Epithelial Barrier Function to Antigens An Overview W. ALLAN WALKER AND IAN R. SANDERSON Departments of Nutrition and Pediatrics Harvard Medical School and Combined Program in Pediatric Gastroenterology and Nutrition Massachusetts General Hospital and The Children’s Hospital Boston, Massachusetts 02115 INTRODUCTION Compared to other organs in the body, the intestine is exposed to an enormous number of antigens. These antigens are of limitless variety and derive from many sources, including ingested food, resident bacteria and viruses. Some antigens are harmful to the host while others pose no threat, and the intestine must deal with this diversity. An important mechanism by which the intestine surveys these antigens in the intestinal lumen is by allowing some to permeate the epithelium and interact with the intestinal and systemic immune systems. However, excessive or inappropriate exposure of antigens to the intestinal immune system may lead to gastrointestinal disease. Two important diseases of childhood underscore this concept. In the first, coeliac disease, the appearance of an enteropathy sensitive to gluten is dependent on the integrity of the cereal protein; partial digestion by papain of any of the gluten-related family of macromolecules will abrogate its acti~ity.~,’ The second, cow’s milk-sensitive enteropathy, responds to a diet in which the casein4 or whey5 have been hydrolysed commercially. Thus the pathogenic nature of these foods lies within their antigenic structure. An understanding of the physiology of the uptake is therefore central to an appreciation of the pathogenesis of disease. For it is possible that the limited exposure to antigens (which constitute a mechanism of surveying the contents of the intestinal lumen) may at times lead to damage to the mucosal barriers and excessive uptake allowing a chronic immune mechanism to develop. Thus we must also consider how uptake is limited so that immune reactions do not flare up. The mechanisms whereby oral exposure to antigens leads to a suppression of immune responsiveness (oral tolerance) may depend, in part, on the pathways of antigen uptake. The intestinal tract is one way in which vaccinations can be given and is the preferred route for polio vaccination in some countries including the United States. However, the opportunities for preventive medicine through oral immunizations have hardly been realized. Progress in this field will depend on our understanding of mechanisms of uptake of antigens (TABLE1). In this paper, we shall consider the pathways by which antigens penetrate the intestinal epithelium. Some mechanisms are specific for various types of antigens and constitute a physiological process by which the molecules can perform their beneficial function. Other mechanisms are nonspecific and may constitute potentially damaging (pathologic)processes that could cause disease. As an introduction to this section, we shall review the relationship of antigen entry to disease and 10
WALKER & SANDERSON: ANTIGENS & EPITHELIUM TABLE I.
11
Importance of Complete Understanding of Antigen Uptake 1. Effective use of oral vaccines 2. Prevent autoimmune reactions 3. Prevent propagation of inflammatoryhmmunologic reactions
explore in particular the possibility that increased or inappropriate uptake may be a prerequisite for disease. Exposure of Antigens in Human Disease States Disease States that Increase Antigen Uptake of the Intestine
A number of conditions are now known to increase the permeability of the intestine to antigens (TABLE2). Acute gastroenteritis is associated with increased antigen transfer. Grusky and Cooke6demonstrated that egg albumin concentrations were fivefold higher in serum of infants with diarrhea than in children with diseases outside the gastrointestinal tract. This study was done before the aetiology of acute diarrhea had been characterized; more recent studies have correlated macromolecules uptake with the onset of well defined infections, particularly in animal models. Transmissible gastroenteritis (TGE) virus causes an acute self limiting disease in piglets similar to human rotavirus gastroenteritis.’ The mucosa to serosa uptake rate of horse radish peroxidase (HRP), a protein enzyme, have been measured in jejunal mucosal segments from piglets infected with TGE virus.8 Uptake across epithelium containing a Peyer’s patch (which is normally high) was not significantly affected; however, uptake increased in areas of jejunal tissue without a Peyer’s patch to the level seen in Peyer’s patch tissue. This increased uptake occurred only early (12 hr) after inoculation with virus. In mice infected with rotavirus, however, intact HRP transfer in vitro was maximal 2-3 days after i n f e ~ t i o nTransfer .~ of antigens (ova1bumin)’Ointo the circulation of mice examined 3 days after inoculation with rotavirus was also significantly greater than uninfected
TABLE 2.
