Increase of lymphocytes bearing the gamma/delta T cell receptor in the jejunum of patients with dermatitis herpetiformis.

The densities of T cells and of cells bearing the T cell receptors gamma/delta and alpha/delta and the surface antigens CD4 and CD8 in jejunal specimens from 21 patients with dermatitis herpetiformis were compared with those in specimens from 13 untreated adults with coeliac disease and 13 control subjects. In the lamina propria of the jejunum the median density of gamma/delta+ cells was significantly (p less than 0.001) greater in untreated patients with dermatitis herpetiformis than in control subjects (114 v 36 cells/mm2) and similar to that found in the patients with coeliac disease (115 cells/mm2). The difference in gamma/delta+ cell density between patients with dermatitis herpetiformis and control subjects was much greater in the surface epithelium of the jejunum: the median density for 14 untreated patients with dermatitis herpetiformis was 39 cells/mm, for seven patients with dermatitis herpetiformis on a gluten free diet 34 cells/mm, and for control subjects 2 cells/mm; the coeliac patients had the same density as the patients with dermatitis herpetiformis (45 cells/mm). The higher density of cells bearing the alpha/delta T cell receptor in the epithelium (median 77 cells/mm) of untreated patients with dermatitis herpetiformis was associated with a gluten containing diet; in specimens taken from patients with dermatitis herpetiformis on a gluten free diet the median density was similar to that in the control subjects (44 v 39 cells/mm). The increase in the number of lymphocytes bearing the T cell receptor gamma/delta, particularly in the epithelium of the jejunum, seems to be a constant marker for these closely related diseases, whereas the density of alpha/delta+ T cells is dependent on the diet.     

Jejunal mucosal immunoglobulin-containing cells and jejunal fluid immunoglobulins in adult coeliac disease and dermatitis herpetiformis

Immunoglobulin-containing plasma cell densities in the jejunal mucosa and serum and jejunal fluid immunoglobulins have been measured in patients with adult coeliac disease and dermatitis herpetiformis with and without jejunal mucosal abnormality. Studies were performed in patients before and after treatment of the jejunal lesion.

Total immunofluorescent plasma cells were increased in untreated adult coeliac disease and dermatitis herpetiformis patients with jejunal lesions, but in general the normal predominance of IgA > IgM > IgG was found. There was no difference from controls in IgA-containing cells in the two conditions before or after treatment. The numbers of IgM-containing cells were significantly increased both before and after treatment in groups of patients with adult coeliac disease and dermatitis herpetiformis who had jejunal lesions. IgG-containing cells were significantly raised in only the before-treatment groups. Patients with dermatitis herpetiformis without jejunal lesions, even whilst on gluten-containing diets, had normal numbers of immunoglobulin-containing cells. IgA and IgM jejunal fluid immunoglobulins were significantly raised in dermatitis herpetiformis and adult coeliac disease.

It is concluded that patients with dermatitis herpetiformis with jejunal morphological abnormality have a comparable immunological disturbance of the jejunal mucosa to that found in adult coeliac disease.

     

Cellular infiltrate of jejunal biopsies in adult coeliac disease in relation to gluten withdrawal

A comparison has been made of inflammatory cell counts in the lamina propria and epithelium of jejunal biopsies in 11 patients with adult coeliac disease with those found in 12 control subjects. In the coeliac patients, there were significant increases in the numbers of total cells, plasma cells, and intraepithelial lymphocytes, but a significant reduction in lamina propria lymphocytes. Following clinical improvement on a strict gluten-free diet, significant changes in cell counts occurred, but with the exception of lymphocytes in the lamina propria, the counts were still abnormal. Analysis of five patients in whom the biopsy improved to near normal morphology and of six in whom there was no such improvement showed that significant falls in plasma cells and rises in lymphocytes in the lamina propria could occur without improvement in other morphological appearances. These results seem relevant to the problem of diagnosing coeliac disease in patients who, on gluten withdrawal, show an unequivocal clinical response, but no gross morphological improvement in the jejunal biopsy. On the basis of the observed changes in cell counts, there seems little justification in questioning the diagnosis of coeliac disease in such patients.     

Effect of diet and age on jejunal and circulating lymphocyte subsets in children with coeliac disease: persistence of CD4-8-intraepithelial T cells through treatment.

