and jejunal fluid immunoglobulins in adult coeliac

Transcription

1 Gut, 1974, 15, Jejunal muosal immunoglobulin-ontaining ells and jejunal fluid immunoglobulins in adult oelia disease and dermatitis herpetiformis M. LANCASTR-SMITH, PARVN KUMAR, R. MARKS, M. L. CLARK, AND A. M. DAWSON From the Department of Gastroenterology, St Bartholomew's Hospital, London, and St John's Hospitalfor Diseases of the Skin, London SUMMARY Immunoglobulin-ontaining plasma ell densities in the jejunal muosa and serum and jejunal fluid immunoglobulins have been measured in patients with adult oelia disease and dermatitis herpetiformis with and without jejunal muosal abnormality. Studies were performed in patients before and after treatment of the jejunal lesion. Total immunofluoresent plasma ells were inreased in untreated adult oelia disease and dermatitis herpetiformis patients with jejunal lesions, but in general the normal predominane of IgA > IgM > IgG was found. There was no differene from ontrols in IgA-ontaining ells in the two onditions before or after treatment. The numbers of IgM-ontaining ells were signifiantly inreased both before and after treatment in groups of patients with adult oelia disease and dermatitis herpetiformis who had jejunal lesions. IgG-ontaining ells were signifiantly raised in only the before-treatment groups. Patients with dermatitis herpetiformis without jejunal lesions, even whilst on gluten-ontaining diets, had normal numbers of immunoglobulin-ontaining ells. IgA and IgM jejunal fluid immunoglobulins were signifiantly raised in dermatitis herpetiformis and adult oelia disease. It is onluded that patients with dermatitis herpetiformis with jejunal morphologial abnormality have a omparable immunologial disturbane of the jejunal muosa to that found in adult oelia disease. The assoiation between adult oelia disease and dermatitis herpetiformis has been further supported by reent reports of similar immunologial findings. Changes in serum (Fry, Keir, MMinn, Cowan, and Hoffbrand, 1967; Fraser, Dik, and Crihton, 1969; Hobbs and Hepner, 1968; Asquith, Thompson, and Cooke, 1969) and jejunal fluid immunoglobulins (Douglas, Crabbe, and Hobbs, 197; MClelland, Bametson, Parkin, Warwik, Heading, and Shearman, 1972), irulating immune omplexes (Mowbray, Hoffbrand, Holborow, Seah, and Fry, 1973; Doe, Booth, and Brown, 1973), and tissue antibodies (Seah, Fry, Hoffbrand, and Holborow, 1971) suggest that immunologial fators may well be involved in the pathogenesis of these two onditions. Abnormalities of the plasma ell densities in the lamina propria of the jejunum using immunofluoresent tehniques have been reported in adult Reeived for publiation 12 February oelia disease (Douglas et al, 197; Sltoft, 197; Pettingale, 1971) but omparable information is not available for dermatitis herpetiformis. In view of the fat that similar though usually less severe jejunal morphologial hanges our in dermatitis herpetiformis (Marks, Shuster, and Watson, 1965; Marks, Whittle, Beard, Robertson, and Gold, 1968; Brow, Parker, Weinstein, and Rubin, 1971), we have ompared the densities of immunoglobulin-ontaining ells in these two onditions and orrelated them with morphology and. treatment. A further study was also made of the onentrations of jejunal fluid and serum immunoglobulins in both diseases Patients and Methods In all studies the diagnosis of adult oelia disease was made on linial and biohemial evidene of malabsorption, villous atrophy of the jejunum, and 371 Gut: first published as /gut on 1 May Downloaded from on 19 July 218 by guest. Proteted by opyright.

