Scientific Review

Inflammatory bowel disease: dysfunction of GALT and gut bacterial flora (II)

P. Chandran* S. Satthaporn* A. Robins** O. Eremin*
*Department of Surgery and **Department of Immunology, Queens Medical Centre, University of Nottingham, Nottingham, NG7 2UH

Correspondence to: P. Chandran, Department of Surgery, Queens Medical Centre, University of Nottingham, Nottingham, NG7 2UH

Introduction Environmental factors Commensal bacteria Quorum sensing molecules Bacteria, viruses and IBD

Antibiotics and IBD Probiotics and IBD Bacterial host interactions Permeability defects in IBD Summary References

Keywords: Crohn’s disease, ulcerative colitis, gut bacterial flora, quorum sensing molecules and probiotics
Surg J R Coll Surg Edinb Irel., 1 June 2003, 125-136

The precise cause(s) of Crohn’s disease and ulcerative colitis are unknown. From animal models and human studies it is well established that gut bacterial flora are essential for inducing the bowel inflammation. Animal models, when kept in a germ-free environment, do not develop colitis until the gut flora is reconstituted. It is not clear whether the bacterial antigens (Ags) from the normal flora or some other pathogenic bacterial Ags induce/propagate the inflammatory process in inflammatory bowel disease (IBD). Despite extensive research it has not been possible to identify any specific bacteria or virus as a credible cause of IBD. Recent understanding of quorum sensing molecules (QSMs) secreted by bacteria helps to explain the community behaviour in bacterial species. When QSMs reach a defined concentration, they activate bacterial proliferation and a number of virulence genes. Also, these molecules have been found to modulate the immune system to the advantage of the gut bacteria. They have not been well studied, however, in the gut. Inappropriate secretion of QSMs may alter the gut-associated lymphoid tissue (GALT) and, thereby, deregulate the immune tolerance normally present. Usefulness of probiotics and their immune modulating effects are being increasingly reported. Probiotics are also being used in the treatment of IBD. The interaction between the epithelial cells and the gut flora is very important as this is the first line of contact; this interaction may determine the induction of tolerance and mucosal integrity or immune activity, tissue inflammation and abnormal permeability. The latter is documented in patients with IBD and their healthy relatives. This may be an important factor in disruption of mucosal integrity and GALT dysfunction

INTRODUCTION
Crohn’s disease and ulcerative colitis are chronic inflammatory bowel diseases (IBDs) of unknown aetiology. Since their first description in 1913 and 1859, respectively, little progress has been made in establishing the precise causes of these diseases. There is substantial evidence to suggest that environmental, genetic and microbial factors (alone or in combination) predispose to and/or induce IBD.¹ Studies carried out in animals have shown that bacterial flora in the gut play a crucial role in inducing IBD.² The T lymphocytes of the gut-associated lymphoid tissue (GALT) become activated and interact with antigens (Ags) derived from gut bacteria. The activated lymphocytes secrete proinflammatory cytokines, which are able to recruit immune cells into the GALT, augmenting and perpetuating the resultant tissue damage and inflammation (see part I Surg J R Coll Surg Edinb Irel 1: 2; 63-75). It is not clear whether it is the Ags derived from the normal bacterial flora or Ags from pathogenic bacteria, which initiate the abnormal activation of the immune cells in GALT. In this review, we will explore the various environmental and microbial factors that have been implicated and their interrelationship in the pathogenesis of IBD. Probiotics are living microorganisms that have been used in the treatment of various gastrointestinal disorders. The beneficial effects of probiotics have been attributed to their role in modulating the epithelial barrier and immune function of the gut. The role of probiotics in IBD will also be discussed. Lastly, alterations in gut barrier function and abnormal permeability to cells, organisms and secretory products, in IBD will be addressed.

ENVIRONMENTAL FACTORS
Two environmental factors, which have been studied extensively, are smoking and appendicectomy. Smoking is negatively correlated with the incidence of ulcerative colitis and positively correlated with Crohn’s disease. The prevalence of ulcerative colitis in Morman communities in Britain and Ireland, has been studied. The Morman community, which discourages smoking, has a five-fold increase of ulcerative colitis, compared with the general population.³ A metaanalysis carried out by Calkins in 1989 of nine suitable case control studies showed that the life-time risk of smokers developing ulcerative colitis was 0.41 and the risk of nonsmokers was 2.9. Meta-analysis of seven studies of looking at the effect of smoking on Crohn’s disease has confirmed a pooled odds ratio of 2.0 with a 95% confidence interval for current smokers, compared with non-smokers, and 1.83 for ex-smokers compared with non-smokers.⁴ Studies have also shown that smoking leads to remission of ulcerative colitis and relapse on cessation of smoking. Smoking also affects the course of Crohn’s disease; patients who continue to smoke have a significantly higher chance of recurrence than lifetime non-smokers. Various mechanisms by which smoking may modulate IBD have been postulated and include the nature of the mucus secreted, suppression of cytokine production, and alteration of blood flow and intestinal motility. Based on the beneficial effects of smoking, randomised placebo-controlled trials have shown that nicotine patches induce remission of ulcerative colitis.^5,6