Monoclonal antibodies were used to determine the relative numbers of T lymphocyte subsets in 61 jejunal biopsies and in peripheral blood of 35 children with coeliac disease, and of 13 healthy controls. The T cell numbers in the lamina propria were unaffected by a change from gluten-free to gluten containing diet in the patients. The number of intraepithelial lymphocytes (where the CD8 cells predominated) were significantly raised in patients taking gluten. Ten to 20% of the patients' intraepithelial CD3 (mature T) cells expressed neither CD8 nor CD4 surface antigens. This CD4 8 T cell population persisted through gluten elimination and challenge. The circulating lymphocyte subsets showed little variation with the diet although there was a marked increase in the proportion (14.9%) of CD4 8 T cells in patients during gluten elimination. In the histologically normal jejunal mucosa from control subjects, the age of the subject showed a positive correlation with villus intraepithelial CD3+ and CD8+ cells, and crypt intraepithelial CD4+ cells. No clear cut effect of age was observed on lamina propria lymphocyte counts of the controls, or on the lymphocyte counts in jejunal mucosa of the coeliac patients. The observed CD3+4-8- lymphocytes may represent activated cells unable to present their surface antigens, or they may be gamma delta-receptor bearing T cells, which could have a significant role in the pathogenesis of coeliac disease.     

Intestinal permeability in patients with coeliac disease and dermatitis herpetiformis

Intestinal permeability was investigated in patients with coeliac disease and dermatitis herpetiformis by a 51Chromium-labelled ethylenediaminetetraacetate (51Cr-EDTA) absorption test and the results correlated with histomorphometric analysis and intraepithelial lymphocyte counts of jejunal biopsies. The mean (+/- SD) 24 hour urine excretion of 51Cr-EDTA in 34 healthy volunteers was 1.9 +/- 0.5% of the orally administered test dose. Patients with untreated coeliac disease (19) or untreated dermatitis herpetiformis (five) excreted significantly more 51Cr-EDTA than controls (5.9 +/- 2.7% and 4.6 +/- 2.1%, respectively, p less than 0.001) and all were outside the normal range of 1.0-2.6%. Patients with coeliac disease (42) treated for 6 months-23 years (mean 5 years) and patients with dermatitis herpetiformis (11) treated for 6 months-8 years (mean 3 years) excreted significantly more 51Cr-EDTA than controls, 4.2 +/- 2.4% p less than 0.0001 and 3.0 +/- 0.9% p less than 0.003 respectively. Eleven of 14 (79%) treated patients with coeliac disease with an entirely normal jejunal mucosae demonstrated abnormal intestinal permeability. Intestinal permeability did not correlate significantly with either the mucosal height/crypt depth ratio or intraepithelial lymphocyte counts in jejunal biopsies from patients with untreated or treated coeliac disease. The demonstration of a persistent increase in intestinal permeability in patients with both coeliac disease and dermatitis herpetiformis may suggest a common pathogenetic mechanism in both disorders. It is postulated that altered permeability may facilitate the entry of gluten or a fraction thereof into the lamina propria where it causes a cascade of immunological events.     

Gluten challenge in children with dermatitis herpetiformis: a clinical, morphological and immunohistological study.

Twenty one children with dermatitis herpetiformis were studied in an attempt to evaluate the response in the skin, in jejunal morphology, and in jejunal immunoglobulin containing cell counts to gluten elimination and subsequent gluten challenge. In all of the 15 patients whose jejunal biopsy was studied after the eventual gluten challenge the jejunal lesion had returned in 2.4 to 28 months. The numbers of IgA- and IgM-containing cells were similarly raised in primary and postchallenge biopsies. In the 13 patients whose skin improved during a gluten free diet and who were challenged with gluten the rash worsened and the dapsone/sulphapyridine requirement increased. The jejunal deterioration was equally marked in the six patients whose gluten challenge was stopped because of an intractable rash as it was in those who completed the preplanned challenge. The specimens of the former, however, had significantly more IgA-containing cells than specimens of the latter. The number of intraepithelial lymphocytes clearly reflected the degree of intestinal damage. IgA-containing cells proved to be the most sensitive indicator of an immune reaction taking place in the gut of these patients. Even in the two children with initially normal or nearly normal jejunal mucosa, the IgA cell counts in the jejunal lamina propria were markedly raised.     

Measuring intraepithelial lymphocytes, surface area, and volume of lamina propria in the jejunal mucosa of coeliac patients.

