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SUPPRESSOR-CELL ACTIVITY IN COELIAC DISEASE INDUCED BY ALPHA-GLIADIN, A DIETARY ANTIGEN
1305
cellular sensitisation to wheat protein has been described in coeliac disease,4-6 it is not known whether these circulating antigens can stimulate specific immune regulatory cell populations. We have investigated the ability of the wheat protein antigen, alpha-gliadin, a 60 000 molecular weight fraction of gluten, to activate suppressorcell mechanisms.
Although
Preliminary
Communication
SUPPRESSOR-CELL ACTIVITY IN COELIAC DISEASE INDUCED BY ALPHA-GLIADIN, A DIETARY ANTIGEN CLIONA O’FARRELLY C. F. FEIGHERY
C. A. WHELAN D. G. WEIR
SUBJECTS AND METHODS
Subjects
Departments of Immunology and Clinical Medicine, Trinity College and St James’s Hospital, Dublin, Ireland
Summary
The wheat
protein antigen alpha-gliadin,
a
fraction derived from gluten of molecular
weight 60 000,
activated suppressor cells from
patients with
coeliac disease but not from normal subjects or patients with Crohn’s disease. Two other dietary antigens, casein and betalactoglobulin, failed to produce suppressor-cell activation. Since this phenomenon appears to be specific to coeliac disease, it may be of pathogenetic significance.
We studied twenty-eight patients with adult coeliac disease, eighteen female, ten male, aged 17-64 years (mean 27). Ten patients were on gluten-free diets and eighteen were untreated when tested. All had the characteristic lesion of coeliac diesease shown by jejunal biopsy, and the treated patients had a clinical response to the diet of least 6 months’ duration. The untreated group subsequently diet. In twenty patients a repeat biopsy was done after treatment; all samples showed histological improvement in response to treatment. The controls were twentytwo normal subjects aged 20-55 years (mean 25) and six patients with ileal Crohn’s disease (four female, two male), mean age 26 years. at
responded to a gluten-free
INTRODUCTION
COELIAC disease is a malabsorptive disorder characterised by a mucosal lesion which reverts to normal on removal of dietary gluten.’ Although the mechanism by which the wheat protein damages the intestinal mucosa is not known, it has been proposed that components of the immune response have a primary pathogenetic role.2 Humoral and cellular sensitisation to wheat protein antigens both in the jejunal mucosa and in the peripheral blood of coeliac patients has been reported.3-8 Furthermore, mucosal changes similar to those in coeliac disease have been described in graft-versushost disease in manand small-intestinal allograft rejection in
mice° .
.
both T-cell-mediated disorders. The coeliac lesion may also be mediated by T lymphocytes. Further evidence for an immunopathogenetic process includes the high frequency in patients with coeliac disease of the haplotype HLA B8, D3, Dr3,]] gene products of the major histocompatibility complex known to be associated with autoimmune disorders. ]2 Abnormalities in regulation of the immune response must be considered when it is proposed that immune mechanisms are involved in disease pathogenesis. Some studies have suggested that immune responsiveness is depressed in coeliac disease’3-15 and we have shown that mitogen-induced suppression is enhanced in coeliac patients.]6 Since the histological lesion in coeliac disease is caused by wheat protein antigens, it is possible that immunological responsiveness in these patients is controlled by immunoregulatory cells sensitised to these antigens. K. W. NEWELL AND OTHERS: 16.
Simpson
BH. An epidemiological study of carcinoma of the small intestine in New Zealand sheep. NZ Vet J 1972; 20: 91-97. 17 Carter AH, Cox EH. Sheep breeds in New Zealand. sheep production. Vol 1 In: Wickham GA, McDonald MF eds. Wellington: New Zealand Institute of Agricultural Science, 1982. 18 Owen JB. Sheep production. London: Baillière Tindall, 1976. 19 Sheep and beef farm survey 1977/8. Wellington: New Zealand Meat and Wool Board’s Economic Service, publication 1816, 1979. 20. Webster WM. Neoplasia in food animals with special reference to high incidence in sheep. NZ Vet J 1966; 14: 203-14. 21. Webster WM. A further survey of neoplasms of abattoir sheep. NZ Vet J 1967; 15: 51-54. 22. Wassom JS, Huff JE, Loprieno N. A review of the genetic toxicology of chlorinated dibenzo-p-dioxins. Mutation Res 1977/8; 47: 141-60. 23. IARC. Evaluation of the carcinogenic risk of chemicals to man. Monograph 15. Lyon: International Agency for Research on Cancer, 1977. 24. Grant WF. The genotoxic effects of 2,4,5-T. Mutation Res 1979; 65: 83-119.