2 372 M. Lanaster-Smith, Parveen Kumar, R. Marks, M. L. Clark, and A. M. Dawson by previous or subsequent response to a gluten-free diet. Dermatitis herpetiformis was diagnosed on the linial and histologial features of the rash and its response to Dapsone. Measurements of immunoglobulin-ontaining ells and jejunal fluid immunoglobulins were performed in different groups of patients.as desribed below. IMMUNOFLUORSCNC STUDIS ON JJUNAL MUCOSA Dermatitis herpetiformis Fourteen biopsies from 11 patients with abnormal jejunal morphology were examined;. 1 showed partial villous atrophy with villous heights ranging from 137 to 28 mirons (normal >33 mirons) and surfae ell heights from 21 to 26 mirons (normal > 29 mirons), and four had subtotal hanges with villous heights ranging from to 1 mirons and surfae ell heights from 15 to 17 mirons. ight were from patients on unrestrited diets, three were from patients who had been on gluten-free diets for three to four months, and three frompatients reeivingprednisolone.of thesepatients, two were studied before and after taking a gluten-free diet for four to six months. Biopsies from three patients with dermatitis herpetiformis with normal jejunal morphology whilst on normal diets were also studied. Adult oelia disease Thirteen biopsies from 11 patients were studied, seven before starting a gluten-free diet and six after gluten restrition of one to six years. Ofthese patients, two were studied before and after taking a gluten-free diet for six and eight months. All of the untreated patients had subtotal villous atrophy with villous heights ranging from to 98 mirons and surfae ell heights from 13 to 22 mirons, and the treated group had partial villous atrophy, with villous heights ranging from 175 to 32 mirons and surfae ell heights from 2 to 26 mirons. Speimens were obtained from eight patients of omparable age to the above groups, who were being investigated for gastrointestinal symptoms. All had normal small bowel histology and no evidene of immunologial disturbane Methods Biopsies were obtained by-cr6sby apsules in fasting subjets from the duodeno-jejunal flexure. Half of the tissue was immediately snap frozen, embedded in Ames OCT ompound and stored at minus 7 C. The remainder of the speimen was proessed for staining with haematoxylin and eosin and methyl green pyronine, whih was used to failitate the identifiation of plasma ells. Setions, 6 mirons thik, were prepared in a Bright's ryostat at minus 25 C. The setions were dried at room temperature for one and a half to two hours. Antisera to human IgA, IgG, and IgM onjugated with fluoresein isothioyanate and diluted with Coons buffer 1:3, 1:2, and 1:2 respetively were plaed on the setions. After 2 minutes the setions were washed in hanges of Coons buffer for a minimum of one hour. xess moisture was removed with filter paper and the setions were mounted in a 1:5 solution of buffered glyerol. The antisera had been shown to be monospeifi to their orresponding antigens before and after onjugation by the manufaturers (Behringwerke, Hoehst Pharmaeutials Ltd). Speifiity of staining by eah onjugate was onfirmed in this study by the bloking tehnique of Nairn (1964). The stained setions were examined with a Leitz Ortholux mirosope fitted with a darkground Tori lens ondenser. A merury vapour light soure was used in onjuntion with Balzer FITC 3 and Leitz K53 filters. A Leitz Orthomat automati amera was attahed to the mirosope. Quantitative measurement of the ellular immunofluoresene was made by taking Kodak ktahrome daylight film slides of at least three high-power fields for eah immunoglobulin lass on eah biopsy. The slides thus obtained were projeted onto graph paper and the areas of total lamina propria and ellular immunofluoresene were traed. The areas of ellular fluoresene were expressed as perentages of the total area of lamina propria. JJUNAL FLUID IMMUNOGLOBULINS Dermatitis herpetiformis Of 14 patients studied, two had subtotal villous atrophy of the jejunum and 12 had partial villous atrophy. Three were on a gluten-free diet and 11 were on a normal diet. Adult oelia disease Fourteen patients were studied and at the time of investigation five patients had been on a gluten-free diet from 1 months to four years and nine were untreated. These onsisted of 14 subjets undergoing investigation for possible gastrointestinal disease and all had normal small bowel morphology. Methods Fasting samples of jejunal juie were olleted from Gut: first published as /gut on 1 May Downloaded from on 19 July 218 by guest. Proteted by opyright.

