doi:10.1006/smim.2001.0308, available online at http://www.idealibrary.com onseminars in IMMUNOLOGY, Vol. 13, 2001: pp. 159–161

Introduction

Tom MacDonald

This issue deals with one of the central problemsof immunology, namely how the immune systemmanages to remain comparatively unresponsive tothe antigens of foods and commensal bacteria yetstill has the capacity to react to pathogens. In thesystemic tissues, the signal-to-noise ratio of non-selfantigens is high, but in the gut there is very highbackground exposure to non-self antigens. For theimmune system the high background creates twomain problems. First, how to identify a pathogenamongst the antigens of foods and normal flora, andsecond how to remain unresponsive to the antigensof foods and the normal flora. That the processeswhich control this unresponsiveness are incompleteis readily seen in patients with gluten sensitiveenteropathy or Crohn’s disease, where there areongoing T cell mediated tissue damaging responsesto wheat and the normal flora respectively. Howeverknowledge of these processes is in its infancy andthere has been very little translation of basic researchinto man.

Several important points need to be emphasizedwhen considering mucosal immune responses.

The gut immune system is designed tomaximize exposure to lumenal antigen and isnot unresponsive

The extent of the organized lymphoid tissue in thegut immune system is often not appreciated. Thesmall intestine of a 14 year old adolescent containsaround 250 Peyer’s patches (defined as having five ormore follicles) scattered along 7–8 metres of bowel.However there is at least 10–20 times as many solitaryfollicles. In the colon there are also many thousandsof solitary follicles. Each follicle has a specialized

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epithelium containing M cells, transporting antigensacross the epithelium and into the dome region. Thusat all levels of the intestine, the immune system issampling the antigens of the lumenal environment.As a consequence of this, normal mucosal lymphoidfollicles have a very high basal level of activation,with prominent T cell zones, activated T cells andlarge reactive germinal centres. It is not possible forthe gut immune system to selectively ignore lumenalantigens. For example, how does a T cell know that apeptide is derived from a milk protein as opposed toan intestinal virus?

Once activated, Peyer’s patch T cells and B cellsenter the blood and migrate to the laminapropria. B cells become plasma cells secretingIgA (4.5 g/day/adult) and the CD4+ T cells lodge inthe lamina propria. For intraepithelial lymphocytesthe situation is still obscure because they are long-lived and the evidence in the normal state that theyare renewed by cells from the Peyer’s patches is notconvincing. However as described in the paper byClaudio Fiocchi, most lamina propria CD4 T cellsdie in situ, being constantly replaced by fresh cellsfrom the Peyer’s patches. Presumably however if theywere initially activated by peptides from a pathogenin the Peyer’s patches and they migrated to thelamina propria and peptides from the pathogen werepresented to the T cells, they would not die and drivea local protective cell-mediated immune response.All of the available evidence therefore suggeststhat the gut immune system is highly responsive toantigens of foods and the normal flora so that innormal individuals there is more immune activity inthe gut than in the rest of the body combined. Theextensive immune activity in the absence of diseasehas been termed ‘physiological inflammation’, forwant of a better term, since the key feature ofthe normal gut immune system is that there is nopathology, i.e. the absence of disease. This editionwould therefore be better termed ‘Maintaining thedisease-free state in the gut’, rather than ‘Maintainingthe unresponsive state’.

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The relevance of oral tolerance tounresponsiveness in the gut is still not clear

Oral tolerance is defined as the comparativeunresponsiveness which occurs after feedingantigens. However the model systems used tostudy the phenomenon invariably use systemicimmunization to elicit a response. Hence it is bestto describe oral tolerance as ‘orally induced systemictolerance’. It may therefore be more relevant tothe control of immune responses to the foodproteins which enter the blood in immunogenicamounts in all normal individuals.There is verylittle evidence for active downregulation of mucosalimmune responses after feeding protein antigens.To the authors knowledge no study has ever shownthat feeding an antigen selectively downregulatessubsequent immune responses in the Peyer’spatches. Likewise there is little evidence that feedingantigens suppresses IgA responses. In a classic paper,Bienenstock and Dolezel1 fed bovine serum albumento hamsters and generated a large secretory IgAresponse in the gut. In the lamina propria there is alsovery little evidence that there is suppression of localT cell responses. The only paper which addressedthis issue showed that the blood T cell proliferativeresponse to E. coli proteins was suppressed by normallamina propria T cells.2 The notion that the gutis unresponsive to protein antigens and that oraltolerance is a major impediment to the generation ofeffective oral vaccines depends largely on perspective.Individuals who work on oral adjuvants and deliverysystems see oral tolerance as a problem, howeverit seems less of an issue to individuals who aredeveloping edible vaccines in potatoes.3