Methods
Mf!M.—Alpha-gliadin was prepared as described previously. 17 Casein and beta-lactoglobulin were obtained commercially. Solutions of the antigens were made up in Hank’s balanced salt solution on the day of use. Lymphocyte transformation. -Peripheral-blood mononuclear cells prepared by density-gradient centrifugation were suspended in RPMI 1640 medium containing 10% fetal calf serum and 50 J.lg/ml gentamicin, at a concentration of 1 x 106 cells/ml. Samples of 200 1 were placed in the wells of microculture plates, covered, and incubated at 37°C with 5% CO2 and 95% humidity for 96 h, with or without alpha-gliadin at final concentrations of 1,2, and 10 J.lg/ml. Each culture was carried out in triplicate. Generation of suppression. -Suppressor-cell activity was demonstrated by a modification of our previous technique.]8 Samples of 4x 106 peripheral-blood mononuclear cells were precultured in RPMI 1640 medium at a concentration of 2 x 106 cells/ml. Antigen (alpha-gliadin, casein, or beta-lactoglobulin) at a concentration of 2 g/ml was added to the test cells and no stimulant was added to control cells. After 24 h incubation, the cells were washed twice in Hank’s balanced salt solution, resuspended in RPMI 1640 medium containing 50 g/ml mitomycin C, and incubated for 1 h. They were then washed three times with Hank’s balanced salt solution. The concentration of each sample of cells was adjusted to 1 x 106/ml and they were dispensed in 100 <1 volumes into the wells of microculture plates. Equal volumes of autologous peripheral-blood mononuclear cells, also at a concentration of 1 x 106 cells/ml, which had been maintained in RPMI 1640 medium at 37°C for 24 h, were added to the mitomycin-C-treated cells. The cells were co-cultured with or without concanavalin A (1, 2, or 5 g/ml) for 72 h. Each test was carried out in triplicate. REFERENCES—continued 25. Pesticides Board. A
guide
to agricultural chemicals. Wellington: New Zealand Fisheries, 1979 food: 1979 evaluations. Rome: Food and Agriculture World Health Organisation, Organisation, 1980. 27. Smith AH, Fisher DO, Giles HJ, Pearce NE. The New Zealand soft tissue sarcoma case-control study: Interview findings concerning phenoxyacetic acid exposure. Chemosphere 1983; 12: 561-71. 28. Johnstone AC, Alley MR, Jolly RD. Small intestinal carcinoma in cattle. NZ Vet J
Ministry of Agriculture
26. FAO Pesticide residues
and
in
1983; 31: 147-49. 29. Ross AD. Small intestinal carcinoma in cattle NZ Vet J1984; 32: 98-99. 30. Head KW. Tumours of the lower alimentary tract. Bull WHO 1976; 53: 167-86.
31. Oliver MF,
32.
Heady JA, Morris JN, Cooper J. WHO Cooperative trial on primary prevention of ischaemic heart disease using clofibrate to lower serum cholesterol: Mortality follow up. Lancet 1980; ii: 379-82. Hardell L. The relation of soft tissue sarcoma, malignant lymphoma and colon cancer to phenoxy acids, chlorophenols, and other agents. Scand J Work Environment Health 1981; 7: 119-30.
1306
Peripheral-blood mononuclear cells from treated and untreated coeliac patients precultured with 2 g/ml alphagliadin suppressed the concanavalin-A-induced proliferative response of autologous peripheral-blood mononuclear cells (see figure). Preculturing cells from normal subjects or from patients with Crohn’s disease with the wheat protein had no significant effect on the mitogenic response of autologous cells. The differences between both groups of coeliac patients and the control groups were significant at concanavalin A With 5 /-lg/ml concentrations of 1 and 2 g/ml (p<0’01). concanavalin A there was a significant difference between untreated coeliac patients and the control groups (p<0’01). Neither casein nor beta-lactoglobulin had any significant effect on cells from normal subjects, Crohn’s disease patients, or coeliac patients, treated or untreated (see table).
)jg Con
DISCUSSION
o/ml
Generation of suppression by alpha-gliadin of mitogenic response in untreated coeliac patients (A), treated coeliac patients (A), normal subjects ( 0 ), and Crohn’s disease patients (.). Bars indicate mean±SEM. Con A=concanavalin A.
Termination
of cultures.-18 h before harvesting 0.3 MCi tritiated thymidine (2 -0 mCi/ml) was added to each well. At the end of the culture period, the cells were washed onto glass-fibre discs by means of an automatic cell harvester. The discs were suspended in a scintillation cocktail and the amount of cell-incorporated tritiated thymidine was measured in a scintillation counter. Calculation of percentage suppression.-The percentage enhancement or suppression by antigen-activated cells of the mitogenic response of autologous peripheral-blood mononuclear cells
was
calculated;
F(cm, - c,) . 1- r
x
-
(CM - C)J
ioo 100 .
where CMA is the counts per min (cpm) of cells precultured in the presence of antigen, and added to autologous cells in the presence of convanavalin A; CA is the cpm of cells precultured in the presence of antigen and added to autologous cells in the absence of concanavalin A; CM is the cpm of cells precultured in the absence of antigen and added to autologous cells in the presence ofconcanavalin A; and C is the cpm of cells precultured in the absence of antigen and added to autologous cells in the absence of concanavalin A. Statistics.-The data were analysed by one-way analysis of variance then by between-group mean t tests. RESULTS
Alpha-gliadin at 2 g/ml stimulated peripheral-blood mononuclear cells from five untreated coeliac patients (mean±SEM 1238±313 cpm) but had no effect on cells from five normal subjects (mean±SEM 301 ±66 cpm; p<0 01). At other concentrations of alpha-gliadin (1 and 10 g/ml) there was a proliferative response in some patients but the mean value was not significantly different from that of the control group. EFFECT OF CASEIN AND BETA-LACTOGLOBULIN ACTIVATED CELLS ON RESPONSE OF AUTOLOGOUS CELLS TO ,
CONCANAVALIN A I
2
tg/ml
The plant mitogen concanavalin A stimulates suppressor cells to secrete inhibitory factors in vitro]9 and this effect is used to measure suppressor function.2o,2] In this study instead of concanavalin A different food antigens were added to peripheral-blood mononuclear cell cultures. Alphagliadin-activated cells from treated and untreated coeliac patients suppressed the response of autologous cells to mitogenic stimulation. In contrast, alpha-gliadin had no significant regulatory effect on cells from normal subjects or patients with Crohn’s disease and control dietary antigens, casein and beta-lactoglobulin, had no such effect on cells from any subjects. The possible association of the HLA B8 and Dr3 antigens with this suppressor system is under
investigation. Since alpha-gliadin-activated immunosuppression is specific for both antigen and disease, a previously sensitised immunoregulatory cell must be intrinsic to this system. Cells of the T-cell lineage are prime candidates for this role.22 Both BCG23 and lepromin24 induced T-suppressor-cell activation in subjects sensitised to these antigens. However, B-cell products, including immunoglobulin molecules, also regulated gastrointestinal immune responses in murine
experiments.25 With the introduction of antigen to the immune system, regulatory cell mechanisms are activated that together decide the nature and intensity of the response. In animals, antigen presented by the enteric route causes stimulation of mucosal immunoregulatory cells,25,26 which commonly results in a local immune response and systemic unresponsiveness .2’ In some circumstances, a systemic immune response occurs. Since food-derived antigens enter the circulation after ingestion,27 regulatory cells controlling the systemic immune response to these antigens are probably stimulated as part of the normal physiological process. Because of the small antigenic load in normal circumstances, food-antigen-specific regulatory cells may remain undetected by existing assay systems. In coeliac disease, a specific hypersensitivity reaction to the highly antigenic wheat gliadins28 is observed ;6,29 it may result in stimulation of systemic regulatory cell populations of sufficient intensity to allow their detection. The increased bowel porosity3° which accompanies coeliac disease may allow food antigens to enter the circulation with greater freedom and thereby could serve to amplify this effect. Although our study provides evidence of the presence of alpha-gliadin-activated suppressor cells, it is likely that cells with other regulatory functions, not sought in this assay,
are
also stimulated.
