5 The normal intestinal mucosa:

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5 The normal intestinal mucosa:

a state of 'controlled inflammation'

CLAUDIO FIOCCHI

Introduction

The focus of investigation in inflammatory bowel disease (IBD) is almost invariably centered on the various cellular and soluble components of the mucosal immune system whose abnormal number or functions presumably underlies the pathogenesis of chronic intestinal inflammation. Although this approach is obviously justified, it is easy to lose sight of the peculiar conditions that allow the normal intestinal immune system to exert a protective rather than an aggressive role. As a matter of fact the intestinal immune system is not only the largest of the body, but primarily a biological shield function- ing in unique ways that distinguish it from all other defense mechanisms. Because it is constantly exposed to the external environment, and carries an extremely rich and varied endogenous flora, the gut mucosa is adapted to work under intense, yet physio- logical, conditions of permanent antigenic pressure.

This pressure requires and brings into action an enormous amount of organized (Peyer's patches and lymphoid follicles) and diffuse (intraepithelial lym- phocytes and lamina propria mononuclear cells) lymphoid cells that respectively form the gut-asso- ciated lymphoid tissue (GALT) and the mucosa- associated lymphoid tissue (MALT) [1]. The existence of this population of mature immunocytes all along the lining of the gastrointestinal tract is quantitatively and qualitatively unparalleled in other organs, including those with a mucosal surface such as the oral cavity, the lungs and airways, the genitourinary tract, and the mammary glands. For this reason the term 'controlled' or 'physiological intestinal inflammation' has been coined to reflect the fact that activated immunocytes are present in large numbers and, rather than causing injury, afford instead an essential protection to the gut and indirectly the rest of the body. Despite its importance this terminology is seldom used outside of the realm

of mucosal immunology and it is difficult to find it even in comprehensive textbooks of mucosal immunology [2].

Interestingly, the majority of components and functions involved in physiological intestinal inflam- mation are the same responsible for pathological inflammation as found in IBD and other conditions.

Thus, the question arises of what distinguishes one from the other, and the answer is the appropriateness or not of the local defense mechanisms. There is not enough information to clearly define what constitu- tes an inappropriate defense response in IBD and why it endures over time, but there is a reasonably good knowledge of the components responsible for physiological intestinal inflammation (Fig. 1). Since avoiding all dietary, microbial or self stimuli that are potentially harmful is practically impossible, the intestine has devised control mechanisms that aUow it to limit the quantity and quality of the antigens presented to the mucosa and GALT. According to Fig. 1 an appropriate response resulting in physiolo- gical inflammation relies on two types of control mechanisms: physical and biological. The first depends on the intrinsic properties of the gut and epithelium, while the latter involves circulatory, h u m o r a l and i m m u n e m e c h a n i s m s . I m m u n e mechanisms rely on both innate and adaptive responses, and when specific immunity is required it can either incite a switch-on response by the induc- tion of effector cells that eliminate the antigen (active immunity), or a switch-off" response resulting in a lack or suppression of response to the antigen (toler- ance). An elaborate discussion of each component and function listed in the diagram is beyond the scope of this review, and some of these topics will be covered more comprehensively in other chapters of this book. Instead, the goal of this review is to provide the reader with an integrated overview of the components that together are presumably

Stephan R. Targan, Fergus Shanahan andLoren C. Karp (eds.J, Inflammatory Bowel Disease: From Bench to Bedside, 2nd Edition, 101-120.

© 2003 Kluwer Academic Publishers. Printed in Great Britain

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Stimuli

Microbes, food antigens, self antigens, toxins

Appropriate response

' ^ ^' ^^

Intestinal inflammation

Inappropriate response

Physiological / Controlled inflammation

Pathological / Uncontrolled inflammation

Stimulus avoidance Stimulus control Infectious, immune-mediated,

autoimmune

Physical control Peristalsis, mucus, epithelial cells, microvilli,

tight junctions

Circulatory Trafficking, homing

cell adhesion

Biological control

Humoral Defensins, lactoferrin, lysozyme, peroxidases

Innate / Non-specific immunity

Neutrophils, macrophages, eosinophils, mast cells, NK cells,

complement, defensins, cytokines, ROM, NO

Immune

\

Adaptive / Specific immunity T-cells, B-cells

Unresponsiveness (Tolerance)

Responsiveness (Active immunity)

Enteric flora Self Food antigens

Figure 1. Diagram of the various components responsible for the induction and maintenance of physiological intestinal inflammation.

The relative thickness of the solid arrows indicates the proportional contribution of single components to each defensive system.

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responsible for the induction and maintenance of physiological inflammation in the human intestine.

Physical control mechanisms

Physical control mechanisms that contribute to physiological intestinal inflammation include primarily those that exclude, limit or select the amount and type of antigenic stimuli that can activate local lymphoid cells. This classification is arbitrary and somewhat artificial because even elements that exert an essentially pure mechanical function, such as peristalsis and the mucus coat, depend on and are integrated with other biological functions. These control mechanisms form what is commonly referred to as intestinal mucosal barrier.

Peristalsis

Peristalsis, the coordinated contraction of the bowel propelling its luminal content aborally, prevents the unnecessary accumulation of food-derived proteins, bacteria, parasites or toxins, and therefore decreases the possibility of absorbing an excessive antigenic load that may induce inflammation [3].

IVIucus

The clinical observation that mucus production and release is enhanced during gut inflammation has long been associated with a protective function.

Intestinal mucus is a viscoelastic gel composed of large mucin molecules made up by a small protein core and a complex array of oligosaccharide chains.

The various mucin gene products are selectively expressed by different cells along the small and large bowel, suggesting that each mucin plays a distinctive role in mucosal protection [4]. Mucus prevents mechanical damage of the mucosa, enables shedding and renewal of mucosal surfaces, functions as a selective barrier for macromolecules and micro- organisms and a trap impeding the penetration of larger microbes, and retains defensive molecules on the luminal surface, such as secretory IgA (sIgA) [5].

Together, these integrated activities generate an important barrier that lowers the damaging and proinflammatory potential of the luminal contents.

Epithelial cells

Just underneath the mucus coat lies a single cell layer of intestinal epithelial cells that offer a variety of physical and functional protective mechanisms.

Intestinal epithelial cells are heterogeneous and include columnar, goblet, Paneth, enteroendocrine and undifferentiated stem cells [6]. Each type has unique morphological features translating distinct physiological and defensive roles. In addition, there are specialized epithelial cells overlying lymphoid follicles called follicle-associated epithelium. Such cells are referred to as M cells; they have less developed microvilli and play a significant role in limiting and selecting antigen sampling [7]. Epithelial cells provide diverse protective systems that include the microvilli with their large surface and negative charge, the lipid bilayer of the plasma membrane, intracellular organelles containing degradative enzymes, and the junctions between adjacent cells.