Insults That Increase Antigen Uptake of the Intestine 1. Intestinal disorders:
Gastrointestinal food allergy Celiac disease Acute gastroenteritis Chronic intestinal infections Inflammatory bowel disease Surgery 2. Systemic insults: Excessive radiation Extensive burns Septicemia shock Hypovolumetric shock Malnutrition 3. Drugs Antiinflammatory drugs
u
ANNALS NEW YORK ACADEMY OF SCIENCES
controls. In developing countries, the contribution of bacteria to the spectrum of infectious agents in gastroenteritis is particularly great. The bacteria that cause acute diarrhea can increase the permeation of macromolecules proteins. In rabbits RDEC-1 increases the in uitro permeation of p-lactoglobulinand HRP." However, this is probably only true of bacteria that cause diarrhea with mucosal damage, as secretory diarrhea caused by cholera-like exotoxins is not associated with increased antigen transport.12In countries where bacterial diarrhea is prevalent, malnutrition is common. Malnutrition can exacerbate the passage of antigens across the intestine during gastrointestinal infection; malnourished mice infected with rotavirus show increased intestinal permeability to ovalbumin (OVA) as compared to well fed infected mice." Indeed malnutrition can by itself lead to increased antigen transfer. Protein-deficient rats show increased intestinal uptake to bovine serum albumin (BSA).13Starvation affects barrier functions extrinsic to the intestine. Significantly less trypsin-like activity was seen in starved newborn rabbits (that had high plasma concentrations of BSA) than in fed ones (that had low concentrations of BSA).14 Intrinsic barrier function is nevertheless also compromised by malnutrition. Jejunal morphology is altered14 and transport of HRP across small intestinal epithelium is increased, as has been shown in biopsies from malnourished children. Many gastrointestinal diseases are associated with malnutrition and this adds to the complexity of interpreting results in studies of irlflammatory bowel disease and celiac disease. Serum immunoglobulins to food antigens have been found in IBD including p-lactoglobulin,16wheat17and maize,'* so it is probable that protein macromolecules permeate in increased amounts causing systemic immune responsiveness. Nonprotein macromolecules have been extensively studied in Crohn's d i ~ e a s e ' ~ and - ~ ' permeate readily, often returning to normal with therapy.'9.z1 Colonic permeability to PEG-4000 is increased in experimental colitis.zz In celiac disease nonprotein macromolecules permeate more easily than in nondisease controls, and some consider use of sugar markers to be a possible screening test.23*24 The sensitivity of cellobiosehannitol absorption test was %% in 817 consecutive adults suspected of having celiac disease." Antigens are also able to permeate more readily in patients with the c o n d i t i ~ nIt. ~is~ possible that small bowel integrity is not normal even after treatment with gluten-free diet.26 Food allergy can manifest as gastrointestinal diseasez7as well as diseases of other organ systems. Protein macromolecules are capable of entering the circulation of milk-allergic children challenged with milk; transfer of a test dose of alactalbumin (which is not found in cow's milk) was greater in children who showed symptoms following cow's milk challenge than in those who did notz8.a-Lactalbumin transfer was increased in children who had either gastrointestinal or skin manifestations of cow's milk allergies. Nonprotein macromolecules markers too are increased in gastrointestinal food allergyz9and this correlates well with intestinal morphology. Disease States and Their Relationship to Antigen Entry
It is likely that the uptake of immunogenic molecules from the lumen is important in the pathogenesis of some gastrointestinal diseases. Antigens can be the entry site of immunological-mediatedactivity both within the intestine and beyond. Interaction between macromolecules and the immune system could lead to immunologically-mediated damage if compensatory mechanisms did not exist. This tolerance to antigens entering the gut could be broken if the uptake of the gut to
WALKER & SANDERSON: ANTIGENS & EPITHELIUM
l3
certain antigens were altered in some way. It is not clear whether increased uptake of a particular antigen is central to the pathogenic process of immune-mediated gastrointestinal disease. Most gastrointestinal diseases exhibit increased uptake of antigens (see above), yet this could be secondary to the disease process itself. While we cannot answer this question at present, there are a number of approaches to it that might distinguish between primary or secondary antigen permeability defect. The approach to this process can be summarized as follows: there are four ways of discerning whether gastrointestinal diseases are a consequence of increased antigen entry: a) examination of subjects before onset of disease, b) examination after resolution of disease, c) examination of first degree relatives and d) the use of animal models. If increased antigen entry is not a prerequisite to immune-mediated gastrointestinal disease then it is necessary to propose that the immune system of affected individuals is different from healthy subjects at some point before the onset of the disease.