An accurate morphometric method is described for quantifying the intraepithelial lymphocytes (IEL), the area of the epithelium, and the volume of the lamina propria in jejunal mucosa of coeliac patients. All measurements are related to a unit-length of muscularis mucosa which is unaltered by the disease process. The results show a significant decrease in the epithelial surface area and an increase in the volume of the lamina propria in coeliac jejunal mucosa compared with normal levels, even after treatment. The number of IEL is the same as normal before or after treatment. Other workers have shown an apparent increase in IEL in untreated disease which returns to normal levels after therapy. This discordance is explained and the importance of accurate quantitative methodology is stressed.     

Analysis of interleukin-4 and interleukin-10 and their association with the lymphocytic infiltrate in the small intestine of patients with coeliac disease.

BACKGROUND: Concentrations of pro-inflammatory cytokines are raised in the small intestine of patients with coeliac disease after ingestion of gluten but there are equivalent data on interleukin-4 (IL-4) and interleukin-10 (IL-10) producing cells. These cytokines are known to exert important regulatory effects on pro-inflammatory cytokine production from lymphocytes and macrophages. AIMS: To investigate whether there is a primary deficiency of IL-4 and IL-10 producing cells and their site of production in the small intestine of patients with coeliac disease in relation to the changes in inflammatory cell infiltrate. PATIENTS: Jejunal biopsy specimens from patients with coeliac disease (11 untreated, 10 treated) and nine disease controls were studied. METHODS: Immunohistochemical staining of sections for IL-4 and IL-10 cytokines and the cell phenotypic markers CD3 (T lymphocytes) and CD45 (total inflammatory cell infiltrate) was carried out using monoclonal antibodies. Expression of IL-4 and IL-10 messenger RNA was detected by in situ hybridisation with oligonucleotide probe cocktails for each cytokine. RESULTS: IL-4 and IL-10 mRNA and protein were detected in the lamina propria of treated and untreated coeliac patients and disease controls but not in the epithelium. A significant increase in the number of CD45 (p < 0.005) and CD3 (p < 0.05) positive cells was found in the lamina propria of patients with untreated coeliac disease compared with treated coeliac patients and disease controls but there were no differences in IL-4 or IL-10 between these groups with either method. CONCLUSIONS: There is no primary deficiency of IL-4 and IL-10 producing cells in the small intestine of patients with coeliac disease. Detectable concentrations of IL-4 and IL-10 were found in control patients which suggests that these cytokines are involved in normal mucosal immunoregulation. The increased number of T lymphocytes but not IL-4 or IL-10 producing cells in the lamina propria of patients with untreated than in those with treated disease suggests not only that the lamina propria is the major mucosal compartment for cytokine production but that newly recruited mucosal T lymphocytes are directed to a predominant Th1 and not a Th2 cytokine response in coeliac patients on a diet containing gluten.     

Immunohistochemical analysis of mucosal gamma-interferon production in coeliac disease.

The role of gamma-interferon in the pathogenesis of enteropathies with an immunological basis such as coeliac disease, is unclear. Gamma-interferon immunoreactive lymphocytes were quantified in jejunal biopsies from patients with coeliac disease and from normal controls. In coeliac disease, there was an apparent decrease in the percentage of both intraepithelial (3.5% v 13.5%) and lamina propria (10.3% v 47.2%) lymphocytes expressing gamma-interferon compared with controls. In patients successfully treated with a gluten free diet, the percentage of gamma-interferon immunoreactive intra-epithelial lymphocytes was 10.3%. Intraepithelial lymphocytes were immunonegative for class II major histocompatibility complex, while epithelial cells showed increased expression of this product in coeliac disease. The results show that a relatively large proportion of lymphocytes in normal small bowel express gamma-interferon. They also indicate that in coeliac disease the major increase in the numbers of mucosal lymphocytes is the result of infiltration by lymphocytes not expressing gamma-interferon.     

Morphometric electron and light microscope analysis of lymphoid cells in coeliac disease.

Lymphocytes in jejunal biopsies from normal subjects and from untreated, treated, and gluten-challenged coeliac patients were examined by accurate morphometric methods. There was no significant difference between lymphocytes from different sources and those in the jejunum. Transforming lymphocytes were ultrastructurally quite different from non-transforming lymphocytes. The appearance of transforming lymphocytes in the lamina propria in different groups of coeliac patients is in accordance with the increase in the number of plasma cells. In treated patients the results indicate a rapid humoral response to gluten challenge.     

Immunoglobulins in the jejunal mucosa in adult coeliac disease and dermatitis herpetiformis after the reintroduction of dietary gluten.