Prominent among the effector mechanisms mediated by these various systems are enhanced salt and water secretion, expression of antimicrobial proteins and peptides, and production of mucin [8]. Most of these systems are regulated by locally produced cytokines [9], and epithelial cells themselves are a source of proinflammatory cytokines, especially in response to bacterial invasion [10]. This paradoxical phenom- enon has been proposed to actually represent an early protective defense mechanism of the mucosa by limiting bacterial invasion through a repertoire of regulatory molecules including cytokines, chemo- kines, adhesion molecules, prostanoids, nitric oxide and the induction of epithelial cell apoptosis [11, 12].

Antigens can penetrate the epithelium directly

through individual epithelial cells (transcellular

route) or the space between two cells (paracellular

route). When antigens are absorbed transcellularly

they are internally processed by individual cells,

destroyed or degraded for exposure on the cell sur-

face in the context of major histocompatibility com-

plex (MHC) class II antigens for presentation to local

T cells. Although this series of events could be

construed as stimulatory and potentially proinflam-

matory, under normal circumstances the ultimate

effect is a limitation in antigen presentation and the

selective activation of T cell subsets [13]. First,

intestinal epithelial cells are less efficient than profes-

sional antigen-presenting cells (APC) such as macro-

phages and dendritic cells. Second, they activate

preferentially CDS"^ cytotoxic/suppressor T cells or

CD4"^ T cells putatively involved in induction of

tolerance [13, 14]. Thus, the overall response is

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actually one promoting containment of excessive immune reactivity at the mucosal level.

When antigens penetrate the epithelium paracellu- larly the pathway utilized consists of the tight junc- tions (zonulae occludentes) and the subjunctional space. The tight junction is a complex and dynamic structure that constitutes a considerable barrier to large molecules and regulates the frequency, quality and quantity of antigen presentation to the adjacent and underlying immunocytes [15]. Various probes used for clinical measurement of small bowel perme- ability, including L-rhamnose, PEG400, and [^'Cr]EDTA, are believed to permeate the intestinal epithelial barrier through the paracellular tight junc- tions [16]. Their increased absorption in conditions such as Crohn's disease, celiac disease, infections, allergy and food intolerance indicates a loss of the protective function of the tight junctions under inflammatory conditions. The same apparently occurs in uninflamed ileal mucosa of Crohn's dis- ease, and this could increase the antigen load in the mucosa and predispose to intestinal inflammation [17].

Biological control mechanisms

Circulatory control GALT and MALT

The impressive size and diffusiveness of the normal MALT, which forms the anatomical basis for physiological intestinal inflammation, imply the existence of highly efficient mechanisms that direct the circulation of lymphoid cells to the intestine and allow their retention in the mucosa where they can mediate a protective eff'ector function. Under physiological conditions lymphocytes circulate con- stantly throughout the body. This movement does not take place in a random fashion, but rather occurs under the control of a tightly regulated process coordinating the traffic of specific cell subsets to inductive (e.g. lymph nodes) and effector (e.g. muco- sal surfaces) sites. In this regard there is a significant dichotomy in lymphocyte trafficking and distribu- tion between naive (CD45A"^) and memory (antigen- primed CD45RO'^) cells: naive lymphocytes are programmed to circulate mainly among secondary lymphoid tissues (lymph nodes, tonsils, spleen and Peyer's patches) while memory cells preferentially access and recirculate to immune effector sites such as the intestinal lamina propria [18]. This distinction is of major importance to the intestinal immunity

because the vast majority of lymphocytes populating the normal intestinal mucosa is composed of mature memory cells [19]. These derive primarily from naive cells which have been primed by antigens sampled by M cells in the Peyer's patches and other organized GALT and recirculate to finally home in the lamina propria [1]. The implementation of this complex distribution system requires a series of signals and receptors on circulating leukocytes as well as the microvasculature to which immunocytes must adhere in order to penetrate the interstitial tissue.

This task is accomplished through a proposed multi- step paradigm in which leukocyte attachment to the vascular endothelium and the subsequent rolling, activation, arrest, spreading and transendothelial migration are mediated by specific cell adhesion and chemoattractant molecules [20]. Once in the inter- stitium the combined influence of local mesenchymal cells and the extracellular matrix provides a protec- tive anti-apoptotic environment that prolongs the survival of immigrated lymphocytes [21] (Fig. 2).

Cell adhesion molecules

Cell adhesion molecules are a large number of structurally and functionally related and unrelated molecules forming four major families: the selectin family which is primarily responsible for leukocyte-

endothelial cell interactions; the integrin family which mediates cell-cell and cell-extracellular matrix interactions, the immunoglobulin superfamily which mediates homophilic adhesion between an identical cell adhesion molecule and another cell, and the cadherin family which establishes molecular links between adjacent cells [22] (Table 1). The process of lymphocyte migration to and retention in the intestinal mucosa has two basic requirements.

The first is the expression of a combination of

adhesion molecules that impart tissue specificity to

memory/effector cells, which for mucosal homing

lymphocytes is represented by high levels of the

integrin (x4p7 and ocL(32 (LFA-1, leukocyte function-

associated molecule) and low levels of L-selectin to

avoid trapping in secondary lymphoid tissues. The

second requirement is the coordinated participation

of several cell adhesion molecules from different

families, particularly those regulating the adhesion

of leukocytes to the vascular endothelial cells (hom-

ing) and subsequent translocation into the interstitial

space. These include L-, E-, and P-selectin of the

selectin family, CD11/CD18, very late activation

antigen (VLA) -4, and oc4p7 of the integrin family,

intercellular cell adhesion molecule (ICAM) 1,

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CD45RA Intravascular space

PBT Rolling

Transmigration PECAM

CD18 Adhesion VLA-4

a4g71

P-selectin E-selectin

ICAM-1 VCAM-1 MAd-CAM-1

Interstitial space

SMF

Figure 2. Major chemokines, chemokine receptors, and cell adhesion molecules involved in the multiple steps necessary to attract and translocate (rolling, adhesion and transmigration) T cells from the intravascular to the Interstitial space, and to distribute and retain T cells in the intraepithelial and lamina propria compartments. In the upper part of the figure chemokines (TECK, IL-8, MCP-1, RANTES, GROa, TARC, and SLC) are shown below their respective cellular source: intestinal epithelial cells (lEC) and mucosal microvascular endothelial cells (MMEC). Receptors for CC and CXC chemokines (CCR2, CCR5, CCR7, CCR9, CXCR1, CXCR2, and CXCR3) are shown around the translocated T cells C indicates the expression and"" indicates the absence of expression of each chemokine receptor). In the lower part of the figure cell adhesion molecules expressed by T cells are shown to their left and cell adhesion molecules expressed by MMEC are shown below them. ECM, extracellular matrix; GRO, growth-related oncogene; ICAM, intercellular adhesion molecule; lEL, intraepithelial lymphocyte; IL-8, interleukin-8; LPT, lamina propria T cell; PBT, peripheral blood T cell; PECAM, platelet-endothelial cell adhesion molecule; RANTES, regulated on activation, normal T cell expressed and secreted;