1307
Evidence of specific cellularb’31 and humoral32 sensitisation has been recorded in coeliac disease. Our lymphocyte transformation to is further evidence of cellular hypersensitivity alpha-gliadin to this antigen. It can be conjectured that in the face of
to
Reviews of Books
alpha-gliadin
demonstration of in-vitro
immunological reactivity physiological immunosuppression is required to control the immune response to wheat protein antigens. In coeliac disease, immunosuppression could be mediated by alpha-gliadinactivated suppressor cells. It has been suggested that the primary stance of the immune system is suppression to guard against excessive immune reactivity.33 This situation
The
pronounced
be exaggerated in coeliac disease. ]6 Increased immunosuppression could explain such features of coeliac disease as skin anergy,15 lymphoreticular dysfunction,34 depressed mitogen responsivenessI5,]6 and a predisposition to gastrointestinal malignant disease. 35 Enhanced immunosuppression in the mucosa of the small intestine may also interfere with normal antigen handling which in turn could contribute to the disease pathogenesis. Organ culture studies3,36,37 and the evaluation of smallintestinal cellular infiltrate after in-vivo challenge with gluten38 indicate that lymphocytes are intimately involved in the development of the histological lesion. The infiltrating intra-epithelial lymphocytes show the OKT8 phenotype.38 may
Since both cytotoxic and suppressor T lymphocytes express this antigen,39 alpha gliadin may be able to activate not only lymphocytes with suppressor function but also those with
cytotoxic capabilities. -
.
We thank the Medical Research Council of Ireland and the Nutrition Fund of the Provost’s Appeal, Trinity College, Dublin, for financial support during this project.
Correspondence should be addressed to D. G. W., Department of Clinical Medicine, Medical School Building, St James’s Hospital, James’s Street, Dublin 8, Ireland.
215l ed. Edited by A. M. Harvey, R. J. Johns, V. A. McKusick, A. A. Owens, and R. S. Ross, Johns Hopkins University School of Medicine, Baltimore. Norwalk, Connecticut: Appleton-Century-Crofts. Hemel Hempstead: Prentice/Hall. 1984. Pp 1596. jE47.05.
THis 21st edition of this well-known textbook of medicine, which Osler first wrote in 1892, has been written almost entirely by present staff of the Johns Hopkins Hospital, Baltimore, as it has been since the 17th edition. It is a thorough revision with many new contributors, new illustrations, and new tables. The objective was to present medicine through clinical problems rather than through an encyclopaedic text, and this is the key to its format. The book is packed with information, and it can appeal to the more advanced student, the young doctor, or the experienced clinician. The opening section presents a thoughtful analysis of the methods of clinical medicine; it puts emphasis on the hypotheticodeductive approach to history-taking which we all employ whether we realise it or not. This approach is not extended into physical examination, where the emphasis is on systematic thoroughness, to the extent that we are recommended to examine the external auditory meati as part of the initial inspection of the patient. Some recommendations seem at first to be lacking-for example, in the section on measuring blood pressure, palpation of the pulse is not described as a precaution against missing the auscultatory gap, an omission later corrected in the chapter on hypertension. The last three sections are on psychiatry in medicine, medical emergencies in critical care, and special topics in medicine, which includes special aspects of the care of the elderly. In between are sections based C. O’FARRELLY AND OTHERS:
1185-91 20. Shou L, Schwartz 21.
3 4. 5
Ferguson A, McClure JP, MacDonald TT, Holden RJ. Cell-mediated immunity to gliadin within the small-intestinal mucosa in coeliac disease. Lancet 1975; i: 895-97. Holmes GKT, Asquith P, Cooke WT. Cell-mediated immunity to gluten fraction III in adult coeliac disease. Clin Exp Immunol 1976; 24: 259-65. Sikora K, Anand BS, Truelove SC, Ciclitira PJ, Offord RE. Stimulation of lymphocytes from patients with coeliac disease by a subfraction of gluten. Lancet 1976; ii: 389-91.