3 Jejunal muosal immunoglobulin-ontaining ells andjejunalfluid immunoglobulins the duodeno-jejunal flexure via a Crosby apsule before firing. Trasylol was added immediately and samples were snap frozen and stored at minus 3 C. Immunoglobulin onentrations (IgA, IgG, IgM) were measured with Hyland low-level plates, using standards supplied by the manufaturer. The lowest levels of aurate estimation by this tehnique was taken as 4 mg per 1 ml. When immunoglobulin was detetable but below 4 mg per 1 ml an arbitrary value of 2 mg per 1 ml was given to that speimen. The IgA standards were of the 7s ategory. The approximated IgA levels reorded in the results, however, are adjusted (Brandtzaeg, Fjellanger, and Gjeruldsen, 197), assuming the majority of IgA to be in the 1 ls form. This has been done to failitate omparison with previous studies in whih 1 is standards have been used. Serum immunoglobulins were measured in all patients studied by the single radial diffusion tehnique using immunoplates supplied by Hyland Laboratories. Results IMMUNOFLUORSCNC ON JJUNAL MUCOSA Details of the results are shown in figures 1-4. These may be summarized as follows: Compared with ontrols the total numbers of immunofluoresent ells were inreased in both disorders in untreated patients with abnormal jejunal morphology. There was no signifiant differene between patients on gluten-free diets or prednisolone, untreated patients with dermatitis herpetiformis with normal jejunal histology, and ontrols. Biopsies were also stained with methyl green pyronine and quantitation of ells performed by the above tehnique also showed that the areas oupied by plasma ells were inreased in untreated patients with both onditions. IgA-ontaining ells Adult oelia disease and dermatitis herpetiformis groups before or after treatment did not differ signifiantly from eah other or ontrols. IgM-ontaining ells IgM ells were greatly inreased to a omparable degree in patients with adult oelia disease on normal diets and patients with dermatitis herpetiformis with jejunal abnormality on normal diets ompared with ontrols (p < 2). The numbers of ells were redued to the same extent in both groups of patients on a gluten-free diet or prednisolone, but there remained a differene between these patients and ontrols (p <.5). Dermatitis herpetiformis patients without jejunal lesions on normal diets did not differ from ontrols. IgG-ontaining ells These were inreased to a omparable degree in adult oelia disease patients on normal diets and dermatitis herpetiformis patients- with jejunal morphologial abnormality -on normal diets ompared with ontrols (p <.1). There were no signifiant differenes between treated patients with adult oelia disease and dermatitis` herpetiformis, untreated dermatitis herpetiformis patients without jejunal lesions and ontrols. RLATIONSHIP OF IMMUNOFLUORSCNT CLLS TO JJUNAL MORPHOLOGY In adult oelia disease the inrease in IgM and IgG ells was greater in patients with subtotal villous atrophy, all of whom were on normal diets, than in those with partial villous atrophy, who were taking gluten-free diets. In ontrast, in dermatitis herpetiformis the severity of the muosal lesion was not exlusively related to the dietary status of the patient and omparable inreases in IgM and IgG ells were found in patients with subtotal and partial villous atrophy. PATINTS STUDID BFOR AND AFTR TRATMNT IgM ells in the four patients (two in eah 'group) studied before and after treatment dereased markedly in all ases following treatment. The mean for the IgM fluoresent area alulated from these four patients fell from 7.7 to 3.9 % of the total area of lamina propria. For IgA and IgG ells there was a redution in three patients (two adult oelia disease and one dermatitis herpetiformis) after treatment but in one patient with dermatitis herpetiformis the reverse ourred. The respetive means for the IgA and IgG fluoresent areas alulated from all four ases fell from 13 to 9% and 1*8 to 1 3% of the total area of lamina propria. JJUNAL FLUID IMMUNOGLOBULINS The onentrations of jejunal fluid immunoglobulins in the three groups are shown in the table. Immunoglobulins Adult Coelia Dermatitis Disease Herpetiformi. IgA 13.3 j IgG 1.6 ±,' IgM 2-3 ± : Table Immunoglobulins in jejunal fluidl 'Means : standard errors are shown in mg per 1 ml. 373 IgA Patients with dermatitis herpetiformis had sig. Gut: first published as /gut on 1 May Downloaded from on 19 July 218 by guest. Proteted by opyright.