SLC, secondary lymphoid organ chemokine; SMF, subepithelial myofibroblast; TARC, thymus and activation-regulated chemokine;

TECK, thymus expressed chemokine; VCAM, vascular cell adhesion molecule; VLA, very late activation antigen.

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vascular cell adhesion molecule (VCAM) 1, and mucosal addressin cell adhesion molecule (MAd- CAM) 1 of the Ig superfamily, and PECAM (plate- let-endothehal cell adhesion molecule) 1 [23] (Fig. 2).

Among these molecules, some play more dominant roles than others do in lymphocyte migration to the gut. Of pivotal importance is the mucosal vascular addressin MAdCAMl which is expressed by high endothelial venules (HEV), specialized postcapillary microvessels that support high levels of lymphocyte extravasation from the blood in both GALT and MALT [24], and is the target of lymphocytes expres- sing the intestinal homing receptor a4p7. Another molecule specifically involved in intestinal lympho- cyte-epithelial interaction is the integrin ocE(57 which determines the destination of intraepithelial lympho- cytes through binding to E-cadherin present on intestinal epithelial cells [25].

Chemoattractants

In addition to classical adhesion molecules, a series of chemoattractant molecules also triggers the migration and controls the directional movement of leukocytes. They include complement factor C5a, the tripeptide f.Met-Leu-Phe, the leukotriene LTB4 and a vast number of chemokines. Among these groups, considerable experimental evidence indicates that chemokines and their receptors play a previously unrecognized but fundamental role in the direction, selection, entry and retention of lymphocytes in the various tissues of the body under both physiological and pathological conditions [26, 27]. Chemokines are a large group of molecules with considerable amino acid sequence homology that function primarily as chemotactic cytokines but also mediate a variety of other biological activities [28]. They are divided into four families depending on the number of amino acids separating the first two cysteine residues, and they are discussed in greater detail in Chapter 7. Most cells produce chemokines, includ- ing human intestinal microvascular endothelial cells [29]. The response to chemokines is mediated through chemokine receptors, an equally large and complex series of cell surface molecules that can be specific, shared or promiscuous for individual chemokine molecules [30] (Table 2). Perhaps even more important than their chemokine ligands, che- mokine receptors are essential in regulating the direction, selection, entry and retention of lympho- cytes in distinct body compartments.

Table 2. Major human chemokines and their receptors Chemokine ligand Chemokine receptor

C chemokines Lymphotactin/SCIVI-1 a SCM-1 p

CC chemokines 1-309

MCP-1/MCAF MIP-1a MIP-1 (3 RANTES MCP-3 MCP-2 Eotaxin MCP-4 HCC-1 HCC-2/MIP-15 HCC-4/LEC TARC

MIP-Sp/ELC/exodus-S MIP-3a/LARC/exodus-1 SLC/exodus-2 MDC/STCP-1 MPIF-1 MPIF-2/eotaxin-2 TECK

Eotaxin-3 CTACK/ILC

CXC chemokines GROoc

GROp GROy ENA-78 GCP-2 NAP-2 IL-8 MIG IP-10 l-TAC S D F - 1 a / p BLC/BCA-1

CX3C chemokines Fractalkine

XCR1 XCR1

CCR8 CCR2 CCR1,CCR5 CCR5

CCR1,CCR3,CCR5 CCR1,CCR2,CCR3 CCR3

CCR3 CCR2, CCR3 CCR1 CCR1,CCR3 CCR1 CCR4 CCR7 CCR6 CCR7 CCR4 CCR1 CCR3 CCR9 CCR3 CCR10

CXCR1,CXCR2 CXCR2 CXCR2 CXCR2 CXCR1,CXCR2 CXCR2 CXCR1,CXCR2 CXCR3 CXCR3 CXCR3 CXCR4 CXCR5

CX3CR1

BCA, B-cell attracting chemokine; BLC, B lymphocyte chemoattractant; ELC, EBI1-ligand chemokine; ENA-78, epithelial cell-derived neutrophil-activating peptide 78; GCP, granulocyte chemotactic protein; GRO, growth-related oncogene; HCC, hemofiltrate CC chemokine; iL-8, interleukin-8; IP-10, interferon-y- inducible protein-10; l-TAC, interferon-inducible T cell a chemoattractant; LARC, liver and activation-regulated chemokine;

MCP, monocyte chemotactic protein; MDC, macrophage-derived chemokine; MIG, monokine induced by interferon-y; MIP, macrophage inflammatory protein; NAP, neutrophil-activating protein;

RANTES, regulated on activation, normal T cell expressed and

secreted; SDF, stromal cell-derived factor; SLC, secondary lymphoid

organ chemokine; TARC, thymus- and activation-regulated

chemokine; TECK, thymus-expressed chemokine.

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Chemokine receptors

Knowledge in this area is still evolving, but it is clear that chemokines and their receptors have an impact on the overall biology of T cells. By virtue of their diversity, members of the four chemokine families can attract various subsets of T cells, and impart signals that cause them to traffic from the intra- vascular to extravascular compartments and within lymph nodes, as well as recirculate from tissues to lymphatics [31], all events directly relevant to homing to and retention of T cells in the MALT. The effect on a T cell responding to chemokines is strictly depen- dent on the types of chemokine receptors displayed on the cell surface, because this controls the relation- ship between the functional activity of lymphocytes and their migratory and functional properties. The net effect of this relationship includes: (a) distinct migratory patterns of naive and memory cells, (b) the final destination to distinct tissues and organs, and (c) the decision to prime a Thl- or a Th2-mediated response. For instance, the chemokine receptor CCR4 is expressed by memory T cells that migrate to the skin but not the intestine [32]. By the same token the chemokine receptor CCR7 discriminates between two subsets of CD450"^ T cells, and its loss after antigen-induced differentiation generates effec- tor cells homing to the intestinal mucosa [33]. Small intestinal epithelial cells produce selected chemo- kines such as TECK (thymus-expressed chemokine, also expressed in the thymus) whose receptor is the newly d i s c o v e r e d G P R - 9 - 6 / C C R 9 which is expressed by all intestinal lamina propria and intrae- pithelial lymphocytes [34]. TECK is also produced by small bowel but not colonic endothelial cells and a subset of cells in the intestinal crypts and lamina propria [35]. TECK is selectively chemotactic for small bowel but not colonic lamina propria mono- nuclear cells (LPMC), which display high and low CCR9 levels, respectively. This restricted expression of TECK and CCR9 implies the existence of unique chemokine-chemokine receptors pairs involved in the development and maintenance of physiological intestinal inflammation which is compartmentalized to the small or large bowel. There is also evidence that the differential expression of chemokine recep- tors is a strong determinant of Thl vs Th2 responses.