Removal of the appendix has been shown to be an important negative factor in the subsequent development of ulcerative colitis. The appendix is an important component of GALT. Appendicectomy carried out in animals soon after birth disturbs the normal distribution of B cells in the rest of the intestinal mucosa and the subsequent decline in the secretion of immunoglobulin A (IgA).⁷ T cell receptor (TCR) deficient mice develop ulcerative colitis spontaneously, and when these animals underwent appendicectomy at an earlier age they did not develop ulcerative colitis. This suggests that the appendix may prime the putative Ag(s) that is (are) responsible for inducing ulcerative colitis.⁸ Several case control studies have demonstrated the negative association between appendicectomy and ulcerative colitis. Meta-analysis of these studies have shown that appendicectomy offers a 69% reduction in the risk of developing ulcerative colitis.⁹ Koutroubakis et al (2002) postulate various mechanisms to account for this negative association between appendicectomy and ulcerative colitis (Table 1).

COMMENSAL BACTERIA AND THE DEVELOPMENT OF GALT
The normal GI flora is composed of the autochthonous organisms that have colonised the host during its development. The composition of this microflora, in any particular host, remains remarkably constant over the passage of time and is not readily altered by changes in the diet. When the composition was mapped, using molecular techniques, there was a substantial variation between different human subjects.¹⁰ There are about 400-500 different species of bacteria in the gut. ¹¹ The newborn infant, during normal vaginal delivery, comes into contact with maternal enteric bacteria that inhabit the perineum. These bacteria rapidly colonise the infant’s intestine and this process is called conventionalisation.¹²

More than 99.9% of the indigenous microflora are obligate anaerobes. Because of this, there are severe limitations in the methodologies for identifying and culturing these indigenous species.¹³ Even with sophisticated techniques, only 60% of the bacteria seen in human faecal samples can be cultured.¹⁴ The indigenous bacteria are not just randomly distributed in the gut but are made up of characteristic populations in particular regions of the GI tract. The stomach, duodenum and jejunum contain only 103 - 104 bacteria per ml of GI contents. The ileum contains about 108 bacteria per ml of intestinal contents and the colon contains 1010 -1011 bacteria per gram of intestinal contents. There is normally a steady passage (translocation) of a small number of bacteria to the regional mesenteric lymph nodes and subsequently to the systemic circulation.¹⁵

Studies in animals have demonstrated convincingly that commensal bacteria induce anergy in the gut. In rodents, enteric bacterial Ags from normal BALB/c mice, when cocultured in vitro with CD4+T cells isolated from colonic lamina propria, mesenteric lymph nodes and the spleen, did not induce T lymphocyte proliferation and activation. In contrast, mice with severe combined immuno-deficiency (SCID) developed colitis after reconstitution with CD4+/CD45RBhigh (immature) T cells. The CD4+Tcells proliferated under the same in vitro conditions.¹⁶ T lymphocytes isolated from the normal human gut and peripheral blood, when incubated with sonicates of autologous gut flora, did not proliferate. When the same cells were incubated in vitro with sonicates of heterologous intestinal flora, however, they did proliferate. Lymphocytes isolated from the lamina propria of the bowel involved in ulcerative colitis and co-cultured with autologous intestinal sonicates underwent in vitro activation and proliferation. Lymphocytes from noninflamed intestines and the peripheral blood did not undergo these in vitro changes.¹⁷ These results are in agreement with the hypothesis that there is normally tolerance towards commensal bacteria in the gut lumen and the breakdown of this tolerance predisposes to IBD.