4 M. Lanaster-Smith, Parveen Kumar, R. Marks, M. L. Clark, and A. M. Dawson JJUNAL CLLULAR IMMUNOFLUORSCNC JJUNAL CLLULAR IMMUNOFLUORSCNC IgA IgG 3% S 5 p = 2* h- o O _ ^ a 374 2% 6 O o v 1S g (X) rx CD - 1% 31 1% IFig1 L v ; Pre Post Pre Post A.C.D. D.H. n-. Pre n_a rpos Pre Drfi D^r Post A. C.D. D. H. Fig 2 Fig 1 JJUNAL CLLULAR IMMUNOFLUORSCNC JJUNAL CLLULAR IMMUNOFLUORSCNC 1gM Toal IIF Cells 3% 1% [. a a 8 2-2% S Q 3 v 9 8 S '6 1N 16 5% *;i z SL -1~~~~4 XL i~l 4-. rs s rre Pro Drets~~~~~~Cotrl A. C. D. D rh. Pre Post PrD Post A. C.D. D.H. Fig 3 Fig 4 Figs 1-4 Immunoglobulin-ontaining ells in the jejunal lamina propria. Adult oelia disease (ACD) and dermatitis herpetiformis (DH)-pretreatment (pre) and posttreatment (post): subtotal villous atrophy, partial villous atrophy, * normal muosa. Gut: first published as /gut on 1 May Downloaded from on 19 July 218 by guest. Proteted by opyright.

5 Jejunal muosal immunoglobulin-ontaining ells andjejunalfluid immunoglobulins nifiantly higher onentrations ompared with those with adult oelia disease (p <.1) and ontrols (p < 2) but the three patients with dermatitis herpetiformis treated with a gluten-free diet were within the normal range. The levels found in adult oelia disease were intermediate between dermatitis herpetiformis and ontrols and were signifiantly higher than the latter (p <.5). IgM The onentrations in dermatitis herpetiformis were higher than those in adult oelia disease and the ontrols (p < 12). The three treated dermatitis herpetiformis patients were within the normal range. The adult oelia disease patients had higher onentrations than ontrols (p < O5), the greatest values ourring in untreated patients. IgG The onentrations in dermatitis herpetiformis were higher than the ontrol levels (p <.5). There was no signifiant differene between both groups of patients nor between adult oelia disease and ontrols. SRUM IMMUNOGLOBULINS Levels of IgA and IgG were within normal limits in all patients but 3% of adult oelia disease and 21 % of dermatitis herpetiformis patients had IgM onentrations below the normal range of 5 to- 17 mg/1 ml. No orrelation was demonstrated between jejunal juie and serum levels of immunoglobulins. Immunoglobulin-ontaining ell densities in the jejunum did not orrelate with immunoglobulin onentrations in the serum. It was not possible to ompare ell densities with immunoglobulins in the jejunal fluid as there were too few subjets ommon to both studies. Wiloxon's sum of ranks test was used throughout. Disussion Total numbers of immunoglobulin-ontaining ells in the jejunal muosa were inreased in untreated adult oelia disease as previously shown by Sltoft (197). Patients on a gluten-free diet, however, had lower values and this agrees with the redution of plasma ells observed after gluten restrition by Holmes, Asquith, Stokes, and Cooke (1973) using routine histologial tehniques. The IgM ell inrease in adult oelia disease onfirms previous reports (Douglas et al, 197; Sltoft, 197) and in two untreated patients reahed predominane over IgA ells, an ourrene also found in other studies (Douglas et al, 197; Pettingale, 1971). IgG-ontaining ells were inreased in untreated adult oelia disease but not to the extent found by Sltoft (197). Douglas and his olleagues (197) and Pettingale (1971) found no signifiant inrease. The most likely explanation for these variations is that although attempts have been made in all studies to redue sampling errors by using areas of maximal ellular immunofluoresene for quantitation, differenes may have arisen beause of wide variations of ell density from one mirosopi field to another. Suh errors are proportionally greatest when ell numbers are small suh as for IgG. The equal numbers of IgA ells found in adult oelia disease and ontrols agrees with Sltoft (197) but it is at variane with the results of Douglas et al (197) and Pettingale (1971) who found varying degrees of redution in adult oelia disease. A possible explanation for the differene between our results for IgA ells and those of Douglas and his olleagues (197) is that their ontrols were normal subjets (Crabbe, Carbonara, and Heremans, 