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healthy first-degree relatives of patients with coeliac disease. Lancet 338:13501353, 1991. 37. Marley NJE, McCarlney JC, Ciclitira PJ: HLA-DR, DP and DQ expression in the small intestine of patients with coeliac disease. Clin Exp Immuno170:386-393, 1987. 38. Niazi NM, Leigh R, Crowe P, Marsh MN: Morphometric analysis of small intestinal mucosa. 1. Methodology, epithelial volume compartments and enumeration of inter-epithelial space lymphocytes. Virchows Arch [Pathol Anaq 404:49-60, 1984. 39. Maki M, Holm K, Collin P, Savilahti E: Increase in ~ T cell receptor bearing lymphocytes in normal small bowel mucosa in latent coeliac disease. Gut 32:1412-1414, 1991. 40. Mantzaris G, Jewell DP: In vivo toxicity of a synthetic dodecapeptide frum A-gliadin in patients with coeliac disease. Scand J Gastro 26:392-398, 1991. 41. Marsh MN, Hinde J: Inflammatory component of celiac sprue mucosa 1. Mast cells, basophils and eosinophils. Gastroenterology 89:92-101, 1985. 42. Marsh MN, Bjamson I, Shaw/, Ellis A, et al.: Studies of intestinal lymphoid tissue. XIV-HLA status, mucosal morphology, permeability, and epithelial lymphocyte populations in fhst degree relatives of patients with coeliac disease. Gut 31:32-36, 1990. 43. Parker CM, Groh V, Band H, et al.: Evidence for extrathymic changes in the T cell repertoire. J Exp Med 171:1597-1612, 1990. 44. Rossipal E: The incidence of childhood coeliac disease in Austria: a study covering the
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period 1969-86. In: Kumar PJ, WalkerSmith JA, (eds): Coeliac Disease 100 Years. Leeds, Leeds University Press, pp 299302, 1990. 45. Savilahti E, Arat A, Verkasalo ML: Intestinal y6 receptor-bearing T lymphocytes in celiac disease and inflammatory bowel diseases in children. Constant increase in celiac disease. Pediatr Res 28:579-581,1990. 46. Scott H, Sollid L M, Fausa O: Expression of major histocompatability Class 11 subregion products by jejunal epithelium in patients with coeliac disease. Scand J Immunol 26:563-571, 1987, 47. Selby WS, Janossy G, Bofill M, Jewell DP: Lymphocyte subpopulations in the human small intestine. The findings in disease. Clin Exp Immuno152:219-228, 1983. 48. Shiner M: Ultrastructural changes suggestive of immune reactions in the jejunal mucosa of children following gluten challenge. Gut 14:1-12, 1973. 49. SoUid LM, Markussen G, Ek J, et al.: Evidence for a primary association of celiac disease to a particular HLA-DQ tx/~ heterodimer. J Exp Med 169:345350, 1989. 50. Spencer J, MacDonald "IF, Diss TC, et al.: Changes in intraepithelial lymphocyte subpopulations in coeliac disease and associated T cell lymphoma (malignant histiocytosis of the intestine). Gut 30:339344, 1989. 51. Spencer J, Isaacson PG, Diss TC, et al.: Expression of disulphide linked and non-disulfide linked forms of the T cell receptor or gamma/delta heterodimer in human intesti-
nal intraepithelial lymphocytes. Eur J hnmunol 19:1335-1338, 1989. 52. Spencer J, Isaacson JA, MacDonald TF, et al.: Gamma]delta T cells and the diagnosis of coeliac disease. Clin Exp Immunol 85:109-113, 1991. 53. Spurkland A, SoUid LM, Ronnmgen KS, et al.: Susceptibility to develop coeliac disease is primarily associated with HLA-DQ alleles. Hum Immunol 29:157-165, 1990. 54. Stevens FM, Egan-Mitchell B, Cryan E, et al.: Decreasing incidence of coeliac disease. Arch Dis Childhood 65:465-468, 1987. 55. Stokes PL, Ferguson R, Holmes GKT, et al.: Familial aspects of coeliac disease. Q J Med 180:567-582,. 1976. 56. Talley NJ, Kephart GM, McGovem TW, et al.: Deposition of eosinophil granule major basic protein in eosinophilic gastroenteritis and celiac disease. Gastroenterology 103:137-145, 1992. 57. Vecchi M, Crosti L, Berti E, et al.: Increased jejunal intraepithelial lymphocytes bearing T-ceil "?6receptor in dermatitis herpetiformis. Gastoenterology 102:14995O5, 1992. 58. Walker-Smith JA, Revised criteria for diagnosis of cocliac disease. Arch Dis Childhood 65:909-911, 1990. 59. Webster ADB, Slavin G, Shiner M, et al.: Coeliac disease with severe hypogammaglobinaemia.Gut 22:153-157, 1981. 60. Wright NA, Watson A, Morly A, et al.: Cell kinetics in flat (avillous) mucosa of the human small intestine. Gut 14:701710, 1973.
Inflammatory Bowel Disease: An Immunological Overview Claudio Fiocchi Department of Gastroenterology and Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio Etiopathogenesis Bowel Disease
of Inflammatory
WO clinical entities are grouped under the denomination of inflammatory bowel disease: Crohn's disease and ulcerative colitis. Although clearly dis-
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tinct in many clinical and pathological aspects, they share several other features, including an unknown cause, a tendency for familial clustering, a chronic and unpredictable clinical course, the need for surgical intervention, and an unsatisfactory Illllllllllll
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response to treatment. These features, common to many other chronic diseases, have generated a wide variety o f hypotheses to explain the cause o f inflammat~y bowel disease. Infectious, genetic, environmental, metabolic, vascular, and immuI
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nological theories have been proposed, each based on qualitatively and quantitatively variable evidence.: Today, the concept that most chronic inflammatory diseases have an important component of immune disregulation has gained general acceptance. This concept is based on substantial evidence showing that immunologic abnormalities are frequent in patients affected by recurrent, indolent, and unmanageable diseases accompanied by cellular infiltrates in various tissues and organs. This is certainly true for both forms of inflammatory bowel disease, where the small and large bowel carry a chronic overload of T cells, B cells, or macrophages even during periods of clinical remission. Whether these cellular infiltrates and the associated immunological abnormalities are primary or secondary may be impossible to determine as long as the exact cause of Crohn's disease or ulcerative colitis is out of reach. Given the status of present knowledge, an in-depth evaluation of the immune system in inflammatory bowel disease should take precedence over abstract considerations of whether immunological abnormalities are the cause or the result of the disease.
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ters in the peripheral circulation have contributed relatively tittle to inflammatory bowel disease immunopathogenesis. Detection of circulating immune complexes, shifts in peripheral blood T and B cell levels, and assessment of proliferative capacity of mononuclear cells to nonspecific stimuli, while suggesting the existence of systemic immune abnormalities, have provided only indirect information on possible defects relevant to gut inflammation. 3 This situation has changed substantially during the last decade, with the surge of
An in-depth evaluation of the immune system in inflammatory bowel disease should take precedent over abstract considerations of whether immunological abnormalites are the cause or the result of the disease.
Immunopathogenesis of Inflammatory Bowel Disease: Systemic vs. Mucosal Immunity Accepting the very solid premise that exploring immune abnormalities in inflammatory bowel disease is a top priority, the next question is how to carry out such investigation. Both Crohn's disease and ulcerarive colitis can display extraintestinal manifestations, such as arthritis, cholangitis, erythema nodosum, pyoderma gangrenosum, and uveitis/'mtis. These manifestations leave no doubt that systemic reactions are an integral part of inflammatory bowel disease, and could be interpreted as evidence that autoimmunity has a major role in pathogenesis. Common antigens shared by various affected organs have been identified, but whether they are the actual target of autoimmune aggression has yet to be proven. 2Regardless, systemic signs and symptoms are always secondary to gut involvement, where the inflammatory process starts and lingers. Based on this postulate, it is not surprising that earlier studies investigating immune parame-
mucosal immunity as a major new player within the arena of immunology. Praising the eminent importance of mucosal immunity is beyond the scope of this review, but two aspects deserve mentioning. The first is the quantitative predominance of the gastrointesrinal tract as the primary organ in immunity. The amount and the variety of cells in the gut lymphoid tissue is unmatched by any other organ, and the intestinal mucosa is by far the largest surface of the body in contact with the external environment, rendering it essential to immune homeostasis.* The second aspect is even more crucial, i.e., inflammatory bowel disease is fundamentally a disorder of the mucosal immune system, and whatever events are responsible for its cause and pathogenesis should he sought in loco. Fortunately, this is now possible through techniques that allow the isolation of cells from vari© 1992 Elsevier Science Publishing Co., Inc.
