clinical efficacy after 3 months of continued application the advantage disappeared in the steroid-free trial period at 6 months [87]. The trial was questioned by the lack of good design. A promising therapeutic concept is the application of the probiotic strain E. coli Nissle. In two controlled trials it was equivalent to mesalazine in successful remission maintenance in recurrent disease [88,89].

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Pouchitis after ileal pouch–anal anastomosis

In contrast to the disappointing results in UC, antibiotics have a defined role in the special condition of pouchitis. In a controlled trial ciprofloxacin was superior to metronidazole with regard to efficacy and side effects [90]. Chronic pouchitis may be effectively treated with metronidazole alone [91] or with antibiotic combination therapy of metronidazole and ciprofloxacin [92] or rifaximin and ciprofloxacin [93]. Interestingly, the probiotic cocktail VSL#3 was able to maintain remission in chronic pouchitis significantly better than placebo [94].

In conclusion, antibiotics appear to have a limited effect in CD and probiotics in UC. In CD the exogenous antibiotics may compensate for the deficient endogenous antibiotic response to infection or commensal bacterial invasion. In UC the pattern is different with low basal activity but normal induction during inflammation. Therefore antibiotics may not work and the benefit of probiotics may be due to the induction of β-defensins as demonstrated recently in vitro [95]. In contrast to the majority of tested E. coli the Nissle strain potently upregulated HBD-2 expression in colonic cell culture. In pouchitis both approaches work for induction and maintenance, respectively.

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Concluding remarks

Seventy years after Crohn's description of the disease named after him it becomes apparent that CD is not a disease but a syndrome. It is not surprising that the diverse facets of genetic predisposition, where only a minority of patients display a defective NOD2 gene, modified by environmental factors such as childhood hygiene and others, may lead to very different forms of disease with respect to localization, natural course and therapeutic response. Although in no way perfect, the present hypothesis appears to be plausible, for the reasons presented above, but particularly since the multitude of defensins, other antibiotic peptides and related transcription factors or transporters leaves enough room for clinical diversity. UC may indeed not be due to a defensin problem but the fact that anti-neutrophil cytoplasmic antibody is directed against the endogenous antibiotic bactericidal permeability increasing protein [96–98] or that the disease is related to certain MDR-1 polymorphisms [99] which may alter defensin export leaves enough room for speculation and, more importantly, hypothesis driven future work.

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Helpful discussions and joint investigations with J.M. Schröder and J. Harder are gratefully acknowledged. The work was generously supported by the Robert Bosch Foundation, Stuttgart, Germany.

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1.Fellermann K, Stange EF. Defensins – innate immunity at the epithelial frontier. Eur J Gastroenterol Hepatol 2001; 13:771–776.
2.Shivananda S, Lennard-Jones J, Logan R, Fear N, Price A, Carpenter L, et al. Incidence of inflammatory bowel disease across Europe: is there a difference between north and south? Results of the European Collaborative Study on Inflammatory Bowel Diseases (EC-IBD). Gut 1996; 39:690–697.
3.Probert CS, Jayanthi V, Rampton DS, Mayberry JF. Epidemiology of inflammatory bowel disease in different ethnic and religious groups: limitations and aetiological clues. Int J Colorectal Dis 1996; 11:25–28.
4.Gent AE, Hellier MD, Grace RH, Swarbrick ET, Coggon D. Inflammatory bowel disease and domestic hygiene in infancy. Lancet 1994; 343: 766–767.
5.Feeney MA, Murphy F, Clegg AJ, Trebble TM, Sharer NM, Snook JA. A case–control study of childhood environmental risk factors for the development of inflammatory bowel disease. Eur J Gastroenterol Hepatol 2002; 14:529–534.
6.Gila T, Hacohen D, Lilos P, Langman MJ. Childhood factors in ulcerative colitis and Crohn's disease. An international cooperative study. Scand J Gastroenterol 1987; 22:1009–1024.
7.Persson PG, Leijonmarck CE, Bernell O, Hellers G, Ahlbom A. Risk indicators for inflammatory bowel disease. Int J Epidemiol 1993; 22:268–272.
8.Stallmach A, Carstens O. Role of infections in the manifestation or reactivation of inflammatory bowel diseases. Inflamm Bowel Dis 2002; 8:213–218.
9.Moss MT, Sanderson JD, Tizard ML, Hermon-Taylor J, el Zaatari FA, Markesich DC, et al. Polymerase chain reaction detection of Mycobacterium paratuberculosis and Mycobacterium avium subsp silvaticum in long term cultures from Crohn's disease and control tissues. Gut 1992; 33:1209–1213.