Raising experimental animals in a germ-free environment has helped to produce a better understanding of the effect of the indigenous microflora on the development of GALT and gut immunity. The number of lamina propria lymphocytes (LPLs), immunoglobulin A-producing plasma cells and the level of serum immunoglobulins are all decreased in animals raised in a germ-free environment. This is because of lack of Ag stimulation from the commensal bacteria. The Peyer’s patches of normal mice display continuous germinal centre activity. By contrast, the Peyer’s patches of mice raised in a germ-free environment show quiescent B cell follicles.¹⁸ The majority of peripheral blood CD4+T cells in normal mice are CD45RBhigh, indicative of resting or naïve T helper (Th) cells. The Peyer’s patches CD4+T cells, however, are CD45RBlow indicative of activated Th cells. The CD4+ T cells from the Peyer’s patches of germ-free animals are quiescent and are of CD45RBhigh phenotype. When these animals are colonised by bacteria there is a shift in the CD4+ T cell phenotype from CD45RBhigh to CD45RBlow.¹⁹ Hence, the bacteria provide the Ag drive for the development and activation of GALT. The ability to generate IgA producing cells progressively increases on the establishment of the gut flora. The number of bacteria translocating through the epithelial barrier decrease as the IgA production increases. The IgA that is secreted into the bowel lumen will bind with gut Ags and prevent them from translocating.^20,21

QUORUM SENSING MOLECULES
Bacteria exist in nature as single cell organisms. Even though they are unicellular they have evolved a multicellular organisation. They have a highly conserved mechanism of communicating with their fellow members through chemical messengers. The ability of the bacteria to attack and survive in a host depends on their ability to overcome the host defence mechanisms and secrete toxins that are damaging to the host. These advantageous and adaptive traits are activated only when a particular bacterial population density is reached. The mechanism by which the bacteria sense their population density is called quorum sensing and the molecules that bacteria secrete to fulfill this function are called quorum sensing molecules (QSMs).¹¹ At low cell density, the signal molecules are produced at low basal levels. During the growth phase the concentration of the QSMs increases reaching a critical concentration, at which stage they bind to specific receptors and activate transcriptional genes. Such QSMs are also called auto-inducers.

Quorum sensing was first discovered in the marine bacteria Vibrio fischeri that colonises fish and squid in sea water. When a bacterial count of sufficient density is reached they produce luminescence. This was then discovered to be due to autoinducers secreted by Vibrio fischeri activating the luciferase enzyme complex, which is responsible for light production. LuxR and LuxI are the two genes involved in the autoinducer production and activation of luciferase enzyme complex genes in Vibrio fischeri.²² LuxI is the gene coding for the enzyme that synthesizes QSMs; LuxR is the gene which codes for the protein which is the sensor/transducer. The amino terminal regions of LuxR-like proteins are involved in autoinducer binding and the C-terminal region binds to the target gene coding for luciferase enzyme complex (Figure 1). Subsequently, homologues of LuxR and LuxI were discovered in a number of other gram-negative bacteria.^11,23,24 In every described case, research indicates that the fundamental functions and biochemical mechanisms of action of the various LuxI-like and LuxR-like proteins are identical to those of LuxI and LuxR of Vibrio fischeri.

Figure 1: LuxI/LuxR quorum sensing system. LuxI synthesises autoinducers which then bind to LuxR protein. The autoinducer and LuxR complex then activates target genes

Various molecules can function as quorum sensing both in Gram-negative and Gram-positive bacteria. Acyl homoserine lactones (AHLs) are well studied QSMs secreted by Gram-negative bacteria. They contain 4-14 carbon atoms with an acyl side chain (Figure 2). The AHL diversity depends on the length of the acyl chain and the nature of the substitution in the C-3 position. Gram-positive bacteria have evolved a communication system, which is different from the system used by Gram-negative bacteria. The signal molecules here are modified oligopeptides. The detectors of these molecules are called sensor kinases. The sensory information is relayed into the cell, which induces appropriate alteration in gene expression.

Figure 2: An example of a QSM secreted by Pseudomonas aeruginosa. It is called OdDHL [N - (oxo-dodecanoyl) - N - homeserine lactone] and is made up of 12 carbon atoms

Quorum sensing molecules have been shown to modulate the immune system. However, aspects of this property of QSMs are poorly understood.²⁵ Since the GI tract is inhabitated by a vast number of bacterial species it is not surprising that QSMs are secreted in the gut lumen, but this process has been poorly studied. In animal models of IBD, inflammation only occurs in the presence of gut flora. To date, no organism has been convincingly associated with the pathogenesis of IBD. It is possible that bacterial products such as QSMs have an important role to play in deregulating GALT and abrogating the immune tolerance of the gut and, thereby, initiating the tissue damage seen in IBD.