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ous bowel segments, and an impressive amount of data has been accumulated. 5
Immune Components of Inflammatory Bowel Disease: Cells and Cytokines In the preceding section the abundance and heterogeneity of immune cells in the intestinal mucosa, a phenomenon that occurs under physiological and pathological conditions, was inlroduced. In addition to immane cells, other elements are present, including a tight voluminous lamina propria rich in structural and nervous ceils, arteriovenous and lymphatic endothelial cells, and smooth muscle cells? Consequently, immune cells involved in triggering and perpetuating bowel inflammation do not do so alone, but in coordination with other cell types that they modulate and by which they are in turn influenced. This constant mutual interaction is mediated primarily through the secretion of soluble mediators or cytokines, creating an intricate system that calls into question thinking of inflammatory bowel disease strictly in terms of abnormal immune reactivity. There is reasonably good information on the types of immune cells involved in mucosal inflammarion, and the concept that these cells are in a high state of activation is widely accepted. Information on local nonimmune cells is considerably more limited. Finally, information on what are likely to be the most critical pathogenic elements of the Crohn's disease and ulcerative colitis, i.e., mucosal cytokines, is accumulating at an increasingly fast pace. Cellular Components Phenotypic analysis of T and B cells in the inflamed mucosa fails to reveal any significant shifts when compared to normal tissue.7a This is surprising, considering the chronicity of the cellular infdtrates and the fact that other chronic diseases like sarcoidosis and leprosy are characterized by definite abnormalities of helper/inducer (CD4+) or suppressor/cytotoxic (CD8+) lymphocytes. Analyses based purely on morphologic or phenotypic criteria are crude, because cells with identical external markers may mediate entirely diverse functions. For instance, some studies have shown that mucosal plasma cells display quite different profiles of cytoplasmic and secreted immunoglobins in both Crohn's I 0197-1859/92150.00 + 3.00
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disease and ulcerative colitis.9'~°It is unknown what induces such changes, whether they represent a nonspecific response to chronic inflammation, or are directly dependent on the cause of inflammatory bowel disease. Nevertheless, production of immunoglobulin subclasses is remarkably different between Crohn's disease and ulcerative colitis, indicating that dissimilar regulatory events control the local antibody response. When mucosal T lymphocytes are assessed for functional activity, neither help nor suppression is grossly abnormal. N Functional tests are performed in vitro utilizing artificial systems designed to explore a specific effect, and may or may not translate the actual performance of any given type in vivo. In addition, the original concept of abnormal helper and suppressor function is also fairly crude and narrow, and in modern terms function is viewed as an activity primarily related to the patten of cytokine secretion and response. Thus, failure to detect major abnormalities of T cell function by classical criteria by no means rules out the existence of intrinsic immune defects. A novel and attractive approach to the evaluation of mucosal T cells is related to the selective activation of discrete subsets based on their receptor repertoire) 2 T lymphocytes that appear indistinguishable by classical criteria are actually quite unique when their T cell receptor (TCR) ability to recognize antigens is examined. The importance of this phenomenon is that overutilization or deletion of certain variable (VI~) regions of the TCR may be induced by exposure to dominant antigens or superantigens, which may be responsible for autoimmunity or inflammation. Preliminary da!a indicate that abnormal expression of TCR repertoire is present among lamina propria mononuclear cells from Crohn's disease and ulcerative colitis patients? 3 Since the number of VI3 regions is quite large, a systematic screening needs to be performed to uncover all possible abnormalities of TCR usage and assess their significance. Information on other mucosal cell types is still fairly limited, largely because of their numerical scarcity. Morphological and histochemical characterization of macrophages has revealed an extraordinary
heterogeneity, with antigen-presenting, scavenger, or intermediate phenotypes.14 Whether any of these functions is altered and relates to inflammatory bowel disease pathogenesis has not been investigated to any significant degree, and even the commonly held notion that macrophages mediate nonspecific intestinal tissue damage is unproven. Other cells, such as eosinophils, mast cells, and basophils, have received even less attention. As for T cells, B cells, and macrophages, they are also in an enhanced state of activation, but their num-
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H o w close is immunological investigation from discovering the cause, explaining the mechanisms, and providing new treatmentsfor Crohn's disease or ulcerative colitis?
ber does not change much even during severe flare-ups. Thus, their potential for pathogenic role is mere speculation. Finally, polymorphonuclear neutrophils have been conspicuously overlooked, mostly due to the long-held, although erroneous notion, that they are not immune cells. This is rapidly changing for two reasons: first, recent studies on leukocyte-endothelial interaction have dramatically expanded knowledge of how neutrophils migrate into site of active inflammation;Is second, one of the basic functions of these cells is to secrete reactive oxygen metabolites, which are major mediators of localized tissue damage? 6 Until recently an overview of inflammatory bowel disease immunology would be largely complete at the end of the precedIlllllllI © 1992 E l s e v i e r Science P u b l i s h i n g Co., Inc.
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ing paragraph with a review of classical concepts of immune and inflammatory cells. This is no longer acceptable because of what was previously referred to as "immune-nonimmune interactions." This relatively new idea is "based on substantial evidence demonstrating that immune ceils communicate with a wide variety of other cells of ectodermal, mesodermal, and endodermal origin. Cell-to-cell communications occur in all organs of the body, but are especially pronounced in the intestinal mucosa because of the close physical proximity of multiple cellular elements in this unique microenvironment.6 Due to increased permeability to lumenal antigens, anatomical disruption, and changes in the composition and functions of mononuclear cells, cellular interactions are unquestionably altered during inflammation. Epithelial cells have been the first nonimmune cells to attract the attention of mucosal immunologists. They mediate antigen presentation differently from classical antigen-presenting cells (macrophages and dendritic cells) by activating preferentially CD8+ T cells,v A potential defect of this function has been reported in both Crohn's disease and ulcerative colitis, where a predominant stimulation of CIM+ T cells may lead to excessive helper function and predispose to inflammation.~s Also abnormal in the diseased mucosa are neuropeptiderich nerve ceils, which are strong regulators of mucosal mononuclear cell function. Alterations of various neuropeptides have been reported, and hypotheses have been advanced on how this could influence the function of local lymphoid cellsfl Additional cell types are coming under scrutiny for a possible functional relationship with immunocytes, and some cells previously considered as having a pure "structural" function, such as fibroblasts, are now viewed as potential targets for cytokines elaborated by local inflammatory cells) °-2~ Soluble Components Since the earliest investigations, unequivocal evidence implicating any particular immune dysfunction as the direct cause of Crohn's disease or ulcerative colitis has been difficult to obtain. Presently, the role of an immune cell tends to be viewed as a function of the soluble factors they secrete and are exposed to. Adopting this modern Ill
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concept, the most critical events responsible for inflammatory bowel disease may well be primarily dictated by the type and concentration of the various cytokines produced in the gut mucosa. This approach has spurred an unprecedented interest in the role of these mediators in Crohn's disease and ulcerative colitis. An enormous amount of dam has been generated in the last decade, and relevant information has been recently reviewed.22 Cytokine activities have been investigated both at the systemic and intestinal levels. It was initially hoped that measurements using serum samples would reflect events occurring in the gut, and perhaps provide markers of clinical activity, but this is questionable. The most widely investigated so far has been the soluble form of the intedeukin-2 0L-2) receptor, but it may not accurately mirror the degree of inflammation in all patients. 23Preliminary reports suggest that comparative levels of IL-6 or IL-8 can distinguish Crohn's disease from ulcerative colitis, but the reliability and reproducibility of these findings are unclear. ~,zs Contrary to peripheral cytokine concenlrations, those in the mucosa could be ideal indices of inflammation. There is general agreement that production of IL-1 is dramatically increased in inflamed intestine. ~ In contrast, IL-2 activity has been reported as decreased, while its mRNA may be increased. 27,'~IFNy has also been found both decreased and increased. 29'3°The activity of other cytokines with a predominant proinflammatory activity, such as IL-6 and IL-8, is predictably augmented, while mucosal levels of TNFct have been reported as low to very hight z2 If any consensus can be derived from these contradictory data, it is that no clear picture has emerged on the status of cytokines in inflammatory bowel disease, but further investigation is certainly warranted. Two major factors can explain this confusing situation. The first is that the methodology employed to assess cytokines has been as diverse as the cellular sources used to study them. Measurements of bioactivity, immunoreactivity, mRNA, and functional capacity do not translate the same information on the status of a specific mediator. The second is that the number of cytokines is constantly expanding, making it
difficult to explore all of them in reasonable depth and obtain defined patterns of expression. This situation is complicated by the interactive nature of cytokines, and in the so-called "cytokine network" each soluble mediator modulates and is in turn modulated by the surrounding cytokines. These difficulties may be resolved by the recent discovery that particular T lymphocytes produce restricted patterns of cytokines. Fundamental work by Mossmann and Coffman has shown that some T helper (Th) cell clones with defined secretory capacity display discrete immune functions. 33 As originally described, Thl cells produce IL-2, IFNy, and lymphotoxin, and mediate primarily delayed-type hypersensitivity, while Th2 cells secrete IL-4, IL-5, IL-6, and IL-10 and provide help for antibody production. Additional work generated evidence for undifferentiated (ThO) patterns, and it is quite possible that additional patterns (ThX) may exist, particularly in the case of a chronic response to a persistent antigen. Novel patterns of cytokine expression may be identified in the intestinal mucosa, and production of"characteristic" cytokines may be linked to the various stages of inflammatory bowel disease.