10.McFadden JJ, Butcher PD, Chiodini R, Hermon-Taylor J. Crohn's disease-isolated mycobacteria are identical to Mycobacterium paratuberculosis, as determined by DNA probes that distinguish between mycobacterial species. J Clin Microbiol 1987; 25:796–801.
11.Ryan P, Bennett MW, Aarons S, Lee G, Collins JK, O'Sullivan GC, et al. PCR detection of Mycobacterium paratuberculosis in Crohn's disease granulomas isolated by laser capture microdissection. Gut 2002; 51:665–670.
12.Liu Y, van Kruiningen HJ, West AB, Cartun RW, Cortot A, Colombel JF. Immunocytochemical evidence of Listeria, Escherichia coli, and Streptococcus antigens in Crohn's disease. Gastroenterology 1995; 108: 1396–1404.
13.Darfeuille-Michaud A, Neut C, Barnich N, Lederman E, di Martino P, Desreumaux P, et al. Presence of adherent Escherichia coli strains in ileal mucosa of patients with Crohn's disease. Gastroenterology 1998; 115:1405–1413.
14.Neut C, Bulois P, Desreumaux P, Membre JM, Lederman E, Gambiez L, et al. Changes in the bacterial flora of the neoterminal ileum after ileocolonic resection for Crohn's disease. Am J Gastroenterol 2002; 97:939–946.
15.Wakefield AJ, Ekbom A, Dhillon AP, Pittilo RM, Pounder RE. Crohn's disease: pathogenesis and persistent measles virus infection. Gastroenterology 1995; 108:911–916.
16.Janowitz HD, Croen EC, Sachar DB. The role of the fecal stream in Crohn's disease: an historical and analytic review. Inflamm Bowel Dis 1998; 4:29–39.
17.D'Haens G, Geboes K, Peeters M, Baert F, Penninckx F, Rutgeerts P. Early lesions of recurrent Crohn's disease caused by infusion of intestinal contents in excluded ileum. Gastroenterology 1998; 114:262–267.
18.Braun J. Unsettling facts of life: bacterial commensalism, epithelial adherence, and inflammatory bowel disease. Gastroenterology 2002; 122:228–230.
19.Sellon RK, Tonkonogy S, Schultz M, Dieleman LA, Grenther W, Balish E, et al. Resident enteric bacteria are necessary for development of spontaneous colitis and immune system activation in interleukin-10-deficient mice. Infect Immun 1998; 66:5224–5231.
20.Duchmann R, May E, Heike M, Knolle P, Neurath M, Meyer zum Büschenfelde K-H. T cell specificity and cross reactivity towards enterobacteria, Bacteroides, Bifidobacterium, and antigens from resident intestinal flora in humans. Gut 1999; 44:812–818.
21.Landers CJ, Cohavy O, Misra R, Yang H, Lin YC, Braun J, et al. Selected loss of tolerance evidenced by Crohn's disease-associated immune responses to auto- and microbial antigens. Gastroenterology 2002; 123:689–699.
22.Annese V, Andreoli A, Andriulli A, Dinca R, Gionchetti P, Latiano A, et al. Familial expression of anti-Saccharomyces cerevisiae mannan antibodies in Crohn's disease and ulcerative colitis: a GISC study. Am J Gastroenterol 2001; 96:2407–2412.
23.Swidsinski A, Ladhoff A, Pernthaler A, Swidsinski S, Loening-Baucke V, Ortner M, et al. Mucosal flora in inflammatory bowel disease. Gastroenterology 2002; 122:44–54.
24.Linskens RK, Huijsdens XW, Savelkoul PH, Vandenbroucke-Grauls CM, Meuwissen SG. The bacterial flora in inflammatory bowel disease: current insights in pathogenesis and the influence of antibiotics and probiotics. Scand J Gastroenterol 2001; 36 (suppl):29–40.
25.Boman HG. Gene-encoded peptide antibiotics and the concept of innate immunity: an update review. Scand J Immunol 1998; 48:15–25.
26.Cunliffe RN, Mahida YR. Antimicrobial peptides in innate intestinal host defence. Gut 2000; 47:16–17.
27.Lehrer RI, Ganz T. Defensins of vertebrate animals. Curr Opin Immunol 2002; 14:96–102.