In-vitro immunological assay for diagnosis of coeliac disease. Lancet 1978; i: 876. 7. Falchuk ZM, Strober W. Gluten-sensitive enteropathy: synthesis of anti-gliadin antibody in vitro. Gut 1974; 15: 947-52. 8 Ferguson A, Carswell F. Precipitins to dietary proteins in serum and upper intestinal secretions of coeliac children. Br Med J 1972; i: 75-77. 9. Lerner KG, Kao GF, Storb R, et al. Histopathology of graft vs host reaction (GvHR) in human recipients of marrow from HL-A matched sibling donors. Transplant Proc 1974; 6: 367-71. 10 Ferguson A, Parrott DMV. Histopathology and time course of rejection of allografts of mouse small intestine. Transplantion 1973; 15: 546-54 1. Keuning JJ, Pena AS, van Leeuwen A, van HoofJP, van Rood JJ. HLA-Dw3 associated with coeliac disease. Lancet 1976; i: 506-07. 12. Svejgaard A, Platz P, Ryder LP. HLA and disease 1982- a survey. Immunol Rev 1983; 70: 193-218. 13. MacLaurin BP, Cooke WT, Ling NR Impaired lymphocyte reactivity against tumour cells in patients with coeliac disease. Gut 1971; 12: 794-800. 14. Baker PG, Verrier-Jones J, Peacock DB, Read AE. The immune response to &phgr;x 174 in man. III Evidence for an association between hyposplenism and immunodeficiency in patients with coeliac disease. Gut 1975; 16: 538-42. 15 Scott BB, Losowsky MS. Depressed cell-mediated immunity in coeliac disease. Gut 1976; 17: 900-05. 16 O’Farrelly C, McKeever U, Feighery G, Weir DG. Increased concanavalin A induced suppression in treated and untreated coeliac disease. Gut 1984; 25: 644-48. 17. Kendall MJ, Cox PS, Schneider R, Hawkins CF. Gluten subfractions in coeliac disease. Lancet 1972; ii: 1065-67. 18 Feighery C, Whelan CA, Weir DG, Greally JF. In vitro studies of suppressor cell function in human peripheral blood mononuclear cells. Clin Exp Immunol 1978; 32: 459-65. 6.
Haeney MR, Asquith P.
REFERENCES—continued
19. Greene WC, Fleisher TA, Waldmann TA. Soluble suppressor supernatants elaborated by concanavalin A-activated human mononuclear cells. J Immunol 1981; 126:
REFERENCES 1. Falchuk ZM. Update on gluten-sensitive enteropathy. Am J Med 1979; 67: 1085-96. 2 Housley J, Asquith P, Cook WT. Immune response to gluten in adult coeliac disease. Br Med J 1969; ii: 159-61.
Principles and Practice of Medicine
22. 23.
S, Good RA. Suppressor cell activity after concanavalin A treatment of lymphocytes from normal donors. J Exp Med 1976; 143: 1100-10. Dwyer JM, Johnson C. The use of concanavalin A to study the immunoregulation of human T cells. Clin Exp Immunol 1981; 46: 237-49. Benacerraf B. Suppressor T cells and suppressor factor. Hosp Pract 1978; 13: 65-75. Mustafa AS, Godal T. In vitro induction of human suppressor T cells by mycobacterial antigens. BCG activated OKT4 + cells mediate suppression of antigen induced T cell proliferation. Clin Exp Immunol 1983, 52: 29-37.
24. Mehra V, Mason LH, Rothman W, Remherz E, Schlossman SF, Bloom BR. Delineation of a human T cell subset responsible for lepromin-induced suppression in leprosy patients J Immunol 1980; 125: 1183-88. 25. Strober W, Richman L, Elson C. The regulation of gastrointestinal immune responses. Immunol Today 1981; 2: 156-62. 26. Kagnoff MF. Effects of antigen-feeding on intestinal and systemic immune responses. Cell Immunol 1978; 40: 186-203. 27. Paganelli R, Levinsky RJ, BrostoffJ, Wraith DG. Immune complexes containing food proteins in normal and atopic subjects after oral challenge and effect of sodium cromoglycate on antigen absorption. Lancet 1979; i. 1270-72. 28. Kieffer M, Frazier PF, Daniels WWR, Coombs RRA Wheat gliadin fractions and other cereal antigens reactive with antibodies in the sera of coeliac patients. Clin Exp Immunol 1982; 50: 651-60. 29. O’Farrelly C, Hekkens WTJM, Feighery C, Weir DG. The specificity of wheat protein reactivity on coeliac disease. Scand J Gastroenterol 1983; 18: 603-07. 30 Cobden I, Rothwell J, Axon ATR. Intestinal permeability and screening tests for coeliac disease. Gut 1980; 21: 512-18. 31 O’Farrelly C, Feighery C, Greally JG, Weir DG. Cellular response to alpha gliadin in untreated coeliac disease. Gut 1982; 23: 83-87. 32. O’Farrelly C, Kelly J, Hekkens W, et al Alpha gliadin antibody levels. a serological test for coeliac disease. Br Med J 1983; 286: 2007-10. 33. Gershon RK. Suppressor T cells: a miniposition paper celebrating a new decade. In: Fougereau M, Dausset J, eds Immunology 80. Progress in immunology IV. London: Academic Press, 1980: 375-89. 34 McCarthy CF, Fraser ID, Evans KT, Read AE. Lymphoreticular dysfunction in idiopathic steatorrhoea. Gut 1966; 7: 140-48. 35. Stokes PL, Holmes GKT. Malignancy In: Cooke WT, Asquith P, eds. Clinics in gastroenterology. Philadelphia: WB Saunders, 1974: 158-70. 36. Simpson FG, Howdle PD, Robertson DAF, Losowsky MS. Jejunal biopsy and lymphocyte co-culture in coeliac disease. Gut 1981, 22: A420-21 (abstr). 37 Strober W, Falchuk ZM, Rogentine GN, Nelson DL, Kleaveman HL. The pathogenesis of gluten-sensitive enteropathy. Ann Intern Med 1975; 83: 242-56 38 Leigh RJ, Marsh MN. Mucosal lymphocyte response to gluten challenge. Gut 1984; 25: A549 (abstr). 39. Reinherz EL, Kung PC, Goldstein G, Schlossman SF. A monoclonal antibody reactive with the human cytotoxic/suppressor T cell subset previously defined by a heteroantiserum termed TH2. J Immunol 1980; 124: 1301-07.
cellular sensitisation to wheat protein has been described in coeliac disease,4-6 it is not known whether these circulating antigens can stimulate specific immune regulatory cell populations. We have investigated the ability of the wheat protein antigen, alpha-gliadin, a 60 000 molecular weight fraction of gluten, to activate suppressorcell mechanisms.