TABLE 3. Antigen Uptake Processes 1. Physiologic transport
a. Ligand receptor uptake (growth factors) Conduit Enterocyte as target organ b. Antibody uptake-passive immunity c. Microfold cell (“M” cell) transport Normal local immune response 2. Pathologic transport a. Intracellular (excessive) Immature gut; post enteritis; allergic enteropathy b. Paracellular Inflammation Edema associate with anaphylaxis 3. Pathologic-antigen-specific a. Gliadin-celiac disease b. Beta lactoglobulin (BLG) allergic gastroenteropathies c. Bacterial antigens-autoimmune disease (IBD?) d. Primary vs tolerance (intracellular processing)
Alteration of Immune System
If gastrointestinal disease is due to altered immune function, antigen uptake by a variety of mechanisms is still necessary for disease but uptake could (in the premorbid state) be no different from that found in normals (TABLE3). However, the evidence that the immune system is altered in an affected individual prior to the onset of disease is often lacking. In Crohn’s disease there is no clear immunological defect that is independent of the pathogenic process, although some rare immunological diseases are associated with a higher incidence of IBD.30Although there is an increased risk of gastrointestinal food allergy in children with atopy, this is only so in a proportion of cases and most atopic children do not present with food
14
ANNALS NEW YORK ACADEMY OF SCIENCES
allergy. However, it is possible that gastrointestinal infections could “alert” the immune system so that the antigens that cross the intestine in fact are more likely to set up an immune response. There is evidence that allergic sensitization of the respiratory tract follows viral respiratory tract infections.” In a prospective study of 13 children born to allergic parents, 11 became clinically allergic in the first 5 years of life. Laboratory immunological criteria of allergic sensitization were made every 3 months. Respiratory infections (confirmed in 10 out of the 11 by complement-fixing antibodies) occurred in the two months before onset of allergic sensitization. Therefore, it is possible that gastrointestinal food allergy could be commoner after gastroenteritis by a similar mechanism. Celiac disease is closely linked to the MHC class I1 haplotype DQw2 (90to 95% of patient^.^^"^ As class I1 molecules are central to the T cell response, it is possible that celiac disease occurs only in subjects who are able to present a certain epitope of the gliadin molecule to T cells and those subjects have the specific heterodimers (namely DQw2 and its linked class I1 MHC molecules) that are able to present that epitope. If this were so, there would be no need to postulate increased macromolecular entry in affected subjects. However, although most patients with celiac disease have a defined class I1 profile, it is only a small number of people with that profile who develop the disease and some further initiating mechanism is required. In addition, patients who have class I1 molecules capable of presenting peptide fragments to antigen-presentingcells are likely to have similar molecules on the basolateral surface of enterocytes. If enterocytes are capable of transporting peptide fragments in uiuo, one could speculate that those with gliadinrecognizing class I1 molecules are transporting them across the epithelial cell barrier. This rate of transport would be very great in comparison to that in enterocytes who do not have the class I1 that recognized peptides of gliadin. Therefore, even in celiac disease where a particular immune state is clearly implicated in the pathogenesis of the disease, increased antigen uptake may indeed be important.
CONCLUSIONS AND SPECULATION In this overview of epithelial barrier function to antigens we have reiterated the speculation’ that increased antigen uptake of the intestine precedes the onset of a number of immunologically-mediatedgastrointestinal diseases. We have examined possible ways of determining whether such an increase in antigen transfer could occur as a preliminary to disease; but we have also examined the contrary hypothesis that antigen uptake activates an immune system that is altered in some way, either by recurrent infection or because of genetic makeup. These two hypotheses are not mutually exclusive. Indeed the immune system plays a significant role in the barrier to antigens transport. IgE-mediated discharge of histamine results in enhanced goblet cell mucus release.34IgA complexes with antigens and immunization decrease antigen tran~fer.~’ There remain many questions to be answered. We have suggested that antigens might penetrate the mucosal barrier by different pathways, but we do not know the relevant importance of those pathways in immune surveillance or in the initiation of gastrointestinal disease. These pathways will be reviewed in depth as part of this session. In some circumstances antigen passage increases immune activity (as in the initiation of allergic disease); in other circumstances oral antigens suppress immune reactions that are already underway.36For example, mice that develop the neurological consequences of the immune response to myelin basic protein are