Impact of Immunology on the Etiology, Pathogenesis, and Therapy of Inflammatory Bowel Disease Hopefully the preceding paragraphs have convinced the reader of how fundamental immune phenomena are in inflammatory bowel disease. Taking this assumption to be true, how close is immunological investigation from discovering the cause, explaining the mechanisms, and providing new treatments for Crohn's disease or ulcerative colitis? As far as etiology, even the most comprehensive studies are unlikely to unveil the specific cause. The immune system is said to exhibit exquisite specificity, and this is true in the context of a response to a defined antigen. In contrast, the cells stimulated by the antigen may be many, the signals they produce multiple, and the action generated nonspecific. Additionally, a myriad of antigens may elicit similar responses, indistinguishable from one another, all occurring independently of the original trigger An example of this common situation is provided by studies of se© 1992 Elsevier Science Publishing Co., Inc.
rum levels of antibodies to bacterial antigens. They may be abnormally high in Crohn's disease and ulcerative colitis patients as compared to normal individuals, but still reflect exposure to the same microorganism. ~ Current trends are centered on the sophisticated identification of the various portions of the TCR that "see" the antigen, and knowing which V[3 region sees it, inferences can be made about its nature. Even assuming that a complete mapping of the TCR with inflammatory bowel disease cells is done, chances that a single or very few VIi regions are consistently utilized in all patients are probably very few considering that specific epitopes from a putative etiological agent may be obscured by an overwhelming amount of dietary and enteric antigens. More promising is immunology's potential for improving the understanding of pathogenic mechanisms. For a long time it has been impossible to immunologically differentiate Crohn's disease from ulcerative colitis. This was due to the limited power of experiments based on the phenotypic characterization of inflammatory cells, and utilization of fairly crude functional assays. Major advances in the mechanisms associated with several chronic inflammatory diseases have occurred only after establishing the fundamental regulatory role of cytokines, and particularly after the discovery of Thl and Th2 cells. Initially, Thl or Th2 cytokine patterns have been detected in animal models and correlated with different phases of the disease. 35Subsequently, similar patterns were identified in humans, Th2 in allergic, and Th I-like in delayed-type sensitivity reactions. 36As alluded before, novel and perhaps characteristic spectra of mucosal cytokine secretion may be expressed during the various stages of mucosal inflammation. However, detecting "inflammatory bowel disease-specific" ThX patterns will become reality only by prospectively cloning gut T cells in patients who go through the sequential stages of their illness. This seemingly overwhelming task may yield crucial clues if fluctuations of cytokine activities show a close correlation with clinical activity. The greatest rewards can be expected when cytokine studies are applied to revo0197-1859/92/$0.00 + 3.00
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lutionize therapy. A thorough understanding of the activity of T cell-derived low-level immunoregulatory cytokines (IL2, IL-4, IL-5, IL-10, etc.) may generate clues to unlocking the intimate mechanisms of disease. However, correcting abnormalities of these cytokines is unlikely to result in cure as long as the cells responsible for their production are not turned off, and this escapes present capabilities. In contrast, if we learn how to modulate pro-inflammatory cytokines OL- 1, IL-6, IL-8, etc.), produced in abundance by cells of the monocyte/macrophage lineage, our ability to downmgulate or even block their tissue damaging effects could improve dramatically. Production of these cytokines is exaggerated at sites of injury. Stopping their secretion by inhibiting macrophages is difficult, and even the most potent drugs have limited effect because of redundant cell circuitry. However, rather than directing therapy at the producing cells, pro-inflammatory cytokines themselves could be targeted for blockade, and today this can be done with tremendous precision and efficacy. This could be achieved by using soluble receptors or other natural antagonists spontaneously produced by the body as part of the normal mechanisms that keep agonistic and antagonistic activities in balance to preserve homeostasis. An excellent example of this critical balance is IL-1 and its natural receptor antagonist (iL-lra). Both molecules compete for the same receptor, but while doing so ILl evokes an inflammatory response, IL-lra does it without triggering such response. 37 Under physiological circumstances, the body produces enough IL-lra to prevent an excessive inflammatory response by ILl. In a disease like Crohn's disease or ulcerative colitis, the amount of ILl produced in the gut may far exceed the body's ability to produce IL- lra, and inflammation may ensue. Assuming that a pathological response is caused by an excessive production of a single cytokine, supplying enough antagonist should reestablish the lost balance and eliminate inflammation. This situation is far less hypothetical than it appears to be. Animal m o d d s of experimental colitis have objectively demonstrated that administration of IL-lra prevents or improves gut inflammation, ss Current experimental ffials based on this IIlllll
principle are now under way in subjects with ulcerative colitis. In practice, Crohn's disease or ulcerative colitis are caused by abnormalities of multiple cytokines. No matter how numerous, ultimately all the relevant antagonists will be identified, cloned and produced in unlimited quantities. Once this is achieved, they can be administered to block the mediators responsible for mucosal inflammation. Once successful, this tailored approach will represent a major breakthrough in therapy because it allows for both total specificity and the use of safe recombinant products instead of nonselective, toxic immunosuppressants. CIN References
1. Fiocchi C: Pathogenesis and clinical implications: where do we stand, where do we go? In: Goebell H, Ewe K, Malchow H, Koelbel C (ed): Inflammatory Bowel Disease. Progress in Basic Research and Clinical implications. Lancaster. Kluwer Academic Publishers, pp 237-254, 1991. 2. Das KM, Vecchi M, Sakamaki S: A shared and unique epitope(s) on human colon, skin, and biliary epithelium detected by a monoclonal antibody. Gastroenterology 98:464--469, 1990. 3. Elson CO: The immunology of inflammatory bowel disease. In: Kirsner JB, Shorter RG (ed): Inflammatory Bowel Disease. Philadelphia: Lea & Febiger, 97-164, 1988. 4. Brandtzaeg P, Halstensen TS, Kett K, et al.: Immunobiology and immunopathology of the human gut mucosa: humoral immunity and intraepithelial lymphocytes. Gastroenterology 97:1562-1584, 1989. 5. MacDermott RP, Elson CO (ed): Mucosal Immunology I. Basic Principles. Philadelphia: W.B. Saunders Company, 1991. 6. Bienenstock J: Cellular communications networks: implications for our understanding of gastrointestinal physiology. Ann NY Acad Sci 664:1--9, 1992. 7. Brandtzaeg P, Halsensten TS, Kett K, Rognum TO: Local immunopathology in inflammatory bowel disease. In: Jamerot G (ed): Inflammatory Bowel Disease. New York: Raven Press, pp 21-35, 1987. 8. James SP, Fiocchi C, Graeff AS, Strober W: Phenotypic analysis of lamina propria lymphocytes. Predominance of helper-inducer and cytolytic T-cell phenotyp¢ and deficiency of suppressor-inducer phenotype in Crohn's disease and control patients. G astroenterology 91:1483-1489, 1986.