Quorum sensing is also the language which allow the different species of bacteria to interact with each other. Burkholderia cepacia is a Gram-negative bacteria which co-infects with Pseudomonas aeruginosa in patients with cystic fibrosis; it cannot infect on its own. Acyl homoserine lactones produced by Pseudomonas aeruginosa communicates with Burkholderia cepacia and induces the organisms to express virulent factors and formation of biofilms. This allows Burkholderia cepacia to establish as a pathogen in the host.^26,27

BACTERIA, VIRUSES AND IBD
Deregulation of the GALT is believed to be a key component of the pathogenesis of IBD. What brings about this deregulation is not clear. Attempts to isolate or detect microorganisms in the tissues affected by IBD have failed. Establishment of the relationship between Helicobacter pylori and peptic ulcer disease clearly demonstrates that it is still possible that unidentified microorganism(s) are involved. Indirect evidence suggests that the Ags from the gut bacterial flora may play a role. The anatomic site of the intestine in which bacteria are present in high numbers such as the distal ileum and colon, are the sites frequently affected in IBD.

Experimental animal models of IBD, can be produced by deleting the genes that codes for cytokines such as interleukin-10 (IL-10) or transgenic mice expressing HLA-B27. In these experimental animal models, when the mice are raised in germ-free conditions, they do not develop IBD. However, when the mice are colonised with their normal gut flora they develop colitis. This suggests that gut bacterial flora produce the Ag drive which causes the activation and proliferation of T lymphocytes which probably interact with gut mucosa and also secrete cytokines which are injurious to the bowel.^28-30 The sera of patients with IBD have antibodies reactive with a range of Ags including gut bacterial Ags. Antibodies directed against the bacteriodes species have been reported in patients with IBD.^29-31

Mycobacterial species have been implicated in the pathogenesis of Crohn’s disease.³² Intestinal infections of mycobacterium tuberculosis in humans cause lesions in the bowel similar to Crohn’s disease.³³ Both affect the distal ileum and produce characteristic granulomas. There is continuing dispute regarding the association of mycobacterial species and Crohn’s disease. A review of 18 studies looked for the mycobacterial species in tissues affected by Crohn’s disease; nine studies detected the organism and in the other nine studies there was no convincing evidence for the presence of mycobacteriae.³⁴

Epidemiological data also do not support a mycobacterial cause because there is no evidence of increased incidence of Crohn’s disease in physicians treating patients with Crohn’s disease, spouses of patients with Crohn’s disease, farmers and veterinary physicians who have close contact with animals infected with mycobacteriae. Koch’s postulate for a bacterial cause of a disease is to isolate the organisms from the diseased tissue and obtain a cure of the disease by elimination of the organisms. Culture of mycobacteria from diseased tissue has been difficult because the organisms have special media requirements and incubation periods of several months.³⁵ Mycobacterium paratuberculosis causes intestinal infection similar to Crohn’s disease in animals. When the tissues from the infected animals are analysed there is great diversity with some of the tissues heavily contaminated with mycobacteria and others lacking any organisms, comparable to the findings in human leprosy. This may be a reason why in only 5% of the patients with Crohn’s disease the mycobacteria can be isolated. Fujita et al (2002) used real-time quantitative polymerase chain reaction (PCR) to detect and estimate mycobacterial bacterial genome IS900 in formalin-fixed, paraffin-embedded tissue from patients with Crohn’s disease.³⁶ None of their patients had any mycobacteria in the tissues affected by Crohn’s disease. In paraffin-embedded tissue fragmentation of DNA can occur, thereby, making identification by PCR difficult.37 Sechi et al (2001) believe that detection of IS900 genome by in situ hybridisation is a better method than PCR. They found nearly 70% of patients with Crohn’s disease were positive when tested by in situ hybridisation, but were not detected by PCR.³⁷

Mycobacteria as a group, are difficult to treat. They require a combination of antibiotics and develop drug resistance readily. Borody et al (2002) treated patients with severe Crohn’s disease with triple macrolide-based antimycobacterial therapy and obtained remissions and cure in half of the patients treated. Three of their patients had a complete cure with no further relapses.³⁸ Shafran et al (2002) treated patients with Crohn’s disease, who had mycobacterial antibody in their serum, with combinations of macrolid antibiotics and found that more than half of the patients had significant clinical improvement.³⁹ These studies, however were not randomised and small numbers of patients were involved. Large, randomised, multicentre trials are required to clarify this further.