CXCR3 and CCR5 are preferentially expressed by human Thl cells, whereas Th2 preferentially express the CCR4 and CCR3 receptors [36]. This has obvious implications not only in the physical posi- tioning of T cells in the immune response, but also the composition and function of cell-mediated

immune responses in the gut. Supporting this notion is recent evidence showing that normal human lamina propria and intraepithelial lymphocytes express the CXCR2, CCR3 and CCR5 receptors which are associated with Thl/ThO responses, but not the CCRl, CXCR2 and CXCR7 receptors that are associated withTh2 responses [37] (Fig. 2).

Under inflammatory conditions the normal traffic of mucosal lymphocytes is drastically disrupted by hemodynamic changes combined with the abnormal expression of adhesion molecules and receptors by both leukocytes and microvascular cells. Levels of MAdCAMl, ICAMl, E- and L-selectins are up- regulated, resulting in an aberrant and enhanced influx of activated and naive lymphocytes in the mucosa [23, 38]. Production of mucosal chemokines is markedly increased in the inflamed intestine and presumably the level of expression and the types of chemokine receptors are also altered [39]. A direct evaluation of all cell surface and secreted molecules in normal and inflamed gut mucosa will be needed to fully understand all migratory patterns and their implication in physiological intestinal inflammation.

Humoral control

Several organs, particularly those with mucosal surfaces, contain a variety of substances with intrinsic antimicrobial activity. In the intestinal tract the most prominent of these substances are the defensins, peptides similar to the ones found in granules of phagocytic leukocytes [40]. The two major classes of defensins are the oc- and (J-defensins;

a-defensins are abundant constituents of Paneth

cells, where they are stored in and released from

granules in response to bacterial stimuli [41]. Paneth

cell a-defensins exhibit selective activity against

several Gram-positive and Gram-negative bacteria,

but also against mycobacteria and spirochetes, fungi

and certain viruses. There is also considerable

evidence that defensins may play other safeguarding

roles including immunomodulation, opsonization,

and stimulation of wound repair [42]. Because of

their multiple defensive functions and spontaneous

presence in normal intestine defensins can also be

c o n s i d e r e d c o m p o n e n t s of i n n a t e intestinal

immunity. In reality the protective functions of

defensins probably extend beyond the natural ability

to kill bacteria, in view of recent evidence that human

defensins are chemotactic for dendritic cells and

memory T cells [43], an observation that establishes

a link between innate and adaptive immunity.

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In addition to defensins a whole host of other substances exists with properties potentially relevant to mucosal protection. The best known is lactoferrin, a single-chain glycoprotein bound to one or two molecules of ferric iron [44]. Among other several functions, lactoferrin has antimicrobial activity against viruses, bacteria, fungi and parasites and can bind sIgA. Additional substances that can contribute to mucosal defenses are lysozyme, peroxidases, histatins, cystatins, calprotectin, and secretory phospholipase A2, all of which display different degrees of microbicidal activity [42].

Immune control

Although systemic and local immunity are often interrelated, the health of each organ depends fundamentally on regulatory events which have been developmentally adapted to optimize and maintain the unique physiological conditions of each specific tissue microenvironment. In view of the preponder- ance of the immune system in the gut, and its lifelong stimulation by dietary and microbial antigens, robust but finely tuned immunomodulatory mechanisms must be constantly at work to preserve the state of physiological intestinal inflammation. The response of the immune system against any aggression has been traditionally divided in two separate but complementary compartments, innate immunity and adaptive immunity [45]. Mechanisms of innate immunity, such as phagocytosis, microbial killing and complement-dependent lysis, are primarily employed against infectious agents, become active immediately, have a limited receptor repertoire, and are largely non-specific [46]. In contrast, adaptive immunity, organized around T cells and B cells, has an almost unlimited target and receptor repertoire, requires a delayed onset, is strictly dependent on clonal expansion, and displays exquisite specificity.

This separation is somewhat arbitrary in view of the sharing of several mediator and eJBfector molecules, and the mutual interplay between innate and adaptive immunity [47]. Knowledge of innate and adaptive immunity in the intestine is fairly uneven, more abundant information existing in regard to the latter than the former. The following two sections will review the basic elements of innate intestinal immunity and provide a more extended discussion on adaptive immunity, with particular emphasis on tolerance as a key controller of physiological intestinal inflammation.

Innate immunity

Components of innate immunity relevant to normal gut physiology include both cellular and soluble mediators. Macrophages, eosinophils, mast cells, natural killer (NK) cells and neutrophils are the cellular mediators. Soluble mediators include the complement system, mannose-binding lectin (collec- tin), C-reactive protein (pentraxin), coagulation actors, defensins, various cytokines and chemokines, and reactive oxygen (ROM) and nitrogen (nitric oxide, NO) metabolites. It should be noted that very few studies have investigated in detail the role of the above components in the normal human intestine, and existing knowledge is almost exclusively derived from control data in studies of inflammatory condi- tions such as IBD or celiac disease. The paucity of information in this area will be supplemented by more extensive discussion of several of the same mediators in Chapters 7, 8 and 9, dealing with their involvement in pathological intestinal inflammation.

Cellular mediators

Macrophages are important to innate immunity because of their role as phagocytic and scavenger cells, but they are also involved in adaptive immunity because of their essential role in antigen presenta- tion. Macrophages are blood-derived monocytes that have differentiated into very efficient cells for recognition and elimination of invading microbes, processes mediated by specialized receptor systems.