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9. MacDermott RP, Nash GS, Bertovich M J, et al.: Alterations oflgM, IgG, and IgA synthesis and secretion by peripheral bl[xxl and intestinal mononuclear ceils from patients with ulcerative colitis and Crohn's disease. Gastroenterology 81:844 852~ 1981. 10. Kett K, Brandtzaeg P: Local IgA subclass alteration in ulcerative colitis and Crohn's disease of the colon. Gut 28:101.3--1021, 1987. 11. Janaes SP, Fiocchi C, Graeff AS, Strober W: Immunoregulation of lamina propria T ceils in Crohn's disease. Gastroenterology 88:1143-1150, 1985. 12. Marguerie C, Lunardi C, So A: PCR-based analysis of the TCR repertoire in human autoimmune diseases. Immunol Today 13:336-338, 1992. 13. Duchmarm R, Strober W, Fiocchi C, James SP: TCR V[~2 gene expression is selective in control but not in IBD lamina propria lymphocytes. Gastroenterology 102:A617, 1992. 14. Mahida Y: Macrophage function in inflammatory bowel disease. Eur J Gastroenterol Hepatol 2:251-255, 1990. 15. Zimmerman GA, Prescott SM, Mcintyre TM: Endothelial ceil interactions with granulocytes: tethering and signal molecules. Immunol Today 13:93--99, 1992. 16. Grisham MB, Yamada T: Neutrophils, nitrogen oxides, and inflammatory bowel disease. Ann NY Acad Sci 664:103-115, 1992. 17. Mayer L, Shlien R: Evidence for function of Ia molecules on gut epithelial cells in man. J Exp Med 166:1471-1483, 1987. 18. Mayer L, Eisenhardt D: Lack of induction of suppressor T cells by intestinal epithelial ceils from patients with inflammatory bowel disease. J Clin Invest 86:1255-1260, 1990. 19. Kubota Y, Petras RE, Ottaway CA, et al.: Colonic vasoactive intestinal peptide nerves in inflammatory bowel disease. A digitized morphometric immunohistochemical study. Gastroenterology 102:1242-1251, 1992. 20. StaUmach A, Schuppan D, Riese HH, et al.: Increased collagen type HI synthesis by fibroblasts isolated from strictures of patients with Crohn's disease. Gastroenterology 102:1920-1929, 1992. 21. Strong SA, West GA, Klein JS, et al.: Inflammatory eytokines stimulate proliferation of intestinal mucosa mesenchymal cells. Gastroenterology 102:A701, 1992. 22. Fiocchi C; Cytokines. In: MacDermott RP, Stenson W (ed): Inflammatory Bowel Disease. New York: Elsevier Publishing CornIIII
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pany, pp 137-162, 1992. 23. Matsuura T, West GA, Klein JS, et al.: Soluble interleukin 2, CD8 and CIM receptors in inflammatory bowel disease. A comparative study of peripheral blood and intestinal mucosal levels. Gastroenterology 102:2006-2014, 1992. 24. Mahida YR, Kurlak L, Gallagher A, Hawkey C J: High circulating levels of interleukin 6 in active Crohn's disease but not ulcerative colitis. Gut 32:1531-1534, 1991. 25. Mahida YR, Ceska M, Effenberger F, et al.: Enhanced synthesis of neutrophil-activating peptide-Ifmterleukin-8 in active ulcerative colitis. Clin Science 82:273 275, 1992. 26. Ligumsky M, Simon PL, Karmeli F, Rachmilewitz D: Role of interleukin 1 in inflammatory bowel disease-enhanced production during active disease. Gut 31:686-689, 1990. 27. Fiocchi C, Hilftker ML, Yotmgman KR, et al.: Interleukin 2 activity of human intestinal mucosal mononuclear ceUs. Decreased levels in inflammatory bowel disease. Gastroenterology 86:734--742, 1984. 28. Mullin GE, Harris ML, Bayless TM, James SP: Increased IL-2 mRNA in the intestinal
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mucosal lesions of Crolm's disease but not ulcerative colitis. Gasa'ocnterology 100:A602, 1991. l.,ieberman BY, Fiocchi C, Youngman KR, et al.: Interferon yproduction by human intestinal mueosal mononuclear cells. Decreased levels in inflammatory bowel disease. Dig Dis Sci 33:1528-1536, 1988. Fais S, Capobianchi MR, Pallone F, et al.: Spontaneous release of interferon y by intestinal lamina propria lymphoeytes in Crohn's disease. Kinetics of in vitro response to interferon yinducers. Gut 32:403407, 1991. Braeggcr CP, Nicholls S, Murch SH, et al.: Tumour necrosis factor alpha in stool as a marker of intestinal inflammation. Lancet 339:89-91, 1992. Isaacs KL, Sartor RB, Haskill S: Cytokine messenger RNA profdes in inflammatory bowel disease mucosa detected by polymerase chain reaction amplification. Gastroenterology 103:1587-1595, 1992. Mosmarm TR, Coffman RL: Thl and Th2 cells different patterns of lymphoklne secretion lead to different functional properties. Ann Rev Immunol 7:145-173, 1989.
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34. Blaser MJ, Miller RA, Laeher J, Singleton JW: Patients with active Crohn's disease have elevated serum antibodies to antigens of seven enteric bacterial pathogens. Gastroenterology 87:888-894, 1984. 35. Heinzel FP, Sadick MD, Holoday B J, et al.: Reciprocal expression of interferon gamma or interleukin 4 during the resolution or progression of murine leishmaniasis: evidence for expansion of distinct helper T cell subsets J Exp Meal 169:5972, 1989. 36. Parronchi P, Macchia D, Piccinini MP, et al.: Allergen- and bacterial antigen-specific T-cell clones established fxom atopic donors show a different prof'de of cytokine production. Proc Natl Acad Sci USA 88:4538-4542, 1991. 37. Dinarello CA, Wolf SM: The role of interleukin-1 in disease. N Engl JMed 328:106113, 1993. 38. Cominelli F, Nast CC, Duchini A, Lee M: Recombinant interleukin-1 receptor antagonist blocks the proinflammatory activity of endogenous interleukin-1 in rabbit immune colitis. Gastroenterology 103:65-71, 1992.
T h y r o t r o p i n Receptor A u t o a n t i b o d i e s i n T h y r o i d A u t o i m m u n e Disease: E p i t o p e s a n d Origin Leonard D. Kohn and Rudolf C. Kuppers
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HHS C a t e g o r i z a t i o n of CLIA Test S y s t e m s i n Laboratory Immunology Deidra B. Abbott and H.A. Homburger
© 1992Elsevier SciencePublishing Co., Inc.
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Information for Contributors
Vol. 12, No. I0/11, 1992
In general, the style adheres to the ASM S~le Manual for Journals and Books. As a backup, follow The Chicago Manual of Style.
Case Reports Reports documenting interesting case presentations are invited for possible publication in the Clinical Immunology Newsletter. Submitted case reports should be written concisely and should contain: a) a brief clinical history summarizing the symptoms and course of the illness: b) a description of immunologic and other laboratory tests performed; and c) the results of these laboratory tests as related to the clinical observation of patients and the outcome of the disease process and therapy, if appropriate.
References Authors are responsible for the accuracy' of references, which should include complete publication information. References should be listed in alphabetical order by the first author's last name, numbered consecutively, and cited in the text by these numbers. Some typical examples of references are listed below. Consult this issue for additional examples of Newsletter reference format. 1. Doone I"~'. Anderson N: Electron Microscopy in Diagnostic Virology. A Practical Guide and Atlas. New York, Cambridge University Press, 1087 2. McCulloch EA: Nomlal stem cells and the clonal hemopathies. In: Cronkite PE (ed): ltematopoietic Stem Cell Physiology, Vol. 184. Nev, York, Alan R. Liss. Inc.. pp. 21-38. 1988. 3. Forsgren A. Binder G. Callow F, el at.: Quinolones affect thymidine incorporation in the DNA of human Iymphocytes. Antimicrob Agents Chemother 29:506-508, 1986.
Letters Letters expressing opinion(s} or offering technical ideas and procedures will be considered for publication (subject to editing) pro',ided they are signed by all authors and do not exceed two typewritten (double-spaced) pages.
Questions and Answers Questions about immunologic techniques, laboratory procedures for immunology, and general problems in clinical inununology are welcome. They v, ill be answered by the editorial beard or by selecled individuals in the field.