WALKER & SANDERSON: ANTIGENS & EPITHELIUM
15
significantly improved by feeding the pr~tein.~’ Do these different responses to oral antigen represent different pathways of permeation? Finally, we need to determine whether antigen uptake is always essential to immune surveillance. Can luminal antigens be recognized without their penetrating the epithelium? If class I1 MHC on the epithelium presents processed luminal peptides on their surface without releasing them, this is indeed a possibility. Can basolateral antigens be recognized within the lumen of the intestine? There is no evidence for this yet, but two observations suggest that uptake may not always be essential. First, lymphocytes can enter the lumen of the intestine from the Peyer’s patch3sand considerably could function as part of the afferent arm of the immune response at that site. Second, the apical surface of mucosal epithelial cells expresses a number of molecules belonging to the immunoglobulinsuperfamily. Their function is at present unknown but they have been identified because they form the attachments for invading viruses. ICAM-1 has been found to be the Ig superfamily poliovirus .~ member that allows rhinovirus attachment in nasal e p i t h e l i ~ r n ,a~ ~ receptor in intestinal epithelium is also a member of the Ig ~uperfamily.~~ As the primary function of these molecules on mucosal surface cannot be to serve as a conduit for viral infection, it is tempting to suggest that they and other similar molecules might play a role in the immunosurveillance of macromolecules in the gastrointestinal tract. If antigens are recognized within the GI tract, is this information correlated with information garnered from the previous permeation of similar antigens? At present we can only speculate on questions such as these, but future research in this area will bring fascinating insights. REFERENCES I . WALKER, W. A. & K. J. ISSELBACHER. 1974. Uptake and transport of macromolecules by the intestine. Gastroenterology 67: 53 1-550. J. H., H. A. WEUERS& W. K. DICKE.1953. Coeliac disease. IV. 2. VANDE KRAMER, An investigation into the injurious constituents of wheat in connection with their action on patients with coeliac disease. Acta Paediatr. 42: 223-231. A. C. 1985. Disordered gastrointestinal function and its relationship to tropical 3. FRAZER, sprue, coeliac disease and idiopathic steatorrhoea. Trans. R. SOC.Trop. Med. Hyg. 46. 576-585. J., M. HARRISON, A. KILBY,A. PHILLIPS & N. FRANCE. 1978. Cow’s 4. WALKER-SMITH, milk-sensitive enteropathy. Arch. Dis. Child. 5 3 375-380. I. A., B. DIGEON& A. D. PHILLIPS.1989. Evaluation of a casein 5. WALKER-SMITH, and a whey hvdrolysate for treatment of cow’s-milk-sensitive enteropathy. . . Eur. J. Pediatr. 149 68-71. 6. GRUSKAY. - ..- - .. F. L. & R. E. COOKE.1955. The gastrointestinal absorotion of unaltered proteinin normal infants and in infants recovering from diarrhea.bediatrics 16:763. 1974. Transmissible 7. BUTLER,D. G., D. G. GALL,M. H. KELLY& J. R. HAMILTON. gastroenteritis; mechanism responsible for diarrhea in an acute viral enteritis in piglets. J. Clin. Invest. 5 3 1335-1342. 8. KEUO, D. J., D. G . BUTLER& J. R. HAMILTON. 1985. Alteredjejunal permeability to macromolecules during viral enteritis in the piglet. Gastroenterology 88: 998-1004. 9. HEYMAN, M., G. CORTHIER, A. PETIT,J. C. MESLIN,C. MOREAU& J. F. DESIEUX. 1987. Intestinal absorption of macromolecules during viral enteritis: an experimental study on rotavirus-infected conventional and germ-free mice. Pediatr. Res. 22: 72-78. M. RIEPENHOFF-TALTY, J. E. FISHER& P. L. OGRA. 10. UHNOO,I. S., J. FREIHORST, 1990. Effect of rotavirus infection and malnutrition on uptake of a dietary antigen in the intestine. Pediatr. Res. 27: 153-159. E., M. GOTTELAND, M. HEYMEN,P. POCHJART & J. F. DESJEUX.1990. 11. ISOLAURI, Antigen absorption in rabbit bacterial diarrhea (RDEC-I). Dig. Dis. Sci. 35: 360-366.