28.Harder J, Siebert R, Zhang Y, Matthiesen P, Christophers E, Schlegelberger B, et al. Mapping of the gene encoding human beta-defensin-2 (DEFB2) to chromosome region 8p22-p23.1. Genomics 1997; 46:472–475.
29.Liu L, Zhao C, Heng HH, Ganz T. The human beta-defensin-1 and alpha-defensins are encoded by adjacent genes: two peptide families with differing disulfide topology share a common ancestry. Genomics 1997; 43:316–320.
30.Liu L, Wang L, Jia HP, Zhao C, Heng HHQ, Schutte BC, et al. Structure and mapping of the human beta-defensin HBD-2 gene and its expression at sites of inflammation. Gene 1998; 222:237–244.
31.Bevins CL, Jones DE, Dutra A, Schaffzin J, Muenke M. Human enteric defensin genes: chromosomal map position and a model for possible evolutionary relationships. Genomics 1996; 31:95–106.
32.Cunliffe RN, Kamal M, Rose FR, James PD, Mahida YR. Expression of antimicrobial neutrophil defensins in epithelial cells of active inflammatory bowel disease mucosa. J Clin Pathol 2002; 55:298–304.
33.Duits LA, Ravensbergen B, Rademaker M, Hiemstra PS, Nibbering PH. Expression of beta-defensin 1 and 2 mRNA by human monocytes, macrophages and dendritic cells. Immunology 2002; 106:517–525.
34.Zhao C, Wang I, Lehrer RI. Widespread expression of beta-defensin hBD-1 in human secretory glands and epithelial cells. FEBS Lett 1996; 396:319–322.
35.Harder J, Bartels J, Christophers E, Schröder JM. A peptide antibiotic from human skin. Nature 1997; 387:861.
36.Becker MN, Diamond G, Verghese MW, Randell SH. CD14-dependent lipopolysaccharide-induced beta-defensin-2 expression in human tracheobronchial epithelium. J Biol Chem 2000; 275:29731–29736.
37.Diamond G, Kaiser V, Rhodes J, Russell JP, Bevins CL. Transcriptional regulation of beta-defensin gene expression in tracheal epithelial cells. Infect Immun 2000; 68:113–119.
38.Tsutsumi-Ishii Y, Nagaoka I. NF-kappa B-mediated transcriptional regulation of human beta-defensin-2 gene following lipopolysaccharide stimulation. J Leukoc Biol 2002; 71:154–162.
39.Takahashi A, Wada A, Ogushi K, Maeda K, Kawahara T, Mawatari K, et al. Production of beta-defensin-2 by human colonic epithelial cells induced by Salmonella enteritidis flagella filament structural protein. FEBS Lett 2001; 508:484–488.
40.Moon SK, Lee HY, Li JD, Nagura M, Kang SH, Chun YM, et al. Activation of a Src-dependent Raf-MEK1/2-ERK signaling pathway is required for IL-1alpha-induced upregulation of beta-defensin 2 in human middle ear epithelial cells. Biochim Biophys Acta 2002; 1590:41–51.
41.Ogura Y, Bonen DK, Inohara N, Nicolae DL, Chen FF, Ramos R, et al. A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease. Nature 2001; 411:603–606.
42.Hugot JP, Chamaillard M, Zouali H, Lesage S, Cezard JP, Belaiche J, et al. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease. Nature 2001; 411:599–603.
43.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 activates NF-kappaB. J Biol Chem 2001; 276:4812–4818.
44.Ghosh D, Porter E, Shen B, Lee SK, Wilk D, Drazba J, et al. Paneth cell trypsin is the processing enzyme for human defensin-5. Nat Immunol 2002; 3:583–590.
45.Wilson CL, Ouellette AJ, Satchell DP, Ayabe T, Lopez-Boado YS, Stratman JL, et al. Regulation of intestinal α-defensin activation by the metalloproteinase matrilysin in innate host defense. Science 1999; 286:113–117.
46.Porter EM, van-Dam E, Valore EV, Ganz T. Broad-spectrum antimicrobial activity of human intestinal defensin 5. Infect Immun 1997; 65: 2396–2401.
47.Harder J, Bartels J, Christophers E, Schröder JM. Isolation and characterization of human β-defensin-3, a novel human inducible peptide antibiotic. J Biol Chem 2001; 276:5707–5713.
48.Garcia JR, Jaumann F, Schulz S, Krause A, Rodriguez-Jimenez J, Forssmann U, et al. Identification of a novel, multifunctional beta- defensin (human beta-defensin 3) with specific antimicrobial activity. Its interaction with plasma membranes of Xenopus oocytes and the induction of macrophage chemoattraction. Cell Tissue Res 2001; 306: 257–264.