Information about Meetings Announcements or reports of meetings and conferences of interest to clinical laboratory immunologists will also be considered for publication. Preparation and Submission of Material All material sulm~itted for publication in the Newsletter should be typed double-spaced (including references) on standard 8-1/2× 11 paper. "l'he original typescript and three copies should be sent to:
Alan L. Landay, Ph.D. Department of Immunology and Microbiology Rush-Presbyterian-St. Luke's Medical Center 1753 West Congress Parkway Chicago, IL 60612
Editors: A l a n L. L a n d a y , P h . D H e n r y A. H o m b u r g e r , M.D.
or
tIenry A. lIomburger, M.D. Dept. of Laboratory Medicine and Pathology Mayo Clinic 920 tlilton Building Rochester. MN 55905
Editorial Board N. Franklin Aclk~son, M.D., Baltimore, MD: Thomas Fleisher, M.D., Bethesda, MD; James Folds, Ph.D., Chapel 1Ii11,NC; A. Bennett Jenson, M.D., Washington DC; David Keren, M.D., Ann Arbor, MI; Robea Lahita, M.D., Ph.D., New York, NY; S. Breanndan Moore, M.D., Rochester, MN; Robert Nakamura, M.D., La Jolla, CA; Bruce Rabin, M.D., Ph.D., Pittsburgh, PA; Daniel Stites, M.D., San Francisco, CA; Russell 1f. Tomar, M.D., Madison, WI. Please address editorial correspondence to either Alan k Landay, Ph.D., Department of Immunology and Microbiology, Rush-Presbytorian-St. Luke's Medical Center, 1753 West Congress Parkway, Chicago, IL 60612, or tlenry Homburger, M.D., Depaflment of Laboratory Medicine and Pathology, Mayo Clinic, 920 Hilton Building, Rochester, MN 55905. General lnformatiotl Clinical Immunology Newsletter is published monthly by Elsevier Science Publishing Co., 655 Avenue of the Americas, New York, NY 10010. See inside front cover for subscription information. This newsletter has been registered with the Copyright Clearance Center, Inc. Consent is given for copying articles for personal or internal use, or for the personal or internal use of specific clients. Tiais consent is given on the condition that the copier pay through the Center the per-page fee stated in the code on each page for copying beyond that permitted by the U.S. Copyright Law. If no code appears on an aaicle, the author has not given broad consent to copy, and permission to copy must be obtained directly from the author. This consent does not extend to other kinds of copying, such as for general distribution, resale, advertising and promotional purposes, or for creating new collective works.
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© 1992 Elsevier Science Publishing Co., Inc.
Newsletter
healthy first-degree relatives of patients with coeliac disease. Lancet 338:13501353, 1991. 37. Marley NJE, McCarlney JC, Ciclitira PJ: HLA-DR, DP and DQ expression in the small intestine of patients with coeliac disease. Clin Exp Immuno170:386-393, 1987. 38. Niazi NM, Leigh R, Crowe P, Marsh MN: Morphometric analysis of small intestinal mucosa. 1. Methodology, epithelial volume compartments and enumeration of inter-epithelial space lymphocytes. Virchows Arch [Pathol Anaq 404:49-60, 1984. 39. Maki M, Holm K, Collin P, Savilahti E: Increase in ~ T cell receptor bearing lymphocytes in normal small bowel mucosa in latent coeliac disease. Gut 32:1412-1414, 1991. 40. Mantzaris G, Jewell DP: In vivo toxicity of a synthetic dodecapeptide frum A-gliadin in patients with coeliac disease. Scand J Gastro 26:392-398, 1991. 41. Marsh MN, Hinde J: Inflammatory component of celiac sprue mucosa 1. Mast cells, basophils and eosinophils. Gastroenterology 89:92-101, 1985. 42. Marsh MN, Bjamson I, Shaw/, Ellis A, et al.: Studies of intestinal lymphoid tissue. XIV-HLA status, mucosal morphology, permeability, and epithelial lymphocyte populations in fhst degree relatives of patients with coeliac disease. Gut 31:32-36, 1990. 43. Parker CM, Groh V, Band H, et al.: Evidence for extrathymic changes in the T cell repertoire. J Exp Med 171:1597-1612, 1990. 44. Rossipal E: The incidence of childhood coeliac disease in Austria: a study covering the
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period 1969-86. In: Kumar PJ, WalkerSmith JA, (eds): Coeliac Disease 100 Years. Leeds, Leeds University Press, pp 299302, 1990. 45. Savilahti E, Arat A, Verkasalo ML: Intestinal y6 receptor-bearing T lymphocytes in celiac disease and inflammatory bowel diseases in children. Constant increase in celiac disease. Pediatr Res 28:579-581,1990. 46. Scott H, Sollid L M, Fausa O: Expression of major histocompatability Class 11 subregion products by jejunal epithelium in patients with coeliac disease. Scand J Immunol 26:563-571, 1987, 47. Selby WS, Janossy G, Bofill M, Jewell DP: Lymphocyte subpopulations in the human small intestine. The findings in disease. Clin Exp Immuno152:219-228, 1983. 48. Shiner M: Ultrastructural changes suggestive of immune reactions in the jejunal mucosa of children following gluten challenge. Gut 14:1-12, 1973. 49. SoUid LM, Markussen G, Ek J, et al.: Evidence for a primary association of celiac disease to a particular HLA-DQ tx/~ heterodimer. J Exp Med 169:345350, 1989. 50. Spencer J, MacDonald "IF, Diss TC, et al.: Changes in intraepithelial lymphocyte subpopulations in coeliac disease and associated T cell lymphoma (malignant histiocytosis of the intestine). Gut 30:339344, 1989. 51. Spencer J, Isaacson PG, Diss TC, et al.: Expression of disulphide linked and non-disulfide linked forms of the T cell receptor or gamma/delta heterodimer in human intesti-
nal intraepithelial lymphocytes. Eur J hnmunol 19:1335-1338, 1989. 52. Spencer J, Isaacson JA, MacDonald TF, et al.: Gamma]delta T cells and the diagnosis of coeliac disease. Clin Exp Immunol 85:109-113, 1991. 53. Spurkland A, SoUid LM, Ronnmgen KS, et al.: Susceptibility to develop coeliac disease is primarily associated with HLA-DQ alleles. Hum Immunol 29:157-165, 1990. 54. Stevens FM, Egan-Mitchell B, Cryan E, et al.: Decreasing incidence of coeliac disease. Arch Dis Childhood 65:465-468, 1987. 55. Stokes PL, Ferguson R, Holmes GKT, et al.: Familial aspects of coeliac disease. Q J Med 180:567-582,. 1976. 56. Talley NJ, Kephart GM, McGovem TW, et al.: Deposition of eosinophil granule major basic protein in eosinophilic gastroenteritis and celiac disease. Gastroenterology 103:137-145, 1992. 57. Vecchi M, Crosti L, Berti E, et al.: Increased jejunal intraepithelial lymphocytes bearing T-ceil "?6receptor in dermatitis herpetiformis. Gastoenterology 102:14995O5, 1992. 58. Walker-Smith JA, Revised criteria for diagnosis of cocliac disease. Arch Dis Childhood 65:909-911, 1990. 59. Webster ADB, Slavin G, Shiner M, et al.: Coeliac disease with severe hypogammaglobinaemia.Gut 22:153-157, 1981. 60. Wright NA, Watson A, Morly A, et al.: Cell kinetics in flat (avillous) mucosa of the human small intestine. Gut 14:701710, 1973.
Inflammatory Bowel Disease: An Immunological Overview Claudio Fiocchi Department of Gastroenterology and Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio Etiopathogenesis Bowel Disease
of Inflammatory
WO clinical entities are grouped under the denomination of inflammatory bowel disease: Crohn's disease and ulcerative colitis. Although clearly dis-
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response to treatment. These features, common to many other chronic diseases, have generated a wide variety o f hypotheses to explain the cause o f inflammat~y bowel disease. Infectious, genetic, environmental, metabolic, vascular, and immuI
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nological theories have been proposed, each based on qualitatively and quantitatively variable evidence.: Today, the concept that most chronic inflammatory diseases have an important component of immune disregulation has gained general acceptance. This concept is based on substantial evidence showing that immunologic abnormalities are frequent in patients affected by recurrent, indolent, and unmanageable diseases accompanied by cellular infiltrates in various tissues and organs. This is certainly true for both forms of inflammatory bowel disease, where the small and large bowel carry a chronic overload of T cells, B cells, or macrophages even during periods of clinical remission. Whether these cellular infiltrates and the associated immunological abnormalities are primary or secondary may be impossible to determine as long as the exact cause of Crohn's disease or ulcerative colitis is out of reach. Given the status of present knowledge, an in-depth evaluation of the immune system in inflammatory bowel disease should take precedence over abstract considerations of whether immunological abnormalities are the cause or the result of the disease.