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M., A. M. DUMONTIER & J. F. DESJEUX.1985. Intestinal barrier to intact 12. HEYMAN, horseradish peroxidase in experimental secretory diarrhea. J. Pediatr. Gastroenterol. Nutr. 5: 463-466. B. S., E. S. BOATMAN & G. E . KENNY.1987. Intestinal absorption of 13. WORTHINGTON, intact proteins in normal and protein-deficient rats. Am. J. Clin. Nutr. 27: 276-286. D., J. N. UDALL,K. Y. PANG,S. E. KIRKHAM & W. A. WALKER.1985. 14. ROTHMAN, The effect of short-term starvation on mucosal barrier function in the newborn rabbit. Pediatr. Res. 19: 727-731. 15. HEYMAN,M., G. BOUDRAA,S. SARRUT,M. GIRAUD,L. EVANS,M. TOUHAMI& J. F. DESJEUX.1984. Macromolecular transport in jejunal mucosa of children with severe malnutrition: a quantitative study. J. Pediatr Gastroenterol. Nutr. J: 357-363. R., F. PALON,S. MONTANO,S. LEMOLI,P. M. MATRICARDIA, S. FAIS, 16. PAGANELLI, P. PAOULUZI, R. D’AMELIO& F. AIUTI.1985. Isotypic analysis of antibody response to a food antigen in inflammatory bowel disease. Int. Arch. Allergy Appl. Immunol. 7 8 81-85. & T. E. 17. ETERMAN,K. P., W. T. HEKKENS,A. S. PENA,P. H. LEMS-VAN-KAN FELTKAMP. 1988. Wheat grains; a substrate for the determination of gluten antibodies in serum of gluten sensitive patients. J. Immunol. Methods 14: 85-92. I. W., R. S. LLOYD, P. J. WHORWELL & R. WRIGHT.1979. Antibodies to 18. DAVIDSON, maize in patients with Crohn’s disease, ulcerative colitis and coeliac disease. Clin. Exp. Immunol. 35: 147-148. I. R., P. BOULTON,I. MENZIES & J. A. WALKER-SMITH. 1987. Improve19. SANDERSON, ment of abnormal lactulosehhamnose permeability in active Crohn’s disease of the small bowel by an elemental diet. Gut 28: 1073-1076. 20. HOLLANDER, D., C. VADHEIM,E. BRETHOLZ, G. M. PETTERSEN, T. DELAHUNTY & J. I. ROTTER.1986. Increased intestinal permeability in patients with Crohn’s disease and their relatives. Ann. Intern. Med. 105: 8831-885. 21. TEAHON,K.,P.SMETHURST,M.PEARSON,A. J. LEVI&I.BJARNASON. 1991.Theeffect of elemental diet on intestinal permeability and inflammation in Crohn’s disease. Gastroenterology 101: 84-89. 22. SEIDMAN, E. G., D. G. HANSON& W. A. WALKER.1986. Increased permeability t o polyethylene glycol 4000 in rabbits with experimental colitis. Gastroenterology 90: 120- 126. 23. COBDEN,I., R. J. DICKENSON & A. T. R. AXON.1978. Intestinal permeability assessed by excretion ratios of two molecules: results in coeliac disease. Br. Med. J. 2 1060. 24. JUBY,L. D., J. ROTHWELL& A. T. AXON.1989. Cellobiose/mannitol sugar test-a sensitive tubeless test for coeliac disease; results on 1010 unselected patients. Gut 30: 476-480. 25. DANNAEUS, A., M. INGANAS,S. G. 0. JOHNSONer al. 1979. Intestinal uptake of ovalbumin in malabsorption and food allergy relation to serum IgG antibody and orally administered sodium cromoglycate., Clin. Allergy 9: 263-270. 26. BJARNASON, I., T. J. PETERS& N. VEALL. 1983. A persistent defect in intestinal permeability in coeliac disease demonstrated by a SICr-labelledEDTA absorption test. Lancet 1: 323-325. 27. WALKER-SMITH, J. A., R. P. FORD & A. D. PHILLIPS. 1984. The spectrumofgastrointestinal allergies to food. Ann. Allergy 53: 629-636. 28. JUVONEN,P., I. JAKOBSSON & T. LINDBERG. 1991. Macromolecular absorption and cow’s milk allergy. Arch. Dis. Child. 66:300-303. 29. FORD, R. P. K., I. S. MENZIES,A. D. PHILIPS,J. A. WALKER-SMITH & M. W. TURNER. 1985. Intestinal sugar permeability relationship to diarrhoea1 disease and small bowel morphology. J. Pediatr. Gastroneterol. Nutr. 4 568-574. 30. FRICK,0. L., D. F. GERMAN& J. MILLS. 1979. Development of allergy in children. 1. Association with virus infections. J. Allergy Clin. Immunol. 63: 228-241. 31. BUGAWAN, G. B. FERRARA & H. A. T. L., G . ANGELINI,J. LARRICK,S. AURICCHIO, ERLICH.1989. A combination of a particular HLA-DP p allele and an HLA-DQ heterodimer confers susceptibility to coeliac disease. Nature 339: 470-473. 32 KAGNOFF,M. F., J. I. HARWOOD,L. T. BUGAWAN & H. A. ERLICH.1989. Structural
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33. 34. 35. 36. 37. 38.
39.
40.
17
analysis of the HLS-DR,-DQ and -DP alleles on the celiac disease-associated HLADR3 (Drwl7) haplotype. Proc. Natl. Acad. Sci. USA 86: 6274-6278. MILLER,H. R. & Y. NAWA.1979. Nippostrongylus brusiliensis: intestinal goblet-cell response in adoptively immunized rats. Exp. Paasitol. 47: 81-90. WALKER, W. A., M. Wu, J. K. ISSELBACHER & K. J. BLOCH.1975. Intestinal uptake of macromolecules. 111. Studies on the mechanisms by which immunization interferes with antigen uptake. J. Immunol. 1U: 8854-861. S. 1990. Immunologically mediated damage to the intestinal mucosa. Acta STROBEL, Paediatr. Scand. Suppl. 365:46-57. MILLER,A., 0. LIDER& H. L. WEINER.1991. Antigen-driven bystander suppression after oral administration of antigen. J. Exp. Med. 174: 791-798. OWEN,R. L., D. K. BHALLA & R. T. APPEL.1982. Migration of Peyer’s patch Lymphocytes through intact M cells into the intestinal lumen in mice, rats, and rabbits. J. Cell Biol. 95:332a. GREVE,J. M., G. DAVIS,A. M. MEYER,C. P. FORTE,S. C. YOST,C. W. MARLOR, M. E. KAMARCK & A. MCCLELLAND. 1989. The major human rhinovirus receptor is ICAM-1. Cell 5 6 839-847. STAUNTON, D. E., V. J. MERLUZZI, R. ROTHLEIN, R. BARTON, D. S. MARLIN& T. A. SPRINGER. 1989. A cell adhesion molecule, ICAM-I , is the major surface receptor for rhinoviruses. Cell 5 6 849-853. MENDELSOHN, C. L., E. WIMMER & V. R. RACANIELLO. 1989. Cellular receptor for poliovirus; molecular cloning, nucleotide sequence, and expression of a new member of this immunoglobulin superfamily. Cell 5 6 855-865.