Cells containing immunoglobulin (IgA, IgG, IgM) have been measured and the distribution of extracellular and epithelial cell immunoglobulin assessed in treated patients with adult coeliac disease (ACD) and dermatitis herpetiformis (DH) before and after gluten was reintroduced to the diet. Patients with ACD and DH frequently had IgM and IgG cells above the normal range even before re-exposure to gluten, although the range of IgA cells was normal. In both diseases IgA and IgM cells increased after gluten with a proportionally greater rise in the latter, so that numbers of IgM cells, but not of IgA, exceeded the control range in all but one patient. There were increased quantities of IgA and IgM extracellularly in the lamina propria and in epithelial cells after challenge with gluten. Third component of complement was also found in some biopsies after re-exposure to gluten. These findings support the suggestion that gluten induces a humoral immunological response within the small intestinal mucosa and that both IgA and IgM systems are involved.     

Detection of interferon gamma mRNA in the mucosa of patients with coeliac disease by in situ hybridisation.

In situ hybridisation has been used to study interferon gamma (IFN gamma) mRNA expression in the small intestine of patients with coeliac disease. Sections of jejunal biopsies were obtained from five patients with treated and five with untreated coeliac disease and five disease controls. These sections were hybridised with radiolabelled specific DNA oligonucleotide probes. The lamina propria of untreated coeliac disease patients contained a significantly increased number of IFN gamma producing cells compared with controls but there was no significant difference between the coeliac patients treated with a gluten free diet and controls. The results suggest that IFN gamma may play a part in the immunopathogenesis of coeliac disease.     

The cell population of the upper jejunal mucosa in tropical sprue and postinfective malabsorption

The cell population of the upper jejunal mucosa has been studied in cases of tropical sprue from the Far East and Middle East, and in similar cases arising in western Europe (`post-infective malabsorption'), and compared with cases of untreated coeliac disease and patients without small bowel disease.

Infiltration of the epithelial layer of the upper jejunal mucosa by lymphocytes was found in tropical sprue to the same extent as in coeliac disease, and, to a lesser extent, in `postinfective malabsorption'.

In the lamina propria, in all forms of acute sprue there was an increased density of lymphocytes. With increasing duration and with increasing mucosal atrophy, the lymphocytes were progressively replaced by plasma cells, and the cellular infiltration in chronic sprue was indistinguishable from that of coeliac disease.

The findings suggest that a humoral antibody response is a feature of sprue, and becomes more prominent as the condition becomes chronic.

     

Dermatitis herpetiformis: diagnosis, diet and demography

We describe a long term study of 76 patients with dermatitis herpetiformis. Unlike patients with coeliac disease, where the peak incidence was during the first and fourth decades, no dermatitis herpetiformis patients presented in the first decade; also, there was a male preponderance in dermatitis herpetiformis which contrasts with the excess of females in coeliac disease. The apparent prevalence of dermatitis herpetiformis was 11 per 100 000 in our population; approximately one fifth of that of coeliac disease. Jejunal villous atrophy was present in 78% of our dermatitis herpetiformis patients, and a single jejunal biopsy was as effective at detecting this as the multiple biopsy technique. A majority of patients were able to stop, or radically reduce their dapsone or sulphapyridine treatment after the institution of a gluten free diet. Spontaneous remission of the skin lesion occurred in only two patients not receiving a gluten free diet. Gastric parietal or thyroid antibodies were detected in 38% of patients, and three cases of thyroid disease and two cases of pernicious anaemia were detected. Lymphoma developed in two patients, one being intestinal in origin. We conclude that a gluten free diet is of therapeutic benefit in dermatitis herpetiformis and that spontaneous remission is uncommon in those not on a diet. Despite patchiness of the enteropathy, a single jejunal biopsy is quite adequate to diagnose the presence of upper intestinal villous atrophy.     

Wheat starch-containing gluten-free flour products in the treatment of coeliac disease and dermatitis herpetiformis. A long-term follow-up study