Products of Gram-negative bacteria such as lipo-

polysaccharide (LPS/endotoxin) bind to a receptor

that stimulates microbicidal activity and cytokine

secretion. This LPS-sensing system consists of a

combination of a plasma LPS-binding protein, the

cell surface receptor CD 14, and a signal-transducing

receptor subunit called Toll-like receptor protein 4

[48]. Additional receptors include seven a-helical

transmembrane receptors for N-formylmethionyl

peptides, lipid mediators and chemokines, as well as

phagocytic receptors such as the mannose and Fc

r e c e p t o r s for o p s o n i z e d and n o n - o p s o n i z e d

microbes. In the normal small and large bowel

mucosa macrophages form a heterogeneous HLA-

DR+ cell population with different morphological

and phenotypic features of interdigitating dendritic

cells or scavenger cells with weak expression of the

B7-1 or B7-2 costimulatory molecules [49-51]. In

vitro they show a limited capacity for phagocytosis

and generation of oxygen radicals [52, 53], but

produce substantial levels of interleukin (IL)-la, IL-

ip, tumor necrosis factor (TNF)-oc and IL-6 in

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response to mitogens, LPS or interferon (IFN)-y [54]. Together, these characteristics suggest that mucosal macrophages are normally involved in base- line antimicrobial and antigen-presenting activities, though they have the capacity to up-regulate both activities under inflammatory conditions [50, 51, 53, 54].

NK cells are a subset of lymphocytes that sponta- neously lyse virus-infected cells and tumor cells. They are rare in the normal human mucosa and their contribution to innate immunity is uncertain. After plasma cells, T cells and macrophages, eosinophils represent the next most common type of mucosal immune cells, followed by mast cells and rare baso- phils. Little is known about these cells in mainte- nance of physiological inflammation, but they are presumably in a readiness state to provide defense against allergic and parasitic insults. Neutrophils are notably absent in a healthy intestine and their presence, no matter how small, should be interpreted as an early sign of infection or pathological inflammation.

Soluble mediators

Knowledge of the various soluble mediators of intestinal innate immunity is heavily skewed towards cytokines, chemokines and reactive metabolites.

Cytokines involved in innate immunity include IL- l a / p a n d TNF-a because of their role in inflamma- tion, IFN-a, and IFN-P because they promote resis- tance against viral infections, IFN-y for its macro- phage-activating activity, IL-12 for stimulating IFN- y production by NK and T cells, IL-15 because of its T cell proliferation inducing ability, and IL-10 and transforming growth factor (TGF)-P due to their anti-inflammatory activity [55]. A comprehensive discussion on cytokines in intestinal immunity and inflammation is found in another chapter entirely dedicated to them.

Reactive metabolites

Reactive metabolites are also discussed in Chapter 8 but they will be briefly mentioned here to point out that these toxic molecules are also produced in the healthy intestine where they contribute to defense against microorganisms and other physiological activities. Using chemiluminescence or histochem- ical techniques low levels of ROM can be detected in the normal colon [56, 57]. The intestine, on the other hand, also produces several antioxidant molecules such as urate, glutathione, a-tocopherol, and ubiqui- nol-10 [58], which help in maintaining a protective

physiological balance between pro- and anti- oxidative activities. The role of NO in intestinal physiology is still somewhat unclear because it participates in both physiological and pathological phenomena. In the healthy mucosa the constitutive endothelial NO synthase (eNOS) is present while the inducible NOS (iNOS) is absent [59], although the latter markedly increases during inflammation [59, 60]. The paradox that NO may be beneficial in physiological inflammation but injurious in patho- logical inflammation may be explained by the ability of different concentrations of NO to produce com- pletely opposite effects in the same tissue [61]. In small amounts NO generally exerts beneficial effects, whereas generation of NO in large amounts is usually detrimental. The beneficial effects of NO that contribute to mucosal defense include increased secretion of mucus, reduction in neutrophil adher- ence, acceleration of wound repair, inhibition of cytokine release and of mast cell degranulation [61].

Complement

The complement system consists of several plasma proteins linking recognition of microbes to micro- bicidal activity and development of inflammation [62]. Recognition of microbes can occur through the classical or alternative pathway, both of which result in sequential recruitment and assembly of additional proteins into protease complexes such as C5, fol- lowed by formation of a complex with C6, C7, C8 and C9 which ultimately cause lysis of cells where complement is activated. Low expression of comple- ment proteins C3d, C5, C9, terminal complement complex and S-protein can be found in submucosal blood vessels of the normal small and large bowel [63], while other proteins such as C3, C4 and factor B can be detected in perfusates of healthy small bowel [64].

Other soluble molecules

Defensins have been previously discussed as part of

humoral control mechanisms but they can also right-

fully belong to innate immunity. The mannose-bind-

ing lectin is a plasma protein that functions as an

opsonin. Like the mannose receptor found on

macrophages, it binds carbohydrates with terminal

mannose and fucose which are typically found in

microbial cell walls. C-reactive protein is another

plasma protein that also functions as a bacterial

opsonin. Finally, coagulation factors contribute to

healing by protecting vessel integrity and limit bac-

terial spreading by walling off'microvasculature.

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Adaptive immunity

The central feature of adaptive immunity is the ability to generate defense mechanisms that respond specifically to the antigen inciting the immune response. Since adaptive immunity is centered around B cells and T cells, often the response ehcited by a single antigen results in antibody production accompanied by a concomitant cell-mediated response. However, one type of reactivity may pre- dominate over the other, depending on the nature of the antigen as well as the conditions and site of stimulation, since the characteristics of the lymphoid tissue in each organ influence the quality and quan- tity of the response. This certainly applies to the intestinal immune system where both the types of antigens present in the lumen and the GALT/MALT display singular attributes resulting in responses entirely different from those that would occur at the systemic level even in response to the same antigen.

Because the achievement of a controlled immune response in the gut is largely dependent on the induction of immune tolerance, a comprehensive review of all elements and phenomena that contri- bute to adaptive immunity in the human intestine is not warranted in view of the goals of this chapter.

Instead, we will outline selected features of humoral and cell-mediated immunity that may help in restraining mucosal immune reactivity, reserving a more detailed discussion for the mechanisms of immune unresponsiveness (tolerance) and their con- tribution to physiological intestinal inflammation.

Active immunity

Humoral immunity

Antibody production at mucosal surface is domi- nated by sIgA, secretory IgM playing only a supple- mentary role. The multiple biological activities of IgA are well known and most of them appear to have evolved in order to provide specialized protection at mucosal surfaces [65]. Secretory IgA is a structurally stable molecule, a quality attributed to the linkage with the secretory component that hides the potential site of proteolytic cleavage. This makes sIgA particu- larly suited to function in the enzymatically rich intestinal mucosa where it can exert its broad protec- tive activities. Prime among these are the inhibition of bacterial adherence [66], a critical first step in bacterial colonization and eventual invasion, the induction of bacterial agglutination, and the ability to trap bacteria within the mucus layer. Secretory IgA has neutralizing activity for viruses, but also for

some enzymes and toxins of bacterial origin [67].