WALKER & SANDERSON: ANTIGENS & EPITHELIUM TABLE I.
11
Importance of Complete Understanding of Antigen Uptake 1. Effective use of oral vaccines 2. Prevent autoimmune reactions 3. Prevent propagation of inflammatoryhmmunologic reactions
explore in particular the possibility that increased or inappropriate uptake may be a prerequisite for disease. Exposure of Antigens in Human Disease States Disease States that Increase Antigen Uptake of the Intestine
A number of conditions are now known to increase the permeability of the intestine to antigens (TABLE2). Acute gastroenteritis is associated with increased antigen transfer. Grusky and Cooke6demonstrated that egg albumin concentrations were fivefold higher in serum of infants with diarrhea than in children with diseases outside the gastrointestinal tract. This study was done before the aetiology of acute diarrhea had been characterized; more recent studies have correlated macromolecules uptake with the onset of well defined infections, particularly in animal models. Transmissible gastroenteritis (TGE) virus causes an acute self limiting disease in piglets similar to human rotavirus gastroenteritis.’ The mucosa to serosa uptake rate of horse radish peroxidase (HRP), a protein enzyme, have been measured in jejunal mucosal segments from piglets infected with TGE virus.8 Uptake across epithelium containing a Peyer’s patch (which is normally high) was not significantly affected; however, uptake increased in areas of jejunal tissue without a Peyer’s patch to the level seen in Peyer’s patch tissue. This increased uptake occurred only early (12 hr) after inoculation with virus. In mice infected with rotavirus, however, intact HRP transfer in vitro was maximal 2-3 days after i n f e ~ t i o nTransfer .~ of antigens (ova1bumin)’Ointo the circulation of mice examined 3 days after inoculation with rotavirus was also significantly greater than uninfected
TABLE 2.
Insults That Increase Antigen Uptake of the Intestine 1. Intestinal disorders:
Gastrointestinal food allergy Celiac disease Acute gastroenteritis Chronic intestinal infections Inflammatory bowel disease Surgery 2. Systemic insults: Excessive radiation Extensive burns Septicemia shock Hypovolumetric shock Malnutrition 3. Drugs Antiinflammatory drugs
u
ANNALS NEW YORK ACADEMY OF SCIENCES
controls. In developing countries, the contribution of bacteria to the spectrum of infectious agents in gastroenteritis is particularly great. The bacteria that cause acute diarrhea can increase the permeation of macromolecules proteins. In rabbits RDEC-1 increases the in uitro permeation of p-lactoglobulinand HRP." However, this is probably only true of bacteria that cause diarrhea with mucosal damage, as secretory diarrhea caused by cholera-like exotoxins is not associated with increased antigen transport.12In countries where bacterial diarrhea is prevalent, malnutrition is common. Malnutrition can exacerbate the passage of antigens across the intestine during gastrointestinal infection; malnourished mice infected with rotavirus show increased intestinal permeability to ovalbumin (OVA) as compared to well fed infected mice." Indeed malnutrition can by itself lead to increased antigen transfer. Protein-deficient rats show increased intestinal uptake to bovine serum albumin (BSA).13Starvation affects barrier functions extrinsic to the intestine. Significantly less trypsin-like activity was seen in starved newborn rabbits (that had high plasma concentrations of BSA) than in fed ones (that had low concentrations of BSA).14 Intrinsic barrier function is nevertheless also compromised by malnutrition. Jejunal morphology is altered14 and transport of HRP across small intestinal epithelium is increased, as has been shown in biopsies from malnourished children. Many gastrointestinal diseases are associated with malnutrition and this adds to the complexity of interpreting results in studies of irlflammatory bowel disease and celiac disease. Serum immunoglobulins to food antigens have been found in IBD including p-lactoglobulin,16wheat17and maize,'* so it is probable that protein macromolecules permeate in increased amounts causing systemic immune responsiveness. Nonprotein macromolecules have been extensively studied in Crohn's d i ~ e a s e ' ~ and - ~ ' permeate readily, often returning to normal with therapy.'9.z1 Colonic permeability to PEG-4000 is increased in experimental colitis.zz In celiac disease nonprotein macromolecules permeate more easily than in nondisease controls, and some consider use of sugar markers to be a possible screening test.23*24 The sensitivity of cellobiosehannitol absorption test was %% in 817 consecutive adults suspected of having celiac disease." Antigens are also able to permeate more readily in patients with the c o n d i t i ~ nIt. ~is~ possible that small bowel integrity is not normal even after treatment with gluten-free diet.26 Food allergy can manifest as gastrointestinal diseasez7as well as diseases of other organ systems. Protein macromolecules are capable of entering the circulation of milk-allergic children challenged with milk; transfer of a test dose of alactalbumin (which is not found in cow's milk) was greater in children who showed symptoms following cow's milk challenge than in those who did notz8.a-Lactalbumin transfer was increased in children who had either gastrointestinal or skin manifestations of cow's milk allergies. Nonprotein macromolecules markers too are increased in gastrointestinal food allergyz9and this correlates well with intestinal morphology. Disease States and Their Relationship to Antigen Entry
It is likely that the uptake of immunogenic molecules from the lumen is important in the pathogenesis of some gastrointestinal diseases. Antigens can be the entry site of immunological-mediatedactivity both within the intestine and beyond. Interaction between macromolecules and the immune system could lead to immunologically-mediated damage if compensatory mechanisms did not exist. This tolerance to antigens entering the gut could be broken if the uptake of the gut to
WALKER & SANDERSON: ANTIGENS & EPITHELIUM
l3
certain antigens were altered in some way. It is not clear whether increased uptake of a particular antigen is central to the pathogenic process of immune-mediated gastrointestinal disease. Most gastrointestinal diseases exhibit increased uptake of antigens (see above), yet this could be secondary to the disease process itself. While we cannot answer this question at present, there are a number of approaches to it that might distinguish between primary or secondary antigen permeability defect. The approach to this process can be summarized as follows: there are four ways of discerning whether gastrointestinal diseases are a consequence of increased antigen entry: a) examination of subjects before onset of disease, b) examination after resolution of disease, c) examination of first degree relatives and d) the use of animal models. If increased antigen entry is not a prerequisite to immune-mediated gastrointestinal disease then it is necessary to propose that the immune system of affected individuals is different from healthy subjects at some point before the onset of the disease.