Helicobacter hepaticus has been linked in the causation of ulcerative colitis. Severe combined immunodeficiency mice, when adoptively transferred with CD4+ T cells, develop colitis. The mice that are kept in a germ-free environment do not develop colitis. When these mice are reconstituted with Helicobacter hepaticus they develop colitis.^40,41 Helicobacter hepaticus also induced coilitis in IL-10 gene knock out mice and T cell receptor gene knock out mice.⁴² In a similar experiment, Jiang et al (2002) showed that SCID mice on transfer of CD4+ T cells from congenic wild type mice and infected with Helicobacter muridarum developed colitis resembling that seen in humans.⁴³

Measles infection in childhood has been linked to the development of IBD, mainly Crohn’s disease in adult life. Measles is an infection of childhood, which causes a selflimiting mucocutaneous infection. The virus persists in a small proportion of individuals in nerve tissue to cause latent infection - subacute sclerosing pan encephalitis (SSPE). There is debate on persistence of measles virus in epithelial cells of the gut causing Crohn’s disease. Ekbom et al (1996) reported that perinatal infection of measles leads to Crohn’s disease in adulthood; others disagree with this postulate.^44,45 A study from Finland found no association between MMR vaccination and IBD.⁴⁶

Demonstration of measles viral genome in the tissues involved in IBD would provide convincing evidence for the association between measles infection and Crohn’s disease similar to SSPE. To date, there are six publications which looked for measles virus in the tissues of patients with IBD using PCR. Of these six studies, five did not find any convincing evidence of the persistent presence of virus. One study did find evidence of persistent measles virus in peripheral blood mononuclear cells.⁴⁶

ANTIBIOTICS AND IBD
In animal models of spontaneous IBD (e.g. IL-10 knock out mice) the use of antibiotics prevents the development of the colitis.⁴⁷ Based on these findings and results from a number of other animal models, antibiotics have been used in the treatment of IBD. The results, however, have been variable. The postulated mechanism of action of these antibiotics is that they reduce luminal bacteria and possibly eliminate certain enteric bacterial subsets. Clinical trials have shown metronidazole to be effective in patients with mild to moderate Crohn’s disease and those with perianal disease.^48,49 Ciprofloxacin has also been used to treat active Crohn’s disease and where there are fistulae.⁵⁰ Various randomised placebo-controlled trials have shown that ciprofloxacin and metronidazole were effective in inducing remissions in patients with Crohn’s disease involving the large bowel.^51-53 A short course of metronidazole following the resection of small bowel strictures due to Crohn’s disease delays the development of recurrent disease. Also, once the recurrence developed it was less severe than the group that did not receive metronidazole.⁵⁴ Ciprofloxacin has also has been reported to be useful in inducing and maintaining remission in patients with ulcerative colitis.⁵⁵ In a small randomised study, rifamixim (broad spectrum non absorbable antibiotic) given orally to patients with acute attacks of ulcerative colitis, not responding to any standard treatment, showed significant improvement in a small group of patients. The patients treated showed reduced stool frequency, rectal bleeding and improvement in sigmoidoscopic appearance.⁵⁶ Pouchitis is one of the complications of patients who undergo surgery for ulcerative colitis and the creation of a pouch and anal anastamoses. Faecal stasis and bacterial overgrowth have been hypothesised to induce the inflammation characteristic of this combination. Various studies have shown that antibiotics such as metronidazole and ciprofloxacin are effective in the treatment of pouchitis.^57-59

PROBIOTICS AND IBD
Bacteria have been considered to be detrimental to good health; however, evidence is accumulating regarding the possible benefits of bacteria. Probiotics are microorganisms that have beneficial effects on health by altering the microbial environment. Lactobacilli, bifidobacteria, and non-pathogenic yeast are some of the organisms, which are found to have a probiotic role. These beneficial probiotic effects are believed to result from the modulation of GALT (Table 2).^60,61 Non probiotic bacteria are known to enhance proliferation of T cells and induce the T cells to produce proinflammatory cytokines.⁶² By contrast, the probiotics are known to inhibit T cell proliferation and inhibit cytokine production.^63,64 Sonicates of probiotic bacteria inhibit the mitogen-induced proliferation of mononuclear cells in a dose dependant manner in vitro. This suppressive effect was comparable to that induced by corticosteroids.⁶⁵

Interleukin -10 deficient mice develop IBD spontaneously and this can be prevented by the administration of lactobacillii. This protective effect was attributed to the production of IL-10 mimic by the probiotic bacteria.⁶⁶ Lactobacilli are a major component of the normal gut microflora. The immune modulating effects of probiotic bacteria have been studied. When the gut flora of children with allergic disorders was analysed, lactobacilli were less often present than in children without allergies.^67,68 Lactobacillii administration has been shown to alleviate food allergy in both children and adults.