CLINICAL IMMUNOLOGY
ters in the peripheral circulation have contributed relatively tittle to inflammatory bowel disease immunopathogenesis. Detection of circulating immune complexes, shifts in peripheral blood T and B cell levels, and assessment of proliferative capacity of mononuclear cells to nonspecific stimuli, while suggesting the existence of systemic immune abnormalities, have provided only indirect information on possible defects relevant to gut inflammation. 3 This situation has changed substantially during the last decade, with the surge of
An in-depth evaluation of the immune system in inflammatory bowel disease should take precedent over abstract considerations of whether immunological abnormalites are the cause or the result of the disease.
Immunopathogenesis of Inflammatory Bowel Disease: Systemic vs. Mucosal Immunity Accepting the very solid premise that exploring immune abnormalities in inflammatory bowel disease is a top priority, the next question is how to carry out such investigation. Both Crohn's disease and ulcerarive colitis can display extraintestinal manifestations, such as arthritis, cholangitis, erythema nodosum, pyoderma gangrenosum, and uveitis/'mtis. These manifestations leave no doubt that systemic reactions are an integral part of inflammatory bowel disease, and could be interpreted as evidence that autoimmunity has a major role in pathogenesis. Common antigens shared by various affected organs have been identified, but whether they are the actual target of autoimmune aggression has yet to be proven. 2Regardless, systemic signs and symptoms are always secondary to gut involvement, where the inflammatory process starts and lingers. Based on this postulate, it is not surprising that earlier studies investigating immune parame-
mucosal immunity as a major new player within the arena of immunology. Praising the eminent importance of mucosal immunity is beyond the scope of this review, but two aspects deserve mentioning. The first is the quantitative predominance of the gastrointesrinal tract as the primary organ in immunity. The amount and the variety of cells in the gut lymphoid tissue is unmatched by any other organ, and the intestinal mucosa is by far the largest surface of the body in contact with the external environment, rendering it essential to immune homeostasis.* The second aspect is even more crucial, i.e., inflammatory bowel disease is fundamentally a disorder of the mucosal immune system, and whatever events are responsible for its cause and pathogenesis should he sought in loco. Fortunately, this is now possible through techniques that allow the isolation of cells from vari© 1992 Elsevier Science Publishing Co., Inc.
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ous bowel segments, and an impressive amount of data has been accumulated. 5
Immune Components of Inflammatory Bowel Disease: Cells and Cytokines In the preceding section the abundance and heterogeneity of immune cells in the intestinal mucosa, a phenomenon that occurs under physiological and pathological conditions, was inlroduced. In addition to immane cells, other elements are present, including a tight voluminous lamina propria rich in structural and nervous ceils, arteriovenous and lymphatic endothelial cells, and smooth muscle cells? Consequently, immune cells involved in triggering and perpetuating bowel inflammation do not do so alone, but in coordination with other cell types that they modulate and by which they are in turn influenced. This constant mutual interaction is mediated primarily through the secretion of soluble mediators or cytokines, creating an intricate system that calls into question thinking of inflammatory bowel disease strictly in terms of abnormal immune reactivity. There is reasonably good information on the types of immune cells involved in mucosal inflammarion, and the concept that these cells are in a high state of activation is widely accepted. Information on local nonimmune cells is considerably more limited. Finally, information on what are likely to be the most critical pathogenic elements of the Crohn's disease and ulcerative colitis, i.e., mucosal cytokines, is accumulating at an increasingly fast pace. Cellular Components Phenotypic analysis of T and B cells in the inflamed mucosa fails to reveal any significant shifts when compared to normal tissue.7a This is surprising, considering the chronicity of the cellular infdtrates and the fact that other chronic diseases like sarcoidosis and leprosy are characterized by definite abnormalities of helper/inducer (CD4+) or suppressor/cytotoxic (CD8+) lymphocytes. Analyses based purely on morphologic or phenotypic criteria are crude, because cells with identical external markers may mediate entirely diverse functions. For instance, some studies have shown that mucosal plasma cells display quite different profiles of cytoplasmic and secreted immunoglobins in both Crohn's I 0197-1859/92150.00 + 3.00
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disease and ulcerative colitis.9'~°It is unknown what induces such changes, whether they represent a nonspecific response to chronic inflammation, or are directly dependent on the cause of inflammatory bowel disease. Nevertheless, production of immunoglobulin subclasses is remarkably different between Crohn's disease and ulcerative colitis, indicating that dissimilar regulatory events control the local antibody response. When mucosal T lymphocytes are assessed for functional activity, neither help nor suppression is grossly abnormal. N Functional tests are performed in vitro utilizing artificial systems designed to explore a specific effect, and may or may not translate the actual performance of any given type in vivo. In addition, the original concept of abnormal helper and suppressor function is also fairly crude and narrow, and in modern terms function is viewed as an activity primarily related to the patten of cytokine secretion and response. Thus, failure to detect major abnormalities of T cell function by classical criteria by no means rules out the existence of intrinsic immune defects. A novel and attractive approach to the evaluation of mucosal T cells is related to the selective activation of discrete subsets based on their receptor repertoire) 2 T lymphocytes that appear indistinguishable by classical criteria are actually quite unique when their T cell receptor (TCR) ability to recognize antigens is examined. The importance of this phenomenon is that overutilization or deletion of certain variable (VI~) regions of the TCR may be induced by exposure to dominant antigens or superantigens, which may be responsible for autoimmunity or inflammation. Preliminary da!a indicate that abnormal expression of TCR repertoire is present among lamina propria mononuclear cells from Crohn's disease and ulcerative colitis patients? 3 Since the number of VI3 regions is quite large, a systematic screening needs to be performed to uncover all possible abnormalities of TCR usage and assess their significance. Information on other mucosal cell types is still fairly limited, largely because of their numerical scarcity. Morphological and histochemical characterization of macrophages has revealed an extraordinary
heterogeneity, with antigen-presenting, scavenger, or intermediate phenotypes.14 Whether any of these functions is altered and relates to inflammatory bowel disease pathogenesis has not been investigated to any significant degree, and even the commonly held notion that macrophages mediate nonspecific intestinal tissue damage is unproven. Other cells, such as eosinophils, mast cells, and basophils, have received even less attention. As for T cells, B cells, and macrophages, they are also in an enhanced state of activation, but their num-
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H o w close is immunological investigation from discovering the cause, explaining the mechanisms, and providing new treatmentsfor Crohn's disease or ulcerative colitis?
ber does not change much even during severe flare-ups. Thus, their potential for pathogenic role is mere speculation. Finally, polymorphonuclear neutrophils have been conspicuously overlooked, mostly due to the long-held, although erroneous notion, that they are not immune cells. This is rapidly changing for two reasons: first, recent studies on leukocyte-endothelial interaction have dramatically expanded knowledge of how neutrophils migrate into site of active inflammation;Is second, one of the basic functions of these cells is to secrete reactive oxygen metabolites, which are major mediators of localized tissue damage? 6 Until recently an overview of inflammatory bowel disease immunology would be largely complete at the end of the precedIlllllllI © 1992 E l s e v i e r Science P u b l i s h i n g Co., Inc.