TABLE 3. Antigen Uptake Processes 1. Physiologic transport
a. Ligand receptor uptake (growth factors) Conduit Enterocyte as target organ b. Antibody uptake-passive immunity c. Microfold cell (“M” cell) transport Normal local immune response 2. Pathologic transport a. Intracellular (excessive) Immature gut; post enteritis; allergic enteropathy b. Paracellular Inflammation Edema associate with anaphylaxis 3. Pathologic-antigen-specific a. Gliadin-celiac disease b. Beta lactoglobulin (BLG) allergic gastroenteropathies c. Bacterial antigens-autoimmune disease (IBD?) d. Primary vs tolerance (intracellular processing)
Alteration of Immune System
If gastrointestinal disease is due to altered immune function, antigen uptake by a variety of mechanisms is still necessary for disease but uptake could (in the premorbid state) be no different from that found in normals (TABLE3). However, the evidence that the immune system is altered in an affected individual prior to the onset of disease is often lacking. In Crohn’s disease there is no clear immunological defect that is independent of the pathogenic process, although some rare immunological diseases are associated with a higher incidence of IBD.30Although there is an increased risk of gastrointestinal food allergy in children with atopy, this is only so in a proportion of cases and most atopic children do not present with food
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ANNALS NEW YORK ACADEMY OF SCIENCES
allergy. However, it is possible that gastrointestinal infections could “alert” the immune system so that the antigens that cross the intestine in fact are more likely to set up an immune response. There is evidence that allergic sensitization of the respiratory tract follows viral respiratory tract infections.” In a prospective study of 13 children born to allergic parents, 11 became clinically allergic in the first 5 years of life. Laboratory immunological criteria of allergic sensitization were made every 3 months. Respiratory infections (confirmed in 10 out of the 11 by complement-fixing antibodies) occurred in the two months before onset of allergic sensitization. Therefore, it is possible that gastrointestinal food allergy could be commoner after gastroenteritis by a similar mechanism. Celiac disease is closely linked to the MHC class I1 haplotype DQw2 (90to 95% of patient^.^^"^ As class I1 molecules are central to the T cell response, it is possible that celiac disease occurs only in subjects who are able to present a certain epitope of the gliadin molecule to T cells and those subjects have the specific heterodimers (namely DQw2 and its linked class I1 MHC molecules) that are able to present that epitope. If this were so, there would be no need to postulate increased macromolecular entry in affected subjects. However, although most patients with celiac disease have a defined class I1 profile, it is only a small number of people with that profile who develop the disease and some further initiating mechanism is required. In addition, patients who have class I1 molecules capable of presenting peptide fragments to antigen-presentingcells are likely to have similar molecules on the basolateral surface of enterocytes. If enterocytes are capable of transporting peptide fragments in uiuo, one could speculate that those with gliadinrecognizing class I1 molecules are transporting them across the epithelial cell barrier. This rate of transport would be very great in comparison to that in enterocytes who do not have the class I1 that recognized peptides of gliadin. Therefore, even in celiac disease where a particular immune state is clearly implicated in the pathogenesis of the disease, increased antigen uptake may indeed be important.