1965), whereas our ontrol group, although having normal jejunal morphology, did have gastrointestinal symptoms and might not have had an entirely normal jejunal IgA ell density. A further possible explanation for these differenes is based on the reent findings that untreated hildhood oelia disease is haraterized by an inrease in IgA ell numbers in the jejunal muosa but falling after treatment with a gluten-free diet (Savilahti, 1972). It has been proposed that the derease in IgA ells found in previous studies in adult oelia disease results from exhaustion of this ell lass (Savilahti, 1972). Presumably intermediate numbers of IgA ells exist at some point in this pathogenesis and it might be that our patients and those of Sltoft (197) were at this stage. This onept is further supported by the demonstration that although numbers of IgA ells in our treated group of adults did not differ from those in untreated ases, three of the four patients studied serially did show a redution in IgA ells when on gluten-free diets, as found in hildhood oelia disease (Savilahti, 1972). There have been no similar previous studies in dermatitis herpetiformis apart from a single ase reported by Douglas et al (197). This patient, with a onvoluted jejunal muosa, had normal numbers of IgM-ontaining ells in the lamina propria. This is similar to the situation found in our dermatitis herpetiformis patients without jejunal lesions and in five of those with jejunal abnormality. Our investigation demonstrates essentially similar abnormalities of immunoglobulin-ontaining ells in dermatitis herpetiformis patients who have jejunal lesions to those found in adult oelia disease, with the same immunologial response to gluten restrition in both onditions. Gut: first published as /gut on 1 May Downloaded from on 19 July 218 by guest. Proteted by opyright.

6 376 M. Lanaster-Smith, Parveen Kumar, R. Marks, M. L. Clark, and A. M. Dawson The omparable inrease in IgM ells in untreated adult oelia disease and dermatitis herpetiformis patients, whether the latter had subtotal or partial villous atrophy, in ontrast to the normal numbers found in dermatitis herpetiformis patients without jejunal abnormality, implies that an inrease in IgM ell infiltration is a fundamental phenomenon in the pathogenesis of the muosal lesion. In addition, the lower IgM ell numbers in treated ases and redution in IgM ells following treatment in the serially studied patients suggests that the IgM ell response is in part due to the presene of gluten in the diet. Inreased IgM ell infiltration of the jejunal muosa is not, however, exlusive to adult oelia disease and dermatitis herpetiformis, having been demonstrated in infetious enteritis (Sltoft and Seberg, 1972) and ulerative olitis (Sltoft, 1969), in both of whih a omparable morphologial abnormality of the jejunal muosa to that of adult oelia disease and dermatitis herpetiformis is only rarely seen. It seems, therefore, that an inreased IgM response alone is not the omplete explanation for the muosal lesion of both these onditions. The signifiantly higher onentration of IgA and IgM in the jejunal fluid in dermatitis herpetiformis ompared with those in adult oelia disease and ontrols onfirms the reent report by MClelland and his olleagues (1972). We have also onfirmed the raised levels of IgA (Thompson, Asquith, and Cooke, 197)andIgM(Thompsonet al, 197; Douglas et al, 197) in adult oelia disease. The inrease in the IgM in the luminal fluid may reflet the inreased numbers of IgM ells in the muosa in these two onditions. Raised IgA onentrations in the jejunal fluid annot be explained by an inrease in IgA ell densities although Douglas et al (197) have pointed out that even a derease in density of IgA ells is ompatible with an absolute inrease in numbers of ells. The greater onentration ofjejunal fluid immunoglobulins in dermatitis herpetiformis than in adult oelia disease is diffiult to explain and has reently been disussed by MClelland and his olleagues (1972) who onsider that oexistent ahlorhydria may be impliated in some oftheir patients. Although no differene ould be demonstrated in the IgA