Due to its ability to agglutinate and entrap in the mucus, sIgA decreases the uptake of antigens gener- ated by prior enteric sensitization and thus limits the subsequent antigenic load stimulating the mucosal lymphoid tissue [68]. Another quality of sIgA, dis- tinct from that of most other immunoglobulins, is that of having a weak or no ability to activate the complement system by either the classical or alter- native pathway [69], a clearly advantageous property with anti-inflammatory implications. Finally, sIgA synergizes with some non-immune components of humoral defenses including lactoferrin, peroxidases and lysozyme. Given all the above characteristics it is fair to conclude that sIgA plays a major contributory role to intestinal immune homeostasis and the local state of controlled inflammation.

Cell-mediated immunity

In the human intestine T lymphocytes can be found at inductive sites, such as Peyer's patches and lymphoid follicles, where naive CD45RA'*' cells are originally primed by antigens, and at effector sites in the intraepithelial compartment and the lamina propria, where memory CD45RO'^ cells reside ready to react upon re-encountering the specific antigen to which they were originally primed. Intraepithelial lymphocytes (lEL) and lamina propria T cells (LPT) are both mature effector T cells, but their phenotype and functional properties are quite distinct.

Intraepithelial lymphocytes

The vast majority of human lEL are CD8"^ T cells bearing the T cell receptor (TCR) a / p and are oligoclonal in nature [70]. This oligoclonality is present all along the epithelium lining, maybe reflect- ing a restricted ability to respond to yet-unidentified antigens present in the gut [71]. This restriction may be useful because it diminishes the frequency of specific immune reactivity and decreases the chance of mounting a response potentially injurious to the surrounding epithelial cells. Some T cells in humans display the y/5 TCR and they are involved in protective immunity against infectious agents [72].

A small number of lEL also display the y/5 TCR and

it is conceivable that they are also involved in a

similar protective activity. The functional capacity

of human lEL is still controversial. They exhibit a

limited proliferative response to mitogens but they

respond vigorously to red blood cells, a reaction that

probably mimics activation through the CD2 recep-

tor [73]. Some of the morphological features of lEL,

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e.g. their resemblance to large granular N K lympho- cytes, and their CDS"^ phenotype, have long sug- gested that lEL may be cytotoxic cells, but some reports deny while others support this contention [74, 75]. Recent evidence shows that lEL have potent chemotactic activity in response to IL-8 and RANTES (regulated on activation, normal T cell expressed and secreted) [76], underlying the ability to be mobilized to sites of inflammation where they could mediate a protective function.

Lamina propria T cells

The lamina propria of the small intestine and colon contains a large and more heterogeneous population of T cells, of which 60-70% are €04"" and 20-30%

are CD8"^. Their predominant phenotype is that of helper and cytolytic cells [77], and they are also oligoclonal in nature, probably reflecting the selec- tive antigen pressure of the intestinal milieu [78].

Compared to their peripheral blood counterparts, LPT are in a higher state of activation, as shown by their cell surface markers and gene expression pat- terns [79, 80]. In addition, they express high levels of Fas antigen and Fas ligand, rendering them more susceptible to Fas-mediated apoptosis than circulat- ing T cells [81, 82] (Fig. 2). This aptness to die could be advantageous in limiting undesirable and poten- tially damaging immune responses. In fact, an inap- propriately low rate of cell death could transform the state of physiological intestinal inflammation into a pathological one, as apparently is the case in IBD [83, 84].

Functionally, LPT proliferate to mitogens, bacter- ial antigens, and LPS [85], but comparatively less than blood T cells, and display various forms of non-specific cytolytic activities including lectin-, cytokine- and anti-CD3-induced cytotoxicity [86- 88], but not spontaneous NK-like activity or mixed lymphocyte reaction-induced lympholysis [86, 89].

LPT produce a broad spectrum of cytokines in response to a variety of stimuli including mitogens, cytokines and receptor stimulation [90]. The types of soluble mediators secreted by LPT include ThO, Thl and Th2 cytokines [91], suggesting that the state of physiological inflammation is independent on an atypically skewed or well-polarized helper T cell response.

An aspect that deserves special attention is the considerable evidence showing that LPT exhibit a distinct diff'erential reactivity when activated through the CD3 or the CD2 receptor pathways.

Compared to peripheral blood T cells, LPT proHf-

erative response and IL-2 receptor expression are reduced following activation of the CD3, but not the CD2 receptor [92, 93]. This may be partially explained by down-regulation of protein kinase C in LPT, perhaps induced by unidentified mucosal fac- tors [94, 95]. On the other hand the CD2 pathway is more efficient than the CD3 pathway in inducing cytokine production by LPT, supporting the conclu- sion that the association of low proliferative reactiv- ity with high cytokine output is a characteristic feature of human mucosal T cells. This could be construed as reflecting a peculiar state of 'selective unresponsiveness' translating an adaptation of T cells to the immunological conditions of the intestine [96]. This notion is strengthened by the proposed role of CD2 signaling in the induction of T cell anergy, which is one of the mechanisms mediating peripheral T cell tolerance [97, 98].

When all the above information is considered together and analyzed in view of results from non- human systems also showing that mucosal T cells provide strong helper function through release of soluble mediators but fail to proliferate in response to antigen-specific stimulation [99], an overall func- tional phenotype emerges for human LPT. Such phenotype is consistent with a highly differentiated effector cell population with a relatively restricted capacity to clonally expand but very efficient at producing regulatory and effector cytokines. How these singular characteristics of mucosal T cells relate to the induction or maintenance of a con- trolled state of intestinal inflammation is not readily apparent. However, highly specialized and very cap- able effector cells are implicitly quicker and more efficient to enact an immune response with a high degree of specificity and able to exert 'damage con- trol' by limiting a number of undesirable collateral effects.