CONCLUSIONS AND SPECULATION In this overview of epithelial barrier function to antigens we have reiterated the speculation’ that increased antigen uptake of the intestine precedes the onset of a number of immunologically-mediatedgastrointestinal diseases. We have examined possible ways of determining whether such an increase in antigen transfer could occur as a preliminary to disease; but we have also examined the contrary hypothesis that antigen uptake activates an immune system that is altered in some way, either by recurrent infection or because of genetic makeup. These two hypotheses are not mutually exclusive. Indeed the immune system plays a significant role in the barrier to antigens transport. IgE-mediated discharge of histamine results in enhanced goblet cell mucus release.34IgA complexes with antigens and immunization decrease antigen tran~fer.~’ There remain many questions to be answered. We have suggested that antigens might penetrate the mucosal barrier by different pathways, but we do not know the relevant importance of those pathways in immune surveillance or in the initiation of gastrointestinal disease. These pathways will be reviewed in depth as part of this session. In some circumstances antigen passage increases immune activity (as in the initiation of allergic disease); in other circumstances oral antigens suppress immune reactions that are already underway.36For example, mice that develop the neurological consequences of the immune response to myelin basic protein are
WALKER & SANDERSON: ANTIGENS & EPITHELIUM
15
significantly improved by feeding the pr~tein.~’ Do these different responses to oral antigen represent different pathways of permeation? Finally, we need to determine whether antigen uptake is always essential to immune surveillance. Can luminal antigens be recognized without their penetrating the epithelium? If class I1 MHC on the epithelium presents processed luminal peptides on their surface without releasing them, this is indeed a possibility. Can basolateral antigens be recognized within the lumen of the intestine? There is no evidence for this yet, but two observations suggest that uptake may not always be essential. First, lymphocytes can enter the lumen of the intestine from the Peyer’s patch3sand considerably could function as part of the afferent arm of the immune response at that site. Second, the apical surface of mucosal epithelial cells expresses a number of molecules belonging to the immunoglobulinsuperfamily. Their function is at present unknown but they have been identified because they form the attachments for invading viruses. ICAM-1 has been found to be the Ig superfamily poliovirus .~ member that allows rhinovirus attachment in nasal e p i t h e l i ~ r n ,a~ ~ receptor in intestinal epithelium is also a member of the Ig ~uperfamily.~~ As the primary function of these molecules on mucosal surface cannot be to serve as a conduit for viral infection, it is tempting to suggest that they and other similar molecules might play a role in the immunosurveillance of macromolecules in the gastrointestinal tract. If antigens are recognized within the GI tract, is this information correlated with information garnered from the previous permeation of similar antigens? At present we can only speculate on questions such as these, but future research in this area will bring fascinating insights. REFERENCES I . WALKER, W. A. & K. J. ISSELBACHER. 1974. Uptake and transport of macromolecules by the intestine. Gastroenterology 67: 53 1-550. J. H., H. A. WEUERS& W. K. DICKE.1953. Coeliac disease. IV. 2. VANDE KRAMER, An investigation into the injurious constituents of wheat in connection with their action on patients with coeliac disease. Acta Paediatr. 42: 223-231. A. C. 1985. Disordered gastrointestinal function and its relationship to tropical 3. FRAZER, sprue, coeliac disease and idiopathic steatorrhoea. Trans. R. SOC.Trop. Med. Hyg. 46. 576-585. J., M. HARRISON, A. KILBY,A. PHILLIPS & N. FRANCE. 1978. Cow’s 4. WALKER-SMITH, milk-sensitive enteropathy. Arch. Dis. Child. 5 3 375-380. I. A., B. DIGEON& A. D. PHILLIPS.1989. Evaluation of a casein 5. WALKER-SMITH, and a whey hvdrolysate for treatment of cow’s-milk-sensitive enteropathy. . . Eur. J. Pediatr. 149 68-71. 6. GRUSKAY. - ..- - .. F. L. & R. E. COOKE.1955. The gastrointestinal absorotion of unaltered proteinin normal infants and in infants recovering from diarrhea.bediatrics 16:763. 1974. Transmissible 7. BUTLER,D. G., D. G. GALL,M. H. KELLY& J. R. HAMILTON. gastroenteritis; mechanism responsible for diarrhea in an acute viral enteritis in piglets. J. Clin. Invest. 5 3 1335-1342. 8. KEUO, D. J., D. G . BUTLER& J. R. HAMILTON. 1985. Alteredjejunal permeability to macromolecules during viral enteritis in the piglet. Gastroenterology 88: 998-1004. 9. HEYMAN, M., G. CORTHIER, A. PETIT,J. C. MESLIN,C. MOREAU& J. F. DESIEUX. 1987. Intestinal absorption of macromolecules during viral enteritis: an experimental study on rotavirus-infected conventional and germ-free mice. Pediatr. Res. 22: 72-78. M. RIEPENHOFF-TALTY, J. E. FISHER& P. L. OGRA. 10. UHNOO,I. S., J. FREIHORST, 1990. Effect of rotavirus infection and malnutrition on uptake of a dietary antigen in the intestine. Pediatr. Res. 27: 153-159. E., M. GOTTELAND, M. HEYMEN,P. POCHJART & J. F. DESJEUX.1990. 11. ISOLAURI, Antigen absorption in rabbit bacterial diarrhea (RDEC-I). Dig. Dis. Sci. 35: 360-366.
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