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ing paragraph with a review of classical concepts of immune and inflammatory cells. This is no longer acceptable because of what was previously referred to as "immune-nonimmune interactions." This relatively new idea is "based on substantial evidence demonstrating that immune ceils communicate with a wide variety of other cells of ectodermal, mesodermal, and endodermal origin. Cell-to-cell communications occur in all organs of the body, but are especially pronounced in the intestinal mucosa because of the close physical proximity of multiple cellular elements in this unique microenvironment.6 Due to increased permeability to lumenal antigens, anatomical disruption, and changes in the composition and functions of mononuclear cells, cellular interactions are unquestionably altered during inflammation. Epithelial cells have been the first nonimmune cells to attract the attention of mucosal immunologists. They mediate antigen presentation differently from classical antigen-presenting cells (macrophages and dendritic cells) by activating preferentially CD8+ T cells,v A potential defect of this function has been reported in both Crohn's disease and ulcerative colitis, where a predominant stimulation of CIM+ T cells may lead to excessive helper function and predispose to inflammation.~s Also abnormal in the diseased mucosa are neuropeptiderich nerve ceils, which are strong regulators of mucosal mononuclear cell function. Alterations of various neuropeptides have been reported, and hypotheses have been advanced on how this could influence the function of local lymphoid cellsfl Additional cell types are coming under scrutiny for a possible functional relationship with immunocytes, and some cells previously considered as having a pure "structural" function, such as fibroblasts, are now viewed as potential targets for cytokines elaborated by local inflammatory cells) °-2~ Soluble Components Since the earliest investigations, unequivocal evidence implicating any particular immune dysfunction as the direct cause of Crohn's disease or ulcerative colitis has been difficult to obtain. Presently, the role of an immune cell tends to be viewed as a function of the soluble factors they secrete and are exposed to. Adopting this modern Ill
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concept, the most critical events responsible for inflammatory bowel disease may well be primarily dictated by the type and concentration of the various cytokines produced in the gut mucosa. This approach has spurred an unprecedented interest in the role of these mediators in Crohn's disease and ulcerative colitis. An enormous amount of dam has been generated in the last decade, and relevant information has been recently reviewed.22 Cytokine activities have been investigated both at the systemic and intestinal levels. It was initially hoped that measurements using serum samples would reflect events occurring in the gut, and perhaps provide markers of clinical activity, but this is questionable. The most widely investigated so far has been the soluble form of the intedeukin-2 0L-2) receptor, but it may not accurately mirror the degree of inflammation in all patients. 23Preliminary reports suggest that comparative levels of IL-6 or IL-8 can distinguish Crohn's disease from ulcerative colitis, but the reliability and reproducibility of these findings are unclear. ~,zs Contrary to peripheral cytokine concenlrations, those in the mucosa could be ideal indices of inflammation. There is general agreement that production of IL-1 is dramatically increased in inflamed intestine. ~ In contrast, IL-2 activity has been reported as decreased, while its mRNA may be increased. 27,'~IFNy has also been found both decreased and increased. 29'3°The activity of other cytokines with a predominant proinflammatory activity, such as IL-6 and IL-8, is predictably augmented, while mucosal levels of TNFct have been reported as low to very hight z2 If any consensus can be derived from these contradictory data, it is that no clear picture has emerged on the status of cytokines in inflammatory bowel disease, but further investigation is certainly warranted. Two major factors can explain this confusing situation. The first is that the methodology employed to assess cytokines has been as diverse as the cellular sources used to study them. Measurements of bioactivity, immunoreactivity, mRNA, and functional capacity do not translate the same information on the status of a specific mediator. The second is that the number of cytokines is constantly expanding, making it
difficult to explore all of them in reasonable depth and obtain defined patterns of expression. This situation is complicated by the interactive nature of cytokines, and in the so-called "cytokine network" each soluble mediator modulates and is in turn modulated by the surrounding cytokines. These difficulties may be resolved by the recent discovery that particular T lymphocytes produce restricted patterns of cytokines. Fundamental work by Mossmann and Coffman has shown that some T helper (Th) cell clones with defined secretory capacity display discrete immune functions. 33 As originally described, Thl cells produce IL-2, IFNy, and lymphotoxin, and mediate primarily delayed-type hypersensitivity, while Th2 cells secrete IL-4, IL-5, IL-6, and IL-10 and provide help for antibody production. Additional work generated evidence for undifferentiated (ThO) patterns, and it is quite possible that additional patterns (ThX) may exist, particularly in the case of a chronic response to a persistent antigen. Novel patterns of cytokine expression may be identified in the intestinal mucosa, and production of"characteristic" cytokines may be linked to the various stages of inflammatory bowel disease.
Impact of Immunology on the Etiology, Pathogenesis, and Therapy of Inflammatory Bowel Disease Hopefully the preceding paragraphs have convinced the reader of how fundamental immune phenomena are in inflammatory bowel disease. Taking this assumption to be true, how close is immunological investigation from discovering the cause, explaining the mechanisms, and providing new treatments for Crohn's disease or ulcerative colitis? As far as etiology, even the most comprehensive studies are unlikely to unveil the specific cause. The immune system is said to exhibit exquisite specificity, and this is true in the context of a response to a defined antigen. In contrast, the cells stimulated by the antigen may be many, the signals they produce multiple, and the action generated nonspecific. Additionally, a myriad of antigens may elicit similar responses, indistinguishable from one another, all occurring independently of the original trigger An example of this common situation is provided by studies of se© 1992 Elsevier Science Publishing Co., Inc.
rum levels of antibodies to bacterial antigens. They may be abnormally high in Crohn's disease and ulcerative colitis patients as compared to normal individuals, but still reflect exposure to the same microorganism. ~ Current trends are centered on the sophisticated identification of the various portions of the TCR that "see" the antigen, and knowing which V[3 region sees it, inferences can be made about its nature. Even assuming that a complete mapping of the TCR with inflammatory bowel disease cells is done, chances that a single or very few VIi regions are consistently utilized in all patients are probably very few considering that specific epitopes from a putative etiological agent may be obscured by an overwhelming amount of dietary and enteric antigens. More promising is immunology's potential for improving the understanding of pathogenic mechanisms. For a long time it has been impossible to immunologically differentiate Crohn's disease from ulcerative colitis. This was due to the limited power of experiments based on the phenotypic characterization of inflammatory cells, and utilization of fairly crude functional assays. Major advances in the mechanisms associated with several chronic inflammatory diseases have occurred only after establishing the fundamental regulatory role of cytokines, and particularly after the discovery of Thl and Th2 cells. Initially, Thl or Th2 cytokine patterns have been detected in animal models and correlated with different phases of the disease. 35Subsequently, similar patterns were identified in humans, Th2 in allergic, and Th I-like in delayed-type sensitivity reactions. 36As alluded before, novel and perhaps characteristic spectra of mucosal cytokine secretion may be expressed during the various stages of mucosal inflammation. However, detecting "inflammatory bowel disease-specific" ThX patterns will become reality only by prospectively cloning gut T cells in patients who go through the sequential stages of their illness. This seemingly overwhelming task may yield crucial clues if fluctuations of cytokine activities show a close correlation with clinical activity. The greatest rewards can be expected when cytokine studies are applied to revo0197-1859/92/$0.00 + 3.00
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lutionize therapy. A thorough understanding of the activity of T cell-derived low-level immunoregulatory cytokines (IL2, IL-4, IL-5, IL-10, etc.) may generate clues to unlocking the intimate mechanisms of disease. However, correcting abnormalities of these cytokines is unlikely to result in cure as long as the cells responsible for their production are not turned off, and this escapes present capabilities. In contrast, if we learn how to modulate pro-inflammatory cytokines OL- 1, IL-6, IL-8, etc.), produced in abundance by cells of the monocyte/macrophage lineage, our ability to downmgulate or even block their tissue damaging effects could improve dramatically. Production of these cytokines is exaggerated at sites of injury. Stopping their secretion by inhibiting macrophages is difficult, and even the most potent drugs have limited effect because of redundant cell circuitry. However, rather than directing therapy at the producing cells, pro-inflammatory cytokines themselves could be targeted for blockade, and today this can be done with tremendous precision and efficacy. This could be achieved by using soluble receptors or other natural antagonists spontaneously produced by the body as part of the normal mechanisms that keep agonistic and antagonistic activities in balance to preserve homeostasis. An excellent example of this critical balance is IL-1 and its natural receptor antagonist (iL-lra). Both molecules compete for the same receptor, but while doing so ILl evokes an inflammatory response, IL-lra does it without triggering such response. 37 Under physiological circumstances, the body produces enough IL-lra to prevent an excessive inflammatory response by ILl. In a disease like Crohn's disease or ulcerative colitis, the amount of ILl produced in the gut may far exceed the body's ability to produce IL- lra, and inflammation may ensue. Assuming that a pathological response is caused by an excessive production of a single cytokine, supplying enough antagonist should reestablish the lost balance and eliminate inflammation. This situation is far less hypothetical than it appears to be. Animal m o d d s of experimental colitis have objectively demonstrated that administration of IL-lra prevents or improves gut inflammation, ss Current experimental ffials based on this IIlllll
principle are now under way in subjects with ulcerative colitis. In practice, Crohn's disease or ulcerative colitis are caused by abnormalities of multiple cytokines. No matter how numerous, ultimately all the relevant antagonists will be identified, cloned and produced in unlimited quantities. Once this is achieved, they can be administered to block the mediators responsible for mucosal inflammation. Once successful, this tailored approach will represent a major breakthrough in therapy because it allows for both total specificity and the use of safe recombinant products instead of nonselective, toxic immunosuppressants. CIN References
1. Fiocchi C: Pathogenesis and clinical implications: where do we stand, where do we go? In: Goebell H, Ewe K, Malchow H, Koelbel C (ed): Inflammatory Bowel Disease. Progress in Basic Research and Clinical implications. Lancaster. Kluwer Academic Publishers, pp 237-254, 1991. 2. Das KM, Vecchi M, Sakamaki S: A shared and unique epitope(s) on human colon, skin, and biliary epithelium detected by a monoclonal antibody. Gastroenterology 98:464--469, 1990. 3. Elson CO: The immunology of inflammatory bowel disease. In: Kirsner JB, Shorter RG (ed): Inflammatory Bowel Disease. Philadelphia: Lea & Febiger, 97-164, 1988. 4. Brandtzaeg P, Halstensen TS, Kett K, et al.: Immunobiology and immunopathology of the human gut mucosa: humoral immunity and intraepithelial lymphocytes. Gastroenterology 97:1562-1584, 1989. 5. MacDermott RP, Elson CO (ed): Mucosal Immunology I. Basic Principles. Philadelphia: W.B. Saunders Company, 1991. 6. Bienenstock J: Cellular communications networks: implications for our understanding of gastrointestinal physiology. Ann NY Acad Sci 664:1--9, 1992. 7. Brandtzaeg P, Halsensten TS, Kett K, Rognum TO: Local immunopathology in inflammatory bowel disease. In: Jamerot G (ed): Inflammatory Bowel Disease. New York: Raven Press, pp 21-35, 1987. 8. James SP, Fiocchi C, Graeff AS, Strober W: Phenotypic analysis of lamina propria lymphocytes. Predominance of helper-inducer and cytolytic T-cell phenotyp¢ and deficiency of suppressor-inducer phenotype in Crohn's disease and control patients. G astroenterology 91:1483-1489, 1986.