49.Fujii G, Selsted ME, Eisenberg D. Defensins promote fusion and lysis of negatively charged membranes. Protein Sci 1993; 2:1301–1312.
    50.Wimley WC, Selsted ME, White SH. Interactions between human defensins and lipid bilayers: evidence for formation of multimeric pores. Protein Sci 1994; 3:1362–1373.
      51.Yang D, Chen Q, Chertov O, Oppenheim JJ. Human neutrophil defensins selectively chemoattract naive T and immature dendritic cells. J Leukoc Biol 2000; 68:9–14.
      52.Yang D, Chertov O, Bykovskaia SN, Chen Q, Buffo MJ, Shogan J, et al. β-Defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science 1999; 286:525–528.
      53.Chertov O, Michiel DF, Xu L, Wang JM, Tani K, Murphy WJ, et al. Identification of defensin-1, defensin-2, and CAP37/azurocidin as T-cell chemoattractant proteins released from interleukin-8-stimulated neutrophils. J Biol Chem 1996; 271:2935–2940.
      54.Lillard JW Jr, Boyaka PN, Chertov O, Oppenheim JJ, McGhee JR. Mechanisms for induction of acquired host immunity by neutrophil peptide defensins. Proc Natl Acad Sci USA 1999; 96:651–656.
      55.Salzman NH, Polin RA, Harris MC, Ruchelli E, Hebra A, Butler SZ, et al. Enteric defensin expression in necrotizing enterocolitis. Pediatr Res 1998; 44:20–26.
      56.Wada A, Mori T, Oishi K, Hojo H, Nakahara Y, Hamanaka Y, et al. Induction of human β-defensin-2 mRNA expression by Helicobacter pylori in human gastric cell line MKN45 cells on cag pathogenicity island. Biochem Biophys Res Commun 1999; 263:770–774.
      57.Hamanaka Y, Nakashima M, Wada A, Ito M, Kurazono H, Hojo H, et al. Expression of human beta-defensin 2 (hBD-2) in Helicobacter pylori induced gastritis: antibacterial effect of hBD-2 against Helicobacter pylori. Gut 2001; 49:481–487.
      58.Wehkamp J, Schmidt K, Herrlinger KR, Baxmann S, Behling S, Wohlschlager C, et al. Defensin pattern in chronic gastritis – HBD-2 is differentially expressed with respect to Helicobacter pylori status. J Clin Pathol 2003; (in press).
      59.Singh PK, Jia HP, Wiles K, Hesselberth J, Liu L, Conway BA, et al. Production of beta-defensins by human airway epithelia. Proc Natl Acad Sci USA 1998; 95:14961–14966.
      60.Hiratsuka T, Nakazato M, Date Y, Ashitani J, Minematsu T, Chino N, et al. Identification of human beta-defensin-2 in respiratory tract and plasma and its increase in bacterial pneumonia. Biochem Biophys Res Commun 1998; 249:943–947.
      61.Bevins CL, Salzman NH, Ghosh D, Huttner, K. Human defensin-5 (HD5) transgenic mice: Paneth cell expression and protection from lethal Salmonella typhimurium infection. Gastroenterology 2002; 122:A34.
      62.Smith JJ, Travis SM, Greenberg EP, Welsh MJ. Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid. Cell 1996; 85:229–236.
      63.Goldman MJ, Anderson GM, Stolzenberg ED, Kari UP, Zasloff M, Wilson JM. Human beta-defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell 1997; 88:553–560.
      64.Ong PY, Ohtake T, Brandt C, Strickland I, Boguniewicz M, Ganz T, et al. Endogenous antimicrobial peptides and skin infections in atopic dermatitis. N Engl J Med 2002; 347:1151–1160.
      65.Cunliffe RN, Rose FRAJ, Keyte J, Abberley L, Chan WC, Mahida YR. Human defensin 5 is stored in precursor form in normal Paneth cells and is expressed by some villous epithelial cells and by metaplastic Paneth cells in the colon in inflammatory bowel disease. Gut 2001; 48: 176–185.
      66.Wehkamp J, Schwind B, Herrlinger KR, Baxmann S, Schmidt K, Duchrow M, et al. Innate immunity and colonic inflammation: enhanced expression of epithelial alpha-defensins. Dig Dis Sci 2002; 47:1349–1355.