Immunological tolerance

Active immunity is generally regarded as the princi-

pal mechanism the body utilizes to ward off offending

agents and ensure an effective protection aimed at

preserving health. While this view is correct, an

immunogenic response is the exception rather than

the rule in the mucosal immune system [100]. Faced

with the continuous onslaught of luminal antigens

the intestine needs very effective mechanisms to

prevent the damaging effects of exceedingly strong

immune responses while concurrently fostering pro-

tection within the limits of a controlled immune

response. Cardinal to these mechanisms is immuno-

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logical tolerance, defined as a state of unresponsive- ness that is specific for a particular antigen and is induced by prior exposure to that antigen. The following paragraphs will provide an overview of the various types of immunological tolerance, how they work, and how their breakdown transforms physiological into pathological intestinal inflamma- tion. It should be noted that, in contrast to the reports mentioned so far, the bulk of knowledge on immunological tolerance is based on animal studies to which we will refer for the sake of understanding and completeness.

Mechanisms of tolerance

Immunological tolerance is a fundamental charac- teristic of the immune system that allows us to discriminate self from non-self, and thus prevents reactions directed at autoantigens that may result in pathogenic events [101]. Two types of immunological tolerance exist: central or thymic tolerance, and peripheral or post-thymic tolerance. Central toler- ance is developed during maturation in the thymus, where immature T cells that recognize high-affinity self-antigens are deleted (clonal deletion). Since diet- ary and microbial antigens are not present during fetal development this form of tolerance is less likely to play a significant role in intestinal immunity.

Peripheral tolerance is developed against antigens not present in the thymus that are met by mature CD4^ T cells later in life. Four different non-exclu- sive cellular mechanisms mediate peripheral toler- ance: clonal ignorance, anergy, deletion, and active suppression. In clonal ignorance self-reactive lymphocytes fail to recognize peripheral auto- antigens such as those sequestered in the eye, thyroid or central nervous system. There is no evidence that this form of tolerance is acting in the intestine and it will not be further considered here.

Anergy

Anergy occurs when antigen recognition occurs under suboptimal costimulatory conditions [98].

This is the case when CD4"*" T cells are presented antigens by APC deficient in costimulatory mole- cules, mainly B7-1 and B7-2, or when T cells use CTLA-4 (cytotoxic T-lymphocyte-associated anti- gen, the inhibitory receptor for the B7 molecules) to recognize costimulatory molecules on APC during the process of antigen presentation [102]. Under these conditions T cells become incapable of recog- nizing antigens even if later presented by competent APC, perhaps due to altered TCR signaling [103].

The nature of the APC (professional APC such as macrophages or dendritic cells vs non-professional APC such as epithelial, endothelial or mesenchymal cells), their state of activation (MHC class II antigen positivity or negativity), and the expression reper- toire of costimulatory molecules (mainly B7-1/

CD80 and B7-2/CD86) are crucial determinants of whether the overall outcome of the immune response will lean toward responsiveness (active immunity) or unresponsiveness (tolerance) [104] (Fig. 3). In addi- tion to lymphoid cells the intestinal mucosa contains epithelial, endothelial and mesenchymal cells that express low or no levels of costimulatory molecules even when MHC class II antigens are up-regulated during inffammation [105]. This results in a limited capacity to respond to local antigens, which further fosters the development of anergy and contributes to restrain immune reactivity and maintain a controlled state of inffammation.

Deletion

Deletion occurs when T cells are repeatedly stimu- lated by exposure to the same antigen resulting in activation-induced cell death (apoptosis) [106]. As a consequence of activation T cells express on their surface the Fas (CD95) receptor, a member of the T N F receptor family, and the Fas ligand, a molecule homologous to T N F [107]. The binding of Fas by Fas ligand on the same or adjacent T cells triggers a cascade of signaling events leading to the activation of intracellular cysteine proteases (caspases) that ultimately cause cell death by apoptosis, resulting in the elimination of the mature T cell population repeatedly stimulated by the original antigen. Con- stant antigen exposure is a typical feature of intest- inal immunity and mucosal T cells normally express high levels of both Fas and Fas ligand [81], making clonal deletion of T cells a likely mechanism of immune homeostasis in physiological intestinal inffammation.

Active suppression

Of all the mechanisms participating in peripheral

tolerance active suppression is probably the most

common and effective. Active suppression is a pro-

cess in which regulatory CD4'^ T cells activated by

exposure to a sensitizing antigen release cytokines

such as IL-14, IL-10 and TGF-p [108]. This release of

cytokines by tolerized T cells blocks the activation

and function of other effector T cells and induces an

important 'bystander suppression' effect in the local

microenvironment, resulting in the inhibition of

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Dietary antigens Enteric flora

\r

CD1 CD28 CDS

Suppression Active immunity

>/ I \

Th1 ThO Th2

i

Anergy (Tolerance)

Th3 Tr1

High antigen

dose

\ Active V suppression

Low antigen

dose

i

Deletion or anergy

Figure 3. Proposed mechanisms of adaptive immunity involved in the induction and maintenance of physiological inflammation in the

normal intestine. Antigens derived from the diet and the commensal enteric flora can access the GALTand MALT through specialized

cells forming the follicle-associated epithelium (FAE), intestinal epithelial cells (lEC), or the paracellular route between lEC. Antigens

processed by non-professional antigen-presenting lEC bearing major histocompatibility complex (MHC) class I and CD1 molecules

will preferentially activate CD8"^ T cells and result in suppression (far left). When antigens are processed by professional antigen-

presenting cells (APC) bearing MHC class II antigens and B7-1/2 costimulatory molecules and are presented to 0028"^ 004"^ T cells

this will result in active immunity with a non-polarized (ThO) or polarized (Th1 orTh2) profile depending on the nature of the antigen

and the surrounding cytokine milieu (center left). When antigens are processed by professional APC bearing MHC class II antigens

but presented through deficient B7-1 /2 costimulation or preferentially to CTLA-4"^ CD4'*' T cells this will result in tolerance mediated

by active suppression (executed by ThS or Tr1 cells) or deletion/anergy depending on the dose of the antigen (center right). Antigens

processed by non-professional antigen-presenting mucosal microvascular endothelial cells (MMEC) or mucosal myofibroblast

(MMF)-bearing major histocompatibility (MHC) class I molecules but no costimulatory molecules will result in the generation of

anergic T cells and the Induction of tolerance (far right).

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immune responses to unrelated, but anatomically co- localized, antigens [109]. Active suppression is prob- ably the most important mechanism of tolerance in the gut, not only because of the high likelihood of local T cells to re-encounter antigens to which they have been previously sensitized, but also because of the abundance of mature effector T cells capable of producing immunosuppressive molecules [110].

Oral tolerance

The tolerance mechanisms discussed so far are based primarily on systemic immune responses to self rather than foreign protein antigens. The nature, dose and way of administration of any foreign protein are key determining factors in deciding whether active immunity or tolerance will ensue.