ell population in groups of patients with both disorders, three patients with dermatitis herpetiformis had values 2 standard deviations above the mean of ontrols and it is possible that these patients represent the muosal ounterpart of the greatly elevated fluid onentrations of IgA found in some patients with dermatitis herpetiformis. In onlusion, it appears likely from our omparative study of adult oelia disease and dermatitis herpetiformis that similar immunologial proesses are involved in the pathogenesis of the muosal lesion in both onditions. We wish to thank Dr Barbara Smith, Mr Adrian Pirson, and Mrs Susan Joye for their tehnial assistane. M.L.S. was on an eletive researh year from the London Hospital. Referenes Asquith, P., Thompson, R. A., and Cooke, W. T. (1969). Serum immunoglobulins in adult oelia disease. Lanet, 2, Brandtzaeg, P., Fjellanger, I., and Gjeruldsen, S. T. (197). Human seretory immunoglobulins. I. Salivary seretions from individuals with normal or low levels of serum immunoglobulins. Sand. J. Haematol., Suppl. 12. Brow, J. R., Parker, F., Weinstein, W. M., and Rubin, C.. (1971). The small intestinal muosa in dermatitis herpetiformis: Severity and distribution of the small intestinal lesion and assoiated malabsorption. Gastroenterology, 6, Crabb6, P. A., Carbonara, A. O., and Heremans, J. F. (1965). The normal human intestinal muosa as a major soure of plasma ells ontaining IgA. Lab. Invest., 14, Doe, W. F., Booth, C. C., and Brown, D. L. (1973). videne for omplement binding immune omplexes in adult oelia disease, Crohn's disease, and ulerative olitis. Lanet, 1, Douglas, A. P., Crabb6, P. A., and Hobbs, J. R. (197). Immunohemial studies of the serum, intestinal seretions, and intestinal muosa in patients with adult elia disease and other forms of the elia syndrome. Gastroenterology, 59, Fraser, N. G., Dik, H. M., and Crihton, W. B. (1969). Immunoglobulins in dermatitis herpetiformis and various other skin diseases. Brit. J. Dermat., 81, Fry, L., Keir, P., MMinn, R. M. H., Cowan, J. D., and Hoffbrand, A. V. (1967). Small intestinal struture and funtion and haematologial hanges in dermatitis herpetiformis. Lanet, 2, Hobbs, J. R., Hepner, G. W. (1968). Defiieny of y M globulin in oelia disease. Lanet, 1, Holmes, G. K. T., Asquith, P., Stokes, P. L., and Cooke, W. T. (1973). Cellular infiltrate of jejunal biopsies in adult oelia disease (ACD) in relation to gluten withdrawal. Gut, 14, 429. MClelland, D. B. L., Barnetson, R. S. C., Parkin, D. M., Warwik, R. R. G., Heading, R. C., and Shearman, D. J. C. (1972). Small intestinal immunoglobulin levels in dermatitis herpetiformis. Lanet, 2, Marks, J., Shuster, S., and Watson, A. (1965). Small bowel hanges in dermatitis herpetiformis. Lanet, 2, Marks, R., Whittle, M. W., Beard, R. J., Robertson, W. B., and Gold, S. C. (1968). Small bowel abnormalities in dermatitis herpetiformis. Brit. med. J., 1, Mowbray, J. F., Hoffbrand, A. V., Holborow,. J., Seah, P. P., and Fry, L. (1973). Cirulating immune omplexes in dermatitis herpetiformis. Lanet, 1, Nairn, R. C. (1964). Fluoresent Protein Traing, 2nd. ed., p. 12. Livingstone, dinburgh. Pettingale, K. W. (1971). Immunoglobulin ontaining ells in the oelia syndrome. Gut, 12, Savilahti,. (1972). Intestinal immunoglobulins in hildren with oelia disease. Gut, 13, Seah, P. P., Fry, L., Hoffbrand, A. V., and Holborow,. J. (1971). Tissue antibodies in dermatitis herpetiformis and adult oelia disease. Lanet, 1, Sltoft, J. (1969). Immunoglobulin ontaining ells in normal jejunal muosa and in ulerative olitis and regional enteritis. Sand. J. Gastroent., 4, Sltoft, J. (197). Immunoglobulin ontaining ells in non-tropial sprue. Clin. exp. Immunol., 6, Soltoft, J., and Soeberg, B. (1972). Imnmunoglobulin ontaining ells in the small intestine during aute enteritis. Gut, 13, Thompson, R. A., Asquith, P., and Cooke, W. T. (197). Immunoglobulin ontent of jejunal fluid in oelia disease and other gastrointestinal diseases. Abstrats of the IVth World Congress of Gastroenterology, Copenhagen, p. 8. Gut: first published as /gut on 1 May Downloaded from on 19 July 218 by guest. Proteted by opyright.