      67.Lawrance IC, Fiocchi C, Chakravarti S. Ulcerative colitis and Crohn's disease: distinctive gene expression profiles and novel susceptibility candidate genes. Hum Mol Genet 2001; 10:445–456.
      68.O'Neil DA, Porter EM, Elewaut D, Anderson GM, Eckmann L, Ganz T, et al. Expression and regulation of the human β-defensins hBD-1 and hBD-2 in intestinal epithelium. J Immunol 1999; 163:6718–6724.
      69.Wehkamp J, Fellermann K, Herrlinger KR, Baxmann S, Schmidt K, Schwind B, et al. Human beta-defensin 2 but not beta-defensin 1 is expressed preferentially in colonic mucosa of inflammatory bowel disease. Eur J Gastroenterol Hepatol 2002; 14:745–752.
      70.Wehkamp J, Harder J, Weichenthal M, Mueller O, Herrlinger KR, Fellermann K, et al. Inducible and constitutive beta-defensins are differentially expressed in Crohn′s disease and ulcerative colitis. Inflamm Bowel Dis 2003; (in press).
      71.Ursing B, Alm T, Barany F, Bergelin I, Ganrot-Norlin K, Hoevels J, et al. A comparative study of metronidazole and sulfasalazine for active Crohn's disease: the cooperative Crohn's disease study in Sweden. II. Result. Gastroenterology 1982; 83:550–562.
      72.Sutherland L, Singleton J, Sessions J, Hanauer S, Krawitt E, Rankin G, et al. Double blind, placebo controlled trial of metronidazole in Crohn's disease. Gut 1991; 32:1071–1075.
      73.Colombel JF, Lemann M, Cassagnou M, Bouhnik Y, Duclos B, Dupas JL, et al. A controlled trial comparing ciprofloxacin with mesalazine for the treatment of active Crohn's disease. Groupe d'Etudes Therapeutiques des Affections Inflammatoires Digestives (GETAID). Am J Gastroenterol 1999; 94:674–678.
      74.Prantera C, Zannoni F, Scribano ML, Berto E, Andreoli A, Kohn A, et al. An antibiotic regimen for the treatment of active Crohn's disease: a randomized, controlled clinical trial of metronidazole plus ciprofloxacin. Am J Gastroenterol 1996; 91:328–332.
      75.Steinhart AH, Feagan BG, Wong CJ, Vandervoort M, Mikolainis S, Croitoru K, et al. Combined budesonide and antibiotic therapy for active Crohn's disease: a randomized controlled trial. Gastroenterology 2002; 123:33–40.
      76.Arnold GL, Beaves MR, Pryjdun VO, Mook WJ. Preliminary study of ciprofloxacin in active Crohn's disease. Inflamm Bowel Dis 2002; 8: 10–15.
      77.Rutgeerts P, Hiele M, Geboes K, Peeters M, Penninckx F, Aerts R, et al. Controlled trial of metronidazole treatment for prevention of Crohn's recurrence after ileal resection. Gastroenterology 1995; 108: 1617–1621.
      78.Prantera C, Scribano ML, Falasco G, Andreoli A, Luzi C. Ineffectiveness of probiotics in preventing recurrence after curative resection for Crohn's disease: a randomised controlled trial with Lactobacillus GG. Gut 2002; 51:405–409.
      79.Jakobovits J, Schuster MM. Metronidazole therapy for Crohn's disease and associated fistulae. Am J Gastroenterol 1984; 79:533–540.
      80.Brandt LJ, Bernstein LH, Boley SJ, Frank MS. Metronidazole therapy for perineal Crohn's disease: a follow-up study. Gastroenterology 1982; 83:383–387.
      81.Solomon MJ, McLeod RS, O'Connor BI, Steinhart AH, Greenberg GR, Cohen Z. Combination ciprofloxacin and metronidazole in severe perianal Crohn's disease. Can J Gastroenterol 1993; 7:571–573.
      82.Burke DA, Axon AT, Clayden SA, Dixon MF, Johnston D, Lacey RW. The efficacy of tobramycin in the treatment of ulcerative colitis. Aliment Pharmacol Ther 1990; 4:123–129.
      83.Mantzaris GJ, Archavlis E, Christoforidis P, Kourtessas D, Amberiadis P, Florakis N, et al. A prospective randomized controlled trial of oral ciprofloxacin in acute ulcerative colitis. Am J Gastroenterol 1997; 92:454–456.