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9. MacDermott RP, Nash GS, Bertovich M J, et al.: Alterations oflgM, IgG, and IgA synthesis and secretion by peripheral bl[xxl and intestinal mononuclear ceils from patients with ulcerative colitis and Crohn's disease. Gastroenterology 81:844 852~ 1981. 10. Kett K, Brandtzaeg P: Local IgA subclass alteration in ulcerative colitis and Crohn's disease of the colon. Gut 28:101.3--1021, 1987. 11. Janaes SP, Fiocchi C, Graeff AS, Strober W: Immunoregulation of lamina propria T ceils in Crohn's disease. Gastroenterology 88:1143-1150, 1985. 12. Marguerie C, Lunardi C, So A: PCR-based analysis of the TCR repertoire in human autoimmune diseases. Immunol Today 13:336-338, 1992. 13. Duchmarm R, Strober W, Fiocchi C, James SP: TCR V[~2 gene expression is selective in control but not in IBD lamina propria lymphocytes. Gastroenterology 102:A617, 1992. 14. Mahida Y: Macrophage function in inflammatory bowel disease. Eur J Gastroenterol Hepatol 2:251-255, 1990. 15. Zimmerman GA, Prescott SM, Mcintyre TM: Endothelial ceil interactions with granulocytes: tethering and signal molecules. Immunol Today 13:93--99, 1992. 16. Grisham MB, Yamada T: Neutrophils, nitrogen oxides, and inflammatory bowel disease. Ann NY Acad Sci 664:103-115, 1992. 17. Mayer L, Shlien R: Evidence for function of Ia molecules on gut epithelial cells in man. J Exp Med 166:1471-1483, 1987. 18. Mayer L, Eisenhardt D: Lack of induction of suppressor T cells by intestinal epithelial ceils from patients with inflammatory bowel disease. J Clin Invest 86:1255-1260, 1990. 19. Kubota Y, Petras RE, Ottaway CA, et al.: Colonic vasoactive intestinal peptide nerves in inflammatory bowel disease. A digitized morphometric immunohistochemical study. Gastroenterology 102:1242-1251, 1992. 20. StaUmach A, Schuppan D, Riese HH, et al.: Increased collagen type HI synthesis by fibroblasts isolated from strictures of patients with Crohn's disease. Gastroenterology 102:1920-1929, 1992. 21. Strong SA, West GA, Klein JS, et al.: Inflammatory eytokines stimulate proliferation of intestinal mucosa mesenchymal cells. Gastroenterology 102:A701, 1992. 22. Fiocchi C; Cytokines. In: MacDermott RP, Stenson W (ed): Inflammatory Bowel Disease. New York: Elsevier Publishing CornIIII
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pany, pp 137-162, 1992. 23. Matsuura T, West GA, Klein JS, et al.: Soluble interleukin 2, CD8 and CIM receptors in inflammatory bowel disease. A comparative study of peripheral blood and intestinal mucosal levels. Gastroenterology 102:2006-2014, 1992. 24. Mahida YR, Kurlak L, Gallagher A, Hawkey C J: High circulating levels of interleukin 6 in active Crohn's disease but not ulcerative colitis. Gut 32:1531-1534, 1991. 25. Mahida YR, Ceska M, Effenberger F, et al.: Enhanced synthesis of neutrophil-activating peptide-Ifmterleukin-8 in active ulcerative colitis. Clin Science 82:273 275, 1992. 26. Ligumsky M, Simon PL, Karmeli F, Rachmilewitz D: Role of interleukin 1 in inflammatory bowel disease-enhanced production during active disease. Gut 31:686-689, 1990. 27. Fiocchi C, Hilftker ML, Yotmgman KR, et al.: Interleukin 2 activity of human intestinal mucosal mononuclear ceUs. Decreased levels in inflammatory bowel disease. Gastroenterology 86:734--742, 1984. 28. Mullin GE, Harris ML, Bayless TM, James SP: Increased IL-2 mRNA in the intestinal
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34. Blaser MJ, Miller RA, Laeher J, Singleton JW: Patients with active Crohn's disease have elevated serum antibodies to antigens of seven enteric bacterial pathogens. Gastroenterology 87:888-894, 1984. 35. Heinzel FP, Sadick MD, Holoday B J, et al.: Reciprocal expression of interferon gamma or interleukin 4 during the resolution or progression of murine leishmaniasis: evidence for expansion of distinct helper T cell subsets J Exp Meal 169:5972, 1989. 36. Parronchi P, Macchia D, Piccinini MP, et al.: Allergen- and bacterial antigen-specific T-cell clones established fxom atopic donors show a different prof'de of cytokine production. Proc Natl Acad Sci USA 88:4538-4542, 1991. 37. Dinarello CA, Wolf SM: The role of interleukin-1 in disease. N Engl JMed 328:106113, 1993. 38. Cominelli F, Nast CC, Duchini A, Lee M: Recombinant interleukin-1 receptor antagonist blocks the proinflammatory activity of endogenous interleukin-1 in rabbit immune colitis. Gastroenterology 103:65-71, 1992.
T h y r o t r o p i n Receptor A u t o a n t i b o d i e s i n T h y r o i d A u t o i m m u n e Disease: E p i t o p e s a n d Origin Leonard D. Kohn and Rudolf C. Kuppers
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HHS C a t e g o r i z a t i o n of CLIA Test S y s t e m s i n Laboratory Immunology Deidra B. Abbott and H.A. Homburger
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168 C L I N I C A L I M M U N O L O G Y Newsletter
Information for Contributors
Vol. 12, No. I0/11, 1992
In general, the style adheres to the ASM S~le Manual for Journals and Books. As a backup, follow The Chicago Manual of Style.
Case Reports Reports documenting interesting case presentations are invited for possible publication in the Clinical Immunology Newsletter. Submitted case reports should be written concisely and should contain: a) a brief clinical history summarizing the symptoms and course of the illness: b) a description of immunologic and other laboratory tests performed; and c) the results of these laboratory tests as related to the clinical observation of patients and the outcome of the disease process and therapy, if appropriate.
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