Although
Preliminary
Communication
SUPPRESSOR-CELL ACTIVITY IN COELIAC DISEASE INDUCED BY ALPHA-GLIADIN, A DIETARY ANTIGEN CLIONA O’FARRELLY C. F. FEIGHERY
C. A. WHELAN D. G. WEIR
SUBJECTS AND METHODS
Subjects
Departments of Immunology and Clinical Medicine, Trinity College and St James’s Hospital, Dublin, Ireland
Summary
The wheat
protein antigen alpha-gliadin,
a
fraction derived from gluten of molecular
weight 60 000,
activated suppressor cells from
patients with
coeliac disease but not from normal subjects or patients with Crohn’s disease. Two other dietary antigens, casein and betalactoglobulin, failed to produce suppressor-cell activation. Since this phenomenon appears to be specific to coeliac disease, it may be of pathogenetic significance.
We studied twenty-eight patients with adult coeliac disease, eighteen female, ten male, aged 17-64 years (mean 27). Ten patients were on gluten-free diets and eighteen were untreated when tested. All had the characteristic lesion of coeliac diesease shown by jejunal biopsy, and the treated patients had a clinical response to the diet of least 6 months’ duration. The untreated group subsequently diet. In twenty patients a repeat biopsy was done after treatment; all samples showed histological improvement in response to treatment. The controls were twentytwo normal subjects aged 20-55 years (mean 25) and six patients with ileal Crohn’s disease (four female, two male), mean age 26 years. at
responded to a gluten-free
INTRODUCTION
COELIAC disease is a malabsorptive disorder characterised by a mucosal lesion which reverts to normal on removal of dietary gluten.’ Although the mechanism by which the wheat protein damages the intestinal mucosa is not known, it has been proposed that components of the immune response have a primary pathogenetic role.2 Humoral and cellular sensitisation to wheat protein antigens both in the jejunal mucosa and in the peripheral blood of coeliac patients has been reported.3-8 Furthermore, mucosal changes similar to those in coeliac disease have been described in graft-versushost disease in manand small-intestinal allograft rejection in
mice° .
.
both T-cell-mediated disorders. The coeliac lesion may also be mediated by T lymphocytes. Further evidence for an immunopathogenetic process includes the high frequency in patients with coeliac disease of the haplotype HLA B8, D3, Dr3,]] gene products of the major histocompatibility complex known to be associated with autoimmune disorders. ]2 Abnormalities in regulation of the immune response must be considered when it is proposed that immune mechanisms are involved in disease pathogenesis. Some studies have suggested that immune responsiveness is depressed in coeliac disease’3-15 and we have shown that mitogen-induced suppression is enhanced in coeliac patients.]6 Since the histological lesion in coeliac disease is caused by wheat protein antigens, it is possible that immunological responsiveness in these patients is controlled by immunoregulatory cells sensitised to these antigens. K. W. NEWELL AND OTHERS: 16.
Simpson
BH. An epidemiological study of carcinoma of the small intestine in New Zealand sheep. NZ Vet J 1972; 20: 91-97. 17 Carter AH, Cox EH. Sheep breeds in New Zealand. sheep production. Vol 1 In: Wickham GA, McDonald MF eds. Wellington: New Zealand Institute of Agricultural Science, 1982. 18 Owen JB. Sheep production. London: Baillière Tindall, 1976. 19 Sheep and beef farm survey 1977/8. Wellington: New Zealand Meat and Wool Board’s Economic Service, publication 1816, 1979. 20. Webster WM. Neoplasia in food animals with special reference to high incidence in sheep. NZ Vet J 1966; 14: 203-14. 21. Webster WM. A further survey of neoplasms of abattoir sheep. NZ Vet J 1967; 15: 51-54. 22. Wassom JS, Huff JE, Loprieno N. A review of the genetic toxicology of chlorinated dibenzo-p-dioxins. Mutation Res 1977/8; 47: 141-60. 23. IARC. Evaluation of the carcinogenic risk of chemicals to man. Monograph 15. Lyon: International Agency for Research on Cancer, 1977. 24. Grant WF. The genotoxic effects of 2,4,5-T. Mutation Res 1979; 65: 83-119.