      84.Mantzaris GJ, Petraki K, Archavlis E, Amberiadis P, Kourtessas D, Christidou A, et al. A prospective randomized controlled trial of intravenous ciprofloxacin as an adjunct to corticosteroids in acute, severe ulcerative colitis. Scand J Gastroenterol 2001; 36:971–974.
      85.Chapman RW, Selby WS, Jewell DP. Controlled trial of intravenous metronidazole as an adjunct to corticosteroids in severe ulcerative colitis. Gut 1986; 27:1210–1212.
      86.Mantzaris GJ, Hatzis A, Kontogiannis P, Triadaphyllou G. Intravenous tobramycin and metronidazole as an adjunct to corticosteroids in acute, severe ulcerative colitis. Am J Gastroenterol 1994; 89:43–46.
      87.Turunen UM, Farkkila MA, Hakala K, Seppala K, Sivonen A, Ogren M, et al. Long-term treatment of ulcerative colitis with ciprofloxacin: a prospective, double-blind, placebo-controlled study. Gastroenterology 1998; 115:1072–1078.
      88.Kruis W, Schutz E, Fric P, Fixa B, Judmaier G, Stolte M. Double-blind comparison of an oral Escherichia coli preparation and mesalazine in maintaining remission of ulcerative colitis. Aliment Pharmacol Ther 1997; 11:853–858.
      89.Rembacken BJ, Snelling AM, Hawkey PM, Chalmers DM, Axon AT. Non-pathogenic Escherichia coli versus mesalazine for the treatment of ulcerative colitis: a randomised trial. Lancet 1999; 354:635–639.
      90.Shen B, Achkar JP, Lashner BA, Ormsby AH, Remzi FH, Brzezinski A, et al. A randomized clinical trial of ciprofloxacin and metronidazole to treat acute pouchitis. Inflamm Bowel Dis 2001; 7:301–305.
      91.Madden MV, McIntyre AS, Nicholls RJ. Double-blind crossover trial of metronidazole versus placebo in chronic unremitting pouchitis. Dig Dis Sci 1994; 39:1193–1196.
      92.Mimura T, Rizzello F, Helwig U, Poggioli G, Schreiber S, Talbot IC, et al. Four-week open-label trial of metronidazole and ciprofloxacin for the treatment of recurrent or refractory pouchitis. Aliment Pharmacol Ther 2002; 16:909–917.
      93.Gionchetti P, Rizzello F, Venturi A, Ugolini F, Rossi M, Brigidi P, et al. Antibiotic combination therapy in patients with chronic, treatment-resistant pouchitis. Aliment Pharmacol Ther 1999; 13:713–718.
      94.Gionchetti P, Rizzello F, Venturi A, Brigidi P, Matteuzzi D, Bazzocchi G, et al. Oral bacteriotherapy as maintenance treatment in patients with chronic pouchitis: a double-blind, placebo-controlled trial. Gastroenterology 2000; 119:305–309.
      95.Wehkamp J, Harder J, Wehkamp-von Meissner B, Schwichtenberg L, Fellermann K, Herrlinger KR, et al. The probiotic E. coli Nissle 1917 (Mutaflor) induces defensins in intestinal epithelial cells: a novel mechanism of action. Gastroenterology 2002; 122:A75.
      96.Vecchi M, Sinico A, Bianchi MB, Radice A, Gionchetti P, Campieri M, et al. Recognition of bactericidal/permeability-increasing protein by perinuclear anti-neutrophil cytoplasmic antibody-positive sera from ulcerative colitis patients: prevalence and clinical significance. Scand J Gastroenterol 1998; 33:1284–1288.
      97.Elzouki AN, Eriksson S, Lofberg R, Nassberger L, Wieslander J, Lindgren S. The prevalence and clinical significance of alpha 1-antitrypsin deficiency (PiZ) and ANCA specificities (proteinase 3, BPI) in patients with ulcerative colitis. Inflamm Bowel Dis 1999; 5:246–252.
      98.Schultz H, Weiss J, Carroll SF, Gross WL. The endotoxin-binding bactericidal/permeability-increasing protein (BPI): a target antigen of autoantibodies. J Leukoc Biol 2001; 69:505–512.
      99.Schwab M, Schaeffeler E, Marx C, Fromm MF, Kaskas B, Metzler J, et al. Association between the C3435T MDR1 gene polymorphism and susceptibility for ulcerative colitis. Gastroenterology 2003; 124:26–33.

      Crohn's disease; ulcerative colitis; innate immunity; defensin

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