There are major species differences in mucosalimmune responses

It has become virtually axiomatic that feedingmoderate amounts of protein antigens to miceelicits a Th2, Th3, Tr response in Peyer’s patchesand that the murine immune system shows a Th2bias.4,5 However there is now increasing evidencethat even in mice, oral antigens can prime for aTh1 response and in some systems Th1 cytokinesare important in mediating orally induced systemicunresponsiveness.6,7 In humans however, thesituation is still not well investigated. Feeding keyholelimpet hemocyanin to healthy adults impairs theskin test response after subsequent parenteral

immunization with KLH in alum.8 However this alsodeals with orally induced systemic unresponsivenessand is not informative of events in the gut.

In man there is abundant evidence now thatlamina propria T cells in healthy individuals showa strongly skewed Th1 cytokine profile, dominatedby a large excess of interferon-γ over IL-4, IL-5 orIL-10, whether measured by RT-PCR, ELISPOT, orin vitro activation.9 Ileal Peyer’s patch T cells fromhealthy children and adolescents display a recallresponse to cows milk proteins that is dominatedby interferon-γ .10 This is probably due to the highexpression of IL-12 p70 in the subepithelial dendriticcells.10 Peyer’s patch T cells also show constitutiveactivation of phospho-STAT4, but phospho-STAT6is undetectable and they also express high numbersof transcripts for the IL-12β2R.11 Functionally, SEBinduces a large Th1 response which is inhibitable byanti-IL-12 p70.11 The available evidence thereforesuggests that ileal Peyer’s patches in man representsan environment where T cell responses to lumenalantigens are driven along the Th1 pathway. While thereason for this is not known, it is highly likely thatthe IL-12 is driven by products of the endogenousbacterial flora, transported by M cells from the lumeninto the dome region.

If Peyer’s patches are a source of Th1 cells whichcan migrate back to the lamina propria via theblood in man, what are the implications of thisobservation? The first is that the Peyer’s patchesthen become a sump of potentially tissue damagingT cells which could cause disease if they recognizeda cross-reactive peptide in the periphery. The secondis that it affirms the notion that the absence oftissue damaging immune responses in the gutof most individuals is due to regulation of theeffector arm of mucosal immunity rather than at itsinduction. In many ways this is obvious; regulatoryT cells make immunosuppressive cytokines whichare indiscriminate in the responses they suppress(bystander tolerance).4,5 It would not make sensefor regulatory cells to operate in Peyer’s patchessince there is the potential for them to suppressa response to a pathogen. Generation of primedeffector Th1 cells in Peyer’s patches would meanthat if the cell migrated into the lamina propriaand encountered a pathogen again, then it couldpotentially respond. If not it would die. It is thisauthors opinion that the most important eventswhich control the development of diseases suchas Crohn’s disease and celiac disease in man arethe factors which control T cell responses (amount

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of antigen, activity of lamina propria antigen-presenting cells, presence of local PGE2, IL-10,TGFβ) in the lamina propria. In general, in healthyindividuals, the lamina propria is an unresponsivesite.