Methods
Mf!M.—Alpha-gliadin was prepared as described previously. 17 Casein and beta-lactoglobulin were obtained commercially. Solutions of the antigens were made up in Hank’s balanced salt solution on the day of use. Lymphocyte transformation. -Peripheral-blood mononuclear cells prepared by density-gradient centrifugation were suspended in RPMI 1640 medium containing 10% fetal calf serum and 50 J.lg/ml gentamicin, at a concentration of 1 x 106 cells/ml. Samples of 200 1 were placed in the wells of microculture plates, covered, and incubated at 37°C with 5% CO2 and 95% humidity for 96 h, with or without alpha-gliadin at final concentrations of 1,2, and 10 J.lg/ml. Each culture was carried out in triplicate. Generation of suppression. -Suppressor-cell activity was demonstrated by a modification of our previous technique.]8 Samples of 4x 106 peripheral-blood mononuclear cells were precultured in RPMI 1640 medium at a concentration of 2 x 106 cells/ml. Antigen (alpha-gliadin, casein, or beta-lactoglobulin) at a concentration of 2 g/ml was added to the test cells and no stimulant was added to control cells. After 24 h incubation, the cells were washed twice in Hank’s balanced salt solution, resuspended in RPMI 1640 medium containing 50 g/ml mitomycin C, and incubated for 1 h. They were then washed three times with Hank’s balanced salt solution. The concentration of each sample of cells was adjusted to 1 x 106/ml and they were dispensed in 100 <1 volumes into the wells of microculture plates. Equal volumes of autologous peripheral-blood mononuclear cells, also at a concentration of 1 x 106 cells/ml, which had been maintained in RPMI 1640 medium at 37°C for 24 h, were added to the mitomycin-C-treated cells. The cells were co-cultured with or without concanavalin A (1, 2, or 5 g/ml) for 72 h. Each test was carried out in triplicate. REFERENCES—continued 25. Pesticides Board. A
guide
to agricultural chemicals. Wellington: New Zealand Fisheries, 1979 food: 1979 evaluations. Rome: Food and Agriculture World Health Organisation, Organisation, 1980. 27. Smith AH, Fisher DO, Giles HJ, Pearce NE. The New Zealand soft tissue sarcoma case-control study: Interview findings concerning phenoxyacetic acid exposure. Chemosphere 1983; 12: 561-71. 28. Johnstone AC, Alley MR, Jolly RD. Small intestinal carcinoma in cattle. NZ Vet J
Ministry of Agriculture
26. FAO Pesticide residues
and
in
1983; 31: 147-49. 29. Ross AD. Small intestinal carcinoma in cattle NZ Vet J1984; 32: 98-99. 30. Head KW. Tumours of the lower alimentary tract. Bull WHO 1976; 53: 167-86.
31. Oliver MF,
32.
Heady JA, Morris JN, Cooper J. WHO Cooperative trial on primary prevention of ischaemic heart disease using clofibrate to lower serum cholesterol: Mortality follow up. Lancet 1980; ii: 379-82. Hardell L. The relation of soft tissue sarcoma, malignant lymphoma and colon cancer to phenoxy acids, chlorophenols, and other agents. Scand J Work Environment Health 1981; 7: 119-30.
1306
Peripheral-blood mononuclear cells from treated and untreated coeliac patients precultured with 2 g/ml alphagliadin suppressed the concanavalin-A-induced proliferative response of autologous peripheral-blood mononuclear cells (see figure). Preculturing cells from normal subjects or from patients with Crohn’s disease with the wheat protein had no significant effect on the mitogenic response of autologous cells. The differences between both groups of coeliac patients and the control groups were significant at concanavalin A With 5 /-lg/ml concentrations of 1 and 2 g/ml (p<0’01). concanavalin A there was a significant difference between untreated coeliac patients and the control groups (p<0’01). Neither casein nor beta-lactoglobulin had any significant effect on cells from normal subjects, Crohn’s disease patients, or coeliac patients, treated or untreated (see table).
)jg Con
DISCUSSION
o/ml
Generation of suppression by alpha-gliadin of mitogenic response in untreated coeliac patients (A), treated coeliac patients (A), normal subjects ( 0 ), and Crohn’s disease patients (.). Bars indicate mean±SEM. Con A=concanavalin A.
Termination
of cultures.-18 h before harvesting 0.3 MCi tritiated thymidine (2 -0 mCi/ml) was added to each well. At the end of the culture period, the cells were washed onto glass-fibre discs by means of an automatic cell harvester. The discs were suspended in a scintillation cocktail and the amount of cell-incorporated tritiated thymidine was measured in a scintillation counter. Calculation of percentage suppression.-The percentage enhancement or suppression by antigen-activated cells of the mitogenic response of autologous peripheral-blood mononuclear cells
was
calculated;
F(cm, - c,) . 1- r
x
-
(CM - C)J
ioo 100 .
where CMA is the counts per min (cpm) of cells precultured in the presence of antigen, and added to autologous cells in the presence of convanavalin A; CA is the cpm of cells precultured in the presence of antigen and added to autologous cells in the absence of concanavalin A; CM is the cpm of cells precultured in the absence of antigen and added to autologous cells in the presence ofconcanavalin A; and C is the cpm of cells precultured in the absence of antigen and added to autologous cells in the absence of concanavalin A. Statistics.-The data were analysed by one-way analysis of variance then by between-group mean t tests. RESULTS
Alpha-gliadin at 2 g/ml stimulated peripheral-blood mononuclear cells from five untreated coeliac patients (mean±SEM 1238±313 cpm) but had no effect on cells from five normal subjects (mean±SEM 301 ±66 cpm; p<0 01). At other concentrations of alpha-gliadin (1 and 10 g/ml) there was a proliferative response in some patients but the mean value was not significantly different from that of the control group. EFFECT OF CASEIN AND BETA-LACTOGLOBULIN ACTIVATED CELLS ON RESPONSE OF AUTOLOGOUS CELLS TO ,
CONCANAVALIN A I
2
tg/ml
The plant mitogen concanavalin A stimulates suppressor cells to secrete inhibitory factors in vitro]9 and this effect is used to measure suppressor function.2o,2] In this study instead of concanavalin A different food antigens were added to peripheral-blood mononuclear cell cultures. Alphagliadin-activated cells from treated and untreated coeliac patients suppressed the response of autologous cells to mitogenic stimulation. In contrast, alpha-gliadin had no significant regulatory effect on cells from normal subjects or patients with Crohn’s disease and control dietary antigens, casein and beta-lactoglobulin, had no such effect on cells from any subjects. The possible association of the HLA B8 and Dr3 antigens with this suppressor system is under
investigation. Since alpha-gliadin-activated immunosuppression is specific for both antigen and disease, a previously sensitised immunoregulatory cell must be intrinsic to this system. Cells of the T-cell lineage are prime candidates for this role.22 Both BCG23 and lepromin24 induced T-suppressor-cell activation in subjects sensitised to these antigens. However, B-cell products, including immunoglobulin molecules, also regulated gastrointestinal immune responses in murine
experiments.25 With the introduction of antigen to the immune system, regulatory cell mechanisms are activated that together decide the nature and intensity of the response. In animals, antigen presented by the enteric route causes stimulation of mucosal immunoregulatory cells,25,26 which commonly results in a local immune response and systemic unresponsiveness .2’ In some circumstances, a systemic immune response occurs. Since food-derived antigens enter the circulation after ingestion,27 regulatory cells controlling the systemic immune response to these antigens are probably stimulated as part of the normal physiological process. Because of the small antigenic load in normal circumstances, food-antigen-specific regulatory cells may remain undetected by existing assay systems. In coeliac disease, a specific hypersensitivity reaction to the highly antigenic wheat gliadins28 is observed ;6,29 it may result in stimulation of systemic regulatory cell populations of sufficient intensity to allow their detection. The increased bowel porosity3° which accompanies coeliac disease may allow food antigens to enter the circulation with greater freedom and thereby could serve to amplify this effect. Although our study provides evidence of the presence of alpha-gliadin-activated suppressor cells, it is likely that cells with other regulatory functions, not sought in this assay,
are
also stimulated.