Dendritic cells (DCs), which are the key Ag presenting cells (APCs) in GALT and systemic immune system, determine immunity or tolerance depending upon the degree of exposure of activation/maturation signals and the cytokines produced in situ. Christensen et al (2002) studied the expression of surface markers and the production of cytokines by various strains of lactobacillii on DCs. Substantial differences were found among strains in their capacity to induce IL-12 and tumor necrosis factor-a (TNF-a) production by DCs. Similar but less pronounced differences were observed in the induction of IL-6 and IL-10 production by lactobacilli. All strains upregulated expression of surface MHC class II and B7-2 (CD86) costimulating molecules, which is indicative of DC activation and maturation; lactobacilli with the greatest capacity to induce IL-12 were the most effective in upregulation. Thus, different strains of lactobacillii induce different activation/maturation patterns in DCs and cytokine secretion.⁶⁹

Several animal and human studies suggest that probiotics have modulating effects on the course of IBD. Several randomised clinical studies have shown that a non-pathogenic strain of Escherichia coli was effective in preventing relapse of Crohn’s disease and ulcerative colitis.^70-72

BACTERIAL HOST INTERACTIONS
It is clear from animal and human studies that bacteria play a key role in the pathogenesis of IBD. However, to date, no single species has been convincingly shown to be the causative agent in IBD. There is no convincing evidence to show that there is a difference in the microflora of patients with ulcerative colitis and Crohn’s disease, compared with normal healthy individuals. Attention has now focused on understanding the interaction between the host and bacteria. Epithelial cells are the first contact for the commensal bacteria and the way they interact with enterocytes may determine whether inflammation in the gut is induced or not. The colonisation of the T 84 human epithelial cell line with pathogenic microorganisms is associated with increased in vitro production of proinflammatory cytokines such as IL-8. Also, treating these cell lines with TNF-a induces the increased production of IL-8. These cytokine secretory changes are associated with increased production of nuclear factor .B (NF.B), which on binding to DNA activates the genes that code for these proinflammatory cytokines. The T84 epithelial cell line, when incubated with non-pathogenic commensal organisms, does not produce proinflammatory cytokines. Also, when T84 epithelial cell lines are pre-treated with nonpathogenic commensal organisms and subsequently exposed to TNF-a they showed a marked reduction in the production of IL-8. Thus, commensal bacteria- epithelial interactions have an antiinflammatory action through modulation of NF.B.⁷³

Mucosal epithelial cells harbour a number of antimicrobial factors that form a biochemical barrier to microbial colonisation. Epithelial surfaces produce bactericidal/ permeability-increasing protein (BPI), an antibacterial and endotoxin-neutralising molecule. It was originally described as a constituent of primary granules in neutrophils and eosinophils. Bactericidal/permeability-increasing protein is a 55-60-kDa protein which selectively exerts multiple antiinfective actions against Gram-negative bacteria, including damage to bacterial inner/outer membranes, neutralisation of bacterial lipopolysaccharide (LPS), as well as serving as an opsonin for phagocytosis of Gram-negative bacteria by neutrophils.⁷⁴ It binds to LPS, inhibiting its binding to the Toll like receptors (TLRs) and, thereby, preventing the activation of innate immunity. Newborns show less expression of BPI than adults suggesting that it is induced as an individual’s mucosa is more exposed to the gut microbial environment. Bactericidal/ permeability-increasing protein is inducible on the surface of epithelial cells by antiinflammatory lipid mediators such as lipoxin analogue that is produced in vivo by aspirin.⁷⁵ Due to its endotoxin neutralising effect, it is being tested in treating patients with menningococcal sepsis.

The concentration of BPI was measured in the mucosa of patients with IBD and found to be increased, compared with normal mucosal samples, and it correlated with the severity of inflammation.^76,77 Systemic autoantibodies against BPI were found in patients with IBD suggesting that this might have a neutralising effect on the endogenous BPI. The increased production of BPI is possibly a reflection of a defect in one aspect of innate immunity, which induces increased production of BPI.