When a foreign protein enters the body via the gastrointestinal tract this usually leads to a marked suppression of systemic humoral and cell-mediated immune responses upon rechallenge by the same protein. This phenomenon is called oral tolerance, and is believed to be essential in preventing deleter- ious immune responses against proteins that need to be absorbed for nutritional purposes, and develop- ing tolerance against the antigens of the normal enteric flora that is indispensable to energy metabo- hsm, nutrient absorption and general health [109, 111, 112]. Various mechanisms mediate oral toler- ance but generally, based on experimental animal systems, a high antigen dose induces anergy or deletion [113, 114], whereas a low antigen dose induces active T cell-mediated suppression (Fig. 3).

This latter form of regulation is mediated the immu- nosuppressive cytokines IL-4, IL-10 and TGF-P produced by various types of CDA^ T cells with functional phenotypes of Th2, Th3, Treg (T regula- tory cell) or Trl (T regulatory cell 1), all of which are involved in systemic or organ-specific autoimmune phenomena [115-117]. There is good evidence that these regulatory T cells exist in the gut of normal humans and animals with experimental intestinal inflammation [118-120], as well as evidence for the anti-inflammatory activity of locally secreted cyto- kines such as IL-10 and TGF-P [120-122].

Broken tolerance: impact on intestinal immune homeostasis

To be fully protective oral tolerance must be devel- oped for the two main components of the luminal content: food antigens and the endogenous flora.

Ingestion of specific proteins and peptides is widely utilized in studies of systemic and organ-specific

immunity and autoimmunity, as well as a form of t r e a t m e n t for a u t o i m m u n e d i s e a s e s [123].

Unfortunately, except for classical food allergies, a form of immediate hypersensitivity r e a c t i o n mediated primarily by mast cells and eosinophils [124], little is known about the role of normal dietary antigen in physiological or pathological intestinal inflammation. In contrast, knowledge on the role of the commensal flora on intestinal immunity and inflammation has greatly expanded in recent years, justifying some discussion on how normal enteric bacteria induce and maintain a controlled state of inflammation in the mucosa, and how loss of tolerance to these bacteria may contribute to chronic intestinal inflammation as observed in IBD.

Gut microbial-immune interactions

The development of a mature intestinal immune system is strictly dependent on the introduction of environmental antigens into the lumen, e.g. food and microorganisms [125]. Immediately after birth the newborn's gastrointestinal tract, previously sterile, is colonized with aerobic and anaerobic bacteria [126].

The ability to induce tolerance requires colonization by Gram-negative bacteria early in life, and germfree conditions or exposure to certain aerobic Gram- positive bacteria may impair development of oral tolerance mechanisms [127]. With advancing age, and feeding of complex diets, the gut acquires a full spectrum of microflora that increases in number and variety from the almost sterile stomach to the luxur- iant milieu of the colon, where Bacteroides, Bifido- bacteria, Peptostreptococcus and Eubacterium spp.

predominate. The type of feeding has a significant impact on colonization: for example, distinct from breast-fed infants, Bacteroides count increases in formula-fed infants, and anaerobic counts remain high after introduction of solid food [128].

The normal human intestinal biota consists of

hundreds of bacterial species that vary considerably

from person to person, though the predominant

phylogenetic groups do not change [129]. The inter-

dependence of the bacterial and immune systems in

the gut is mutual and lifelong, and enteric bacteria

exert a wide range of modulatory effects on specific

and non-specific immune responses. In animals they

range from mitogenic effects on lymphocytes to

induction of N K and cytotoxic cell activity,

enhanced antibody production, stimulation of

macrophage phagocytosis, cytokine production,

and induction of oxygen free radicals [128]. In

humans the enteric bacteria affect both systemic and

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mucosal immunity, including modulation of IL-1, IL-2, IL-6, TNF-a, and IFN-y production, antigen- and mitogen-induced lymphocyte proliferation, and macrophage phagocytic and killing activity [130].

Normal enteric flora and pathological intestinal inflammation

There is compelling evidence for a decisive role of the commensal flora in pathological gut inflammation.

Four lines of evidence support this claim. The strongest evidence for a role of the normal enteric flora in IBD comes from experimental models of bowel inflammation in transgenic and gene-deleted rodents [131]. For instance, HLA-B27/human P2- microglobulin transgenic rats and IL-10-deficient mice fail to develop colitis under germfree condi- tions, but do so when exposed to normal luminal bacteria, and the load and composition of commen- sal bacteria influences the degree of inflammation [132-134]. Additional support comes from human studies in which the fecal content has been removed from or put in contact with intestinal mucosa.

Crohn's disease patients with fecal stream diversion after ileal resection have no inflammation in the neoterminal ileum, but inflammation quickly reappears after reanastomosis [135]. Furthermore, infusion of intestinal luminal contents into histologi- cally normal excluded ileal loops after ileocolonic resection induces inflammatory changes within a few days [136]. Another example of how luminal content affects the health of the intestinal mucosa derives from ulcerative colitis patients with a reconstructed ileo-anal pouch who develop pouchitis, a recurrent inflammation in the remodeled small bowel loops.

This condition is believed to be caused by a bacterial dysbiosis induced by the presence of colonic-type bacteria in the small bowel loops forming the pouch [137]. Finally, antibiotics can be beneficial in the management of IBD and particularly CD [138], and probiotics appear to be a promising new form of treatment for IBD and pouchitis [139, 140].

Loss of tolerance to normal enteric flora in intestinal inflammation

If gut-induced tolerance is crucial to prevent systemic immune responses, the same must be true for local responses, and it seems logical that gut inflammation may result from loss of local tolerance.

This concept finds support in a series of human and animal studies. LPMC isolated from the inflamed, but not uninflamed, intestine of adult IBD patients proliferate strongly in response to autologous, but

not heterologous, intestinal bacteria [141]. LPMC from control and IBD patients in remission fail to proliferate to autologous bacteria. This suggests that tolerance to commensal flora normally exists in the healthy intestine, and that tolerance is broken during inflammation. Loss of tolerance to commensal flora is also demonstrable in murine hapten-induced colitis, and tolerance can be restored by treatment with IL-10 or neutralization of IL-12 [142]. Gut bacteria-reactive T cells are found in patients with IBD and murine models of IBD [119, 143-145], and the adoptive transfer of bacterial antigen-specific CD4"*" T cells from diseased into naive animals induces colitis [119, 145]. Taken together, these results indicate that loss of tolerance to autologous enteric flora is instrumental in the pathogenesis of some forms of pathological intestinal inflammation.