1307
Evidence of specific cellularb’31 and humoral32 sensitisation has been recorded in coeliac disease. Our lymphocyte transformation to is further evidence of cellular hypersensitivity alpha-gliadin to this antigen. It can be conjectured that in the face of
to
Reviews of Books
alpha-gliadin
demonstration of in-vitro
immunological reactivity physiological immunosuppression is required to control the immune response to wheat protein antigens. In coeliac disease, immunosuppression could be mediated by alpha-gliadinactivated suppressor cells. It has been suggested that the primary stance of the immune system is suppression to guard against excessive immune reactivity.33 This situation
The
pronounced
be exaggerated in coeliac disease. ]6 Increased immunosuppression could explain such features of coeliac disease as skin anergy,15 lymphoreticular dysfunction,34 depressed mitogen responsivenessI5,]6 and a predisposition to gastrointestinal malignant disease. 35 Enhanced immunosuppression in the mucosa of the small intestine may also interfere with normal antigen handling which in turn could contribute to the disease pathogenesis. Organ culture studies3,36,37 and the evaluation of smallintestinal cellular infiltrate after in-vivo challenge with gluten38 indicate that lymphocytes are intimately involved in the development of the histological lesion. The infiltrating intra-epithelial lymphocytes show the OKT8 phenotype.38 may
Since both cytotoxic and suppressor T lymphocytes express this antigen,39 alpha gliadin may be able to activate not only lymphocytes with suppressor function but also those with
cytotoxic capabilities. -
.
We thank the Medical Research Council of Ireland and the Nutrition Fund of the Provost’s Appeal, Trinity College, Dublin, for financial support during this project.
Correspondence should be addressed to D. G. W., Department of Clinical Medicine, Medical School Building, St James’s Hospital, James’s Street, Dublin 8, Ireland.
215l ed. Edited by A. M. Harvey, R. J. Johns, V. A. McKusick, A. A. Owens, and R. S. Ross, Johns Hopkins University School of Medicine, Baltimore. Norwalk, Connecticut: Appleton-Century-Crofts. Hemel Hempstead: Prentice/Hall. 1984. Pp 1596. jE47.05.
THis 21st edition of this well-known textbook of medicine, which Osler first wrote in 1892, has been written almost entirely by present staff of the Johns Hopkins Hospital, Baltimore, as it has been since the 17th edition. It is a thorough revision with many new contributors, new illustrations, and new tables. The objective was to present medicine through clinical problems rather than through an encyclopaedic text, and this is the key to its format. The book is packed with information, and it can appeal to the more advanced student, the young doctor, or the experienced clinician. The opening section presents a thoughtful analysis of the methods of clinical medicine; it puts emphasis on the hypotheticodeductive approach to history-taking which we all employ whether we realise it or not. This approach is not extended into physical examination, where the emphasis is on systematic thoroughness, to the extent that we are recommended to examine the external auditory meati as part of the initial inspection of the patient. Some recommendations seem at first to be lacking-for example, in the section on measuring blood pressure, palpation of the pulse is not described as a precaution against missing the auscultatory gap, an omission later corrected in the chapter on hypertension. The last three sections are on psychiatry in medicine, medical emergencies in critical care, and special topics in medicine, which includes special aspects of the care of the elderly. In between are sections based C. O’FARRELLY AND OTHERS:
1185-91 20. Shou L, Schwartz 21.
3 4. 5
Ferguson A, McClure JP, MacDonald TT, Holden RJ. Cell-mediated immunity to gliadin within the small-intestinal mucosa in coeliac disease. Lancet 1975; i: 895-97. Holmes GKT, Asquith P, Cooke WT. Cell-mediated immunity to gluten fraction III in adult coeliac disease. Clin Exp Immunol 1976; 24: 259-65. Sikora K, Anand BS, Truelove SC, Ciclitira PJ, Offord RE. Stimulation of lymphocytes from patients with coeliac disease by a subfraction of gluten. Lancet 1976; ii: 389-91.