Toll was originally identified as a Drosophila gene important for ontogenesis and antimicrobial resistance. Subsequently, receptors for Toll-like protein were found in mammalian cells. The intracellular portion of the TLR is very similar to the cytoplasmic domain of the IL-1R and related molecules. Toll-like receptors have been identified as an essential component of the LPS receptor signaling complex that controls innate immune responses. Lipopolysacchride after binding to the TLR activates a series of protein cascades intracellulary, which ultimately activates NF.B, which regulates a number of transcriptional genes involved in inflammation. Binding of NF.B to the specific gene locii switches on the genes that regulate inflammation. The proinflammatory cytokine genes are regulated by this factor. When the NF.B levels were analysed in the mucosa of patients with IBD they were increased compared with controls (see review I).⁷⁸

PERMEABILITY DEFECTS IN IBD
Deregulation of the GALT response to Ags present in the normal bacterial flora is believed to be an important factor in the pathogenesis of IBD in genetically susceptible individuals (see review I). It is also possible that the failure of the normal regulatory mechanisms that operate in the GALT in response to enteric bacteria induce IBD.⁷⁹ Defects in the normal epithelial barrier increase protein/Ag fluxes into the mucosa and bacterial translocation resulting in enhanced and continuous closer interaction with immune cells in the GALT. This may be an early and important event in the pathogenesis of IBD.

The intestinal epithelium is composed of polarised epithelial cells that provide a crucial barrier function. This single cell layer separates a myriad of bacterial and food Ags in the lumen of the GI tract from the GALT. In addition to its crucial role as a barrier and in transport of nutrients, the intestinal epithelium is an active component in the complex immunoregulation of GALT. Intestinal epithelial cells secrete a wide variety of cytokines, both constitutively and after contact with bacterial pathogens. Moreover, intestinal epithelial cells have extensive cellular contact with several distinct populations of T lymphocytes both within the epithelium and in the underlying lamina propria. Clonal ignorance of T cells could result if immunologically relevant forms of Ags did not gain access to the immune system via the gut. This may result from the destruction of relevant epitopes by the non-specific effects of digestion or because the physical barrier of the epithelium and its associated mucus layer prevented Ag access to APCs. However, this is unlikely as immune reactive proteins can be detected in serum within minutes of their ingestion.^80-82 The barrier effect of the gut is to filter aggregated Ags resulting in only monomeric forms being absorbed systemically. Apart from the mucus layer, the epithelial cell tight junctions prevent access of luminal Ags to the APCs in the subepithelial layer. The disruption of this junction is believed to lead to uncontrolled inflammation in the gut. Patients with ulcerative colitis exhibit increased epithelial permeability that correlates with the induction of nitric oxide synthase (NOS) activity.⁸³ Animal models of IBD, such as in the spontaneous colitis forming C3H/ HeJBir mouse and HLA B27transgenic rat, show primary permeability defects. Also, the DSS and TNBS colitis mouse models acquire early permeability defects following chemical induction.^84-86

The evidence for the importance of intercellular junctions was found in the N-cadherin dominant negative mouse.⁸⁷ These mice develop IBD including aphthoid ulcers and adenomas. A complex of proteins, collectively termed tight junctions, maintain the seal between adjacent cells (Figure 3). Proteins such as hyperphosphorylated occludin, zona occludens (ZO) and the claudin family are known to contribute to the structure of the tight junction. Recent evidence has shown that far from being a static structure, the tight junction is highly regulated. For example, interferon-. (IFN.), a proinflammatory cytokine, can cause down regulation of a component of the junction, ZO-1.⁸⁸ This time-dependant decrease in ZO-1 corresponds with a significant decrease in transepithelial resistance as well as increase in mannitol flux from the gut lumen into the circulation.

Key: t.j. tight junction (occludens), a.b. adhesion belt (adherens), d.s. desmosome (spot), g.j. gap junction (communicating), h.d. hemidesmosome

Figure 3: Schematic representation showing intercellular junctions between epithelial cells of the gut mucosa

Failure of barrier function of the gut epithelium in IBD is important for several reasons. Firstly, defects in barrier function may allow Ags and bacteria and other noxious agents to penetrate the epithelium and possibly initiate and perpetuate the inflammatory process. A loss of barrier function and associated inflammation results in passive leakage of water and ions into the intestinal lumen, producing diarrhoea. When the integrity of the epithelial junctions were tested in patients with IBD, by passing an electrical current in vitro and measuring the impedance, it has been found to be decreased by 50%-80%, compared with the normal epithelium. Electron microscopy showed loss of horizontal strands between the epithelial cells and this correlated very strongly with functional defects. Thus, a small percentage decrease in strand numbers can be associated with a large increase in barrier dysfunction.⁸⁹