The key area is the interaction of the normalflora with the mucosal immune system

Mucosal immune responses have been dominated bystudies on protein antigens, and the flora has beenneglected in recent years. This is because it is moredifficult to work in this area, especially in humans.Nonetheless the growing interest in pro-biotics andthe realization that a disease such as Crohn’s diseasecould be due to an excessive response to the normalflora has focused attention on this area again. Thedemonstration that the normal flora drives thepathology in many rodent models of inflammatorybowel disease underpins this idea even more.12

In humans the normal flora is diverse, complexand very stable. In rodents it is possible to studygerm-free mice or animals with only a few speciesof indigenous microorganisms, but this is expensiveand requires dedication. Even in mice, the bacteriumwhich elicits mucosal T cells and B cells early inlife is a filamentous organism which has not yetbeen cultured, but is clearly highly immunogenic.13

The abundance of bacteria in the lower bowelmeans that recognition of any protein antigentakes place in a situation where natural adjuvantsand immunomodulators are common. Recognitionof bacterial antigens themselves is subject to thesame potential regulation. Gut pathogens producemolecules which have clear immunoregulatoryactivity, such as intimin of enteropathogenic E. coli 14

or invasin of Yersinia spp.15 It is likely that the normalflora also produces immunoregulatory moleculesand it is not entirely unfeasible that the disease-freestate of the gut in normal individuals is caused bythe flora and not by sophisticated immunoregulatorycircuits.

References

1. Dolezel J, Bienenstock J (1971) Immune response of thehamster to oral and parenteral immunization. Cell Immunol2:326–334

2. Khoo UY, Proctor IE, Macpherson AJS (1997) CD4+ T celldown-regulation in human intestinal mucosa. J Immunol158:3626–3634

3. Tacket CO, Mason HS, Losonsky G, Estes MK, Levine MM,Arntzen CJ (2000) Human immune responses to a novelNorwalk virus vaccine delivered in transgenic potatoes.J Infect Dis 182:302–305

4. Strobel S, Mowat AM (1998) Immune responses to dietaryantigens: oral tolerance. Immunol Today 19:173–181

5. Faria AM, Weiner HL (1999) Oral tolerance:mechanisms andtherapeutic applications. Adv Immunol 73:153–264

6. Hoyne GF, Callow MG, Kuhlman J, Thomas WR (1993) T-celllymphokine response to orally administered proteins duringpriming and unresponsiveness. Immunology 78:534–540

7. Lee H-O, Miller SD, Hurst SD, Tan L-J, Cooper CJ,Barrett TA (2000) Interferon-gamma induction during oraltolerance reduces T-cell migration to sites of inflammation.Gastroenterology 119:129–138

8. Husby S, Mestecky J, Moldoveanu Z, Holland S, El-son CO (1994) Oral tolerance in humans: T cell butnot B cell tolerance after antigen feeding. J Immunol152:4663–4670

9. MacDonald TT (1999) Effector and regulatory lymphoidcells and cytokines in mucosal sites, in Defence of MucosalSurfaces: Pathogenesis, Immunity and Vaccines (Neutra M,Kraehenbuhl J-P, eds); Curr Top Microbiol and Immunol236:113–136

10. Nagata S, McKenzie C, Pender SLF, Bajaj-Elliott M, Fair-clough PD, Monteleone G, MacDonald TT (2000) HumanPeyer’s patch T cells are sensitised to dietaryantigen anddisplay a T helper cell type 1 cytokine profile. J Immunol165:5315–5321

11. Monteleone G, Salvati V, Croft NM, MacDonald TT Th1 andTh2 cytokines and related transcription factors in humanPeyer’s patches. (Submitted).

12. Sartor RB (1997) The influence of normal microbial floraon the development of chronic mucosal inflammation. ResImmunol 148:567–576

13. Umesaki Y, Setoyama H, Matsumoto S, Imaoka A,Itoh K (1999) Differential roles of segmentedfilamentous bacteria and Clostridia in development ofthe immune system. Infect Immun 67:3504–3511

14. Higgins LM, Frankel G, Connerton I, Goncalves NS,Dougan G, MacDonald TT (1999) Role of bacterial intimin incolonic hyperplasia and inflammation. Science 285:588–591

15. Ennis E, Isberg RR, Shimizu Y (1993) Very late antigen 4-dependent adhesion and co-stimulation of resting human Tcells by the bacterial beta 1 integrin ligand invasin. J Exp Med177:207–212

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