In-vitro immunological assay for diagnosis of coeliac disease. Lancet 1978; i: 876. 7. Falchuk ZM, Strober W. Gluten-sensitive enteropathy: synthesis of anti-gliadin antibody in vitro. Gut 1974; 15: 947-52. 8 Ferguson A, Carswell F. Precipitins to dietary proteins in serum and upper intestinal secretions of coeliac children. Br Med J 1972; i: 75-77. 9. Lerner KG, Kao GF, Storb R, et al. Histopathology of graft vs host reaction (GvHR) in human recipients of marrow from HL-A matched sibling donors. Transplant Proc 1974; 6: 367-71. 10 Ferguson A, Parrott DMV. Histopathology and time course of rejection of allografts of mouse small intestine. Transplantion 1973; 15: 546-54 1. Keuning JJ, Pena AS, van Leeuwen A, van HoofJP, van Rood JJ. HLA-Dw3 associated with coeliac disease. Lancet 1976; i: 506-07. 12. Svejgaard A, Platz P, Ryder LP. HLA and disease 1982- a survey. Immunol Rev 1983; 70: 193-218. 13. MacLaurin BP, Cooke WT, Ling NR Impaired lymphocyte reactivity against tumour cells in patients with coeliac disease. Gut 1971; 12: 794-800. 14. Baker PG, Verrier-Jones J, Peacock DB, Read AE. The immune response to &phgr;x 174 in man. III Evidence for an association between hyposplenism and immunodeficiency in patients with coeliac disease. Gut 1975; 16: 538-42. 15 Scott BB, Losowsky MS. Depressed cell-mediated immunity in coeliac disease. Gut 1976; 17: 900-05. 16 O’Farrelly C, McKeever U, Feighery G, Weir DG. Increased concanavalin A induced suppression in treated and untreated coeliac disease. Gut 1984; 25: 644-48. 17. Kendall MJ, Cox PS, Schneider R, Hawkins CF. Gluten subfractions in coeliac disease. Lancet 1972; ii: 1065-67. 18 Feighery C, Whelan CA, Weir DG, Greally JF. In vitro studies of suppressor cell function in human peripheral blood mononuclear cells. Clin Exp Immunol 1978; 32: 459-65. 6.
Haeney MR, Asquith P.
REFERENCES—continued
19. Greene WC, Fleisher TA, Waldmann TA. Soluble suppressor supernatants elaborated by concanavalin A-activated human mononuclear cells. J Immunol 1981; 126:
REFERENCES 1. Falchuk ZM. Update on gluten-sensitive enteropathy. Am J Med 1979; 67: 1085-96. 2 Housley J, Asquith P, Cook WT. Immune response to gluten in adult coeliac disease. Br Med J 1969; ii: 159-61.
Principles and Practice of Medicine
22. 23.
S, Good RA. Suppressor cell activity after concanavalin A treatment of lymphocytes from normal donors. J Exp Med 1976; 143: 1100-10. Dwyer JM, Johnson C. The use of concanavalin A to study the immunoregulation of human T cells. Clin Exp Immunol 1981; 46: 237-49. Benacerraf B. Suppressor T cells and suppressor factor. Hosp Pract 1978; 13: 65-75. Mustafa AS, Godal T. In vitro induction of human suppressor T cells by mycobacterial antigens. BCG activated OKT4 + cells mediate suppression of antigen induced T cell proliferation. Clin Exp Immunol 1983, 52: 29-37.
24. Mehra V, Mason LH, Rothman W, Remherz E, Schlossman SF, Bloom BR. Delineation of a human T cell subset responsible for lepromin-induced suppression in leprosy patients J Immunol 1980; 125: 1183-88. 25. Strober W, Richman L, Elson C. The regulation of gastrointestinal immune responses. Immunol Today 1981; 2: 156-62. 26. Kagnoff MF. Effects of antigen-feeding on intestinal and systemic immune responses. Cell Immunol 1978; 40: 186-203. 27. Paganelli R, Levinsky RJ, BrostoffJ, Wraith DG. Immune complexes containing food proteins in normal and atopic subjects after oral challenge and effect of sodium cromoglycate on antigen absorption. Lancet 1979; i. 1270-72. 28. Kieffer M, Frazier PF, Daniels WWR, Coombs RRA Wheat gliadin fractions and other cereal antigens reactive with antibodies in the sera of coeliac patients. Clin Exp Immunol 1982; 50: 651-60. 29. O’Farrelly C, Hekkens WTJM, Feighery C, Weir DG. The specificity of wheat protein reactivity on coeliac disease. Scand J Gastroenterol 1983; 18: 603-07. 30 Cobden I, Rothwell J, Axon ATR. Intestinal permeability and screening tests for coeliac disease. Gut 1980; 21: 512-18. 31 O’Farrelly C, Feighery C, Greally JG, Weir DG. Cellular response to alpha gliadin in untreated coeliac disease. Gut 1982; 23: 83-87. 32. O’Farrelly C, Kelly J, Hekkens W, et al Alpha gliadin antibody levels. a serological test for coeliac disease. Br Med J 1983; 286: 2007-10. 33. Gershon RK. Suppressor T cells: a miniposition paper celebrating a new decade. In: Fougereau M, Dausset J, eds Immunology 80. Progress in immunology IV. London: Academic Press, 1980: 375-89. 34 McCarthy CF, Fraser ID, Evans KT, Read AE. Lymphoreticular dysfunction in idiopathic steatorrhoea. Gut 1966; 7: 140-48. 35. Stokes PL, Holmes GKT. Malignancy In: Cooke WT, Asquith P, eds. Clinics in gastroenterology. Philadelphia: WB Saunders, 1974: 158-70. 36. Simpson FG, Howdle PD, Robertson DAF, Losowsky MS. Jejunal biopsy and lymphocyte co-culture in coeliac disease. Gut 1981, 22: A420-21 (abstr). 37 Strober W, Falchuk ZM, Rogentine GN, Nelson DL, Kleaveman HL. The pathogenesis of gluten-sensitive enteropathy. Ann Intern Med 1975; 83: 242-56 38 Leigh RJ, Marsh MN. Mucosal lymphocyte response to gluten challenge. Gut 1984; 25: A549 (abstr). 39. Reinherz EL, Kung PC, Goldstein G, Schlossman SF. A monoclonal antibody reactive with the human cytotoxic/suppressor T cell subset previously defined by a heteroantiserum termed TH2. J Immunol 1980; 124: 1301-07.