Gut bacterial flora regulates the permeability across the mucosal barrier. García-Lafuente et al (2001) used a rat model to study the effect of luminal bacteria in altering the barrier function. The rats, raised in germ-free conditions had a closed loop of bowel brought out as a colostomy. In these loops, bacteria were introduced and colonies were established. Radioactive carbon-labelled mannitol was instilled into the loops and measured in the blood, producing a measure of mannitol diffusion across the epithelial barrier. Bacteria such as Escherichia coli increased gut permeability, whilst bacteria such as the lactobacillus decreased the permeability across the bowel epithelium.⁹⁰ Hudault et al (2001) also showed that nonpathogenic-Escherichia coli strains reduced the translocation of pathogenic Salmonella typhymurium across the mucosal barrier in mice.⁹¹

It is clear from various studies that abnormal permeability of the gut to bacterial Ags from the luminal bacteria activate the T cells in GALT and cause gut tissue inflammation. Studies have shown that patients with IBD have abnormal permeability across the mucosa. It is very difficult to draw conclusion as to whether the permeability defect is the primary event or secondary to the inflammatory process. Suenaert et al (2002) postulate that the barrier defect is the primary event which allows the gut bacterial Ags to pass through into the gut tissues inducing inflammation. They measured the gut barrier status before and after treating Crohn’s disease patients with chimeric mAbs directed against TNF-a. When the inflammation subsided the gut barrier was restored to near normality.⁹² Many studies investigated the relatives of patients with IBD for permeability defects. Permeability of the bowel was studied using radioactive.⁵¹ Chromium labelled EDTA, which has shown mixed results in IBD and their healthy relatives.^93-97

The evidence from animal studies suggests that cytokines can modify gut barrier function. Madsen et al (1999) studied the permeability defect in IL-10 gene knockout mice and found that they have increased permeability, long before they develop colitis. This permeability defect becomes apparent only when these animals are colonised with normal gut flora. These defects are accompanied by increased production of proinflammatory cytokines such as IFN-.y and TNF-a. When these animals are kept in a germ-free environment they do not develop permeability defects. Thus, the gut flora alters the permeability of the gut mucosa in animals with IL-10 deficiency.⁹⁸ Also, probiotic lactobacillus can restore normal gut permeability and reduce the synthesise of IFN-y and TNF-a.

Astrocytes of the central nervous system, which represent the most abundant glial cells, play a major role in maintaining the blood-brain barrier. They provide metabolic, trophic, and protective support for neurons. In vitro and in vivo experiments have shown that astrocytes express major histocompatibility complex (MHC) class I molecules and can be induced to express class II molecules, as well as CD80 and CD86 costimulatory molecules. Hence, they can function as APCs. They also secrete chemokines and proinflammatory and regulatory cytokines. The enteric nervous system has a similar network of glial cells called enteric glial cells. The latter share many features of astrocytes of the central nervous system and can be expected to participate in the immune functions of the gut. The evidence for this, however, is limited at present.⁹⁹ Genetically engineered animals with defective enteric glial cells developed mucosal inflammation similar to Crohn’s disease. Also, animals with autoimmune targeted deletion of enteric glial cells developed mucosal inflammation resembling Crohn’s disease.¹⁰⁰ Astrocytes form a network over the neuronal tissues preventing the movement of blood components into the brain tissues. In animals with defective astrocytes, oedema and infiltration of immune cells into the brain parenchyma occurs. In animal models with defective enteric glial cells similar defective vascular permeability was noted before the development of inflammation. The enteric glial cells also extend from the mucosa to the tip of the villus and contribute to the integrity of the epithelial barrier. Defective enteric glia allows luminal Ags to pass through the epithelial barrier.¹⁰⁰

SUMMARY
Inflammatory bowel disease is believed to be induced and/or propagated by deregulation and dysfunction of GALT. What factors bring about these changes in the GALT are, as yet, poorly defined. The gut bacterial flora is essential for inducing the disease. Antibiotics have been used successfully in certain studies in treating IBD, which highlights the important role of bacteria in IBD. There are many bacterial species in the gut but attempts to link any particular one to the cause of IBD have been unsuccessful. Defects in gut barrier function are believed to be important, resulting in enhanced fluxes of Ags, cytokines and secretory products into the gut mucosa and increased interaction with GALT. Quorum sensing molecules may be key serectory products but their role has not been fully explored. In vitro studies have shown that they have immune modulating effects. It is possible that inappropriate secretion of QSMs, cytokines etc. significantly modulate GALT, thereby, disrupting the tolerance that normally exists in the gut. This breakdown of tolerance is believed to be a key biological alteration that initiates IBD.

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Copyright: 24 March 2003