BACKGROUND: We investigated whether wheat starch-based gluten-free products are safe in the treatment of gluten intolerance. METHODS: The study involved 41 children and adults with coeliac disease and 11 adults with dermatitis herpetiformis adhering to a gluten-free diet for 8 years on average. Thirty-five newly diagnosed coeliac patients at diagnosis and 6 to 24 months after the start of a gluten-free diet and 27 non-coeliac patients with dyspepsia were investigated for comparison. Daily dietary gluten and wheat starch intake were calculated. Small-bowel mucosal villous architecture, CD3+, alphabeta+, and gammadelta+ intraepithelial lymphocytes, mucosal HLA-DR expression, and serum endomysial, reticulin, and gliadin antibodies were investigated. RESULTS: Forty of 52 long-term-treated patients adhered to a strict wheat starch-based diet and 6 to a strict naturally gluten-free diet; 6 patients had dietary lapses. In the 46 patients on a strict diet the villous architecture, enterocyte height, and density of alphabeta+ intraepithelial lymphocytes were similar to those in non-coeliac subjects and better than in short-term-treated coeliac patients. The density of gammadelta(+)cells was higher, but they seemed to decrease over time with the gluten-free diet. Wheat starch-based gluten-free flour products did not cause aberrant upregulation of mucosal HLA-DR. The mucosal integrity was not dependent on the daily intake of wheat starch in all patients on a strict diet, whereas two of the six patients with dietary lapses had villous atrophy and positive serology. CONCLUSION: Wheat starch-based gluten-free flour products were not harmful in the treatment of coeliac disease and dermatitis herpetiformis.     

Increased jejunal intraepithelial lymphocytes bearing gamma/delta T-cell receptor in dermatitis herpetiformis.

T-cell receptor 1 (gamma/delta) expression was studied in 19 jejunal or duodenal specimens from patients with dermatitis herpetiformis and in 16 jejunal or duodenal specimens showing normal histology. In normal specimens, gamma/delta+ cells represented 10.8% of intraepithelial CD3+ lymphocytes. Around 50% of these cells were recognized by the A13 monoclonal antibody, which detects products of the V gamma 1/V delta 1 gene rearrangement and the non-disulfide-linked form of T-cell receptor 1. The remaining 50% reacted with the BB3 monoclonal antibody, which recognizes products of the V gamma 9/V delta 2 rearrangement and the disulfide-linked form of receptor. Very few gamma/delta+ cells were observed in the lamina propria. In jejunal specimens from patients with dermatitis herpetiformis, a significant increase in the prevalence of gamma/delta+ intraepithelial lymphocytes was observed (P less than 0.001). This finding was largely accounted for by an increase in those cells recognized by the A13 monoclonal antibody, thus possibly expressing the V gamma 1/V delta 1 rearrangement and the nondisulfide-linked form of receptor. These data suggest that similar pathogenetic mechanisms may be active in determining the jejunal damage in celiac disease and dermatitis herpetiformis.     

T cell depletion in untreated adult coeliac disease.

The proportional and absolute numbers of circulating thymus dependent lymphocytes (T cells) were reduced in untreated patients with coeliac disease but were normal after treatment with a gluten free diet. There was an inverse correlation between circulating T cell numbers and jejunal intraepithelial lymphocytes. This evidence suggests a possible role for T cells in the pathogenesis of coeliac disease and is a further example of disturbed cell mediated immunity in this condition.     

Malignancy and survival in dermatitis herpetiformis: a comparison with coeliac disease.

BACKGROUND--Dermatitis herpetiformis is a lifelong, gluten sensitive skin disease. Patients with dermatitis herpetiformis, similar to patients with coeliac disease not adhering to a gluten free diet, seem to have increased risk for lymphoma. AIMS--This study looked at the occurrence of malignancy and survival of patients with dermatitis herpetiformis and compared the results with those seen in patients with coeliac disease or in the general population. PATIENTS--A total of 305 adult patients with dermatitis herpetiformis diagnosed at the University Hospital of Tampere in 1970-1992 were studied. Most patients started a gluten free diet and at the end of the study 93% of the patients were adhering to the diet. A control group comprised 383 adult patients with coeliac disease, 81% of them adhered to a gluten free diet, 6% had a normal diet, and in 13% the diet history remained unknown. METHODS--The occurrence of malignant diseases and survival of the patients were assessed up to the end of 1993. Standardised incidence ratios (SIR) with 95% confidence intervals were used for the malignant diseases. The survival of the patients was compared with that of the general population. RESULTS--Thirteen (4.3%) patients with dermatitis herpetiformis developed 14 malignant disorders during the follow up (SIR 1.25; 95% confidence intervals 0.68 to 2.09). A non-Hodgkin's lymphoma occurred in four patients with dermatitis herpetiformis, significantly more than expected (SIR 10.3; 2.8-26.3). Thirteen (4.3%) patients with dermatitis herpetiformis died during the follow up but there was no increased general mortality. In coeliac disease, 13 (3.4%) patients developed malignancy (SIR 1.16; 0.62 to 1.97), 31 (8.1%) patients died but the survival rate did not differ from that in the general population. CONCLUSIONS--The incidence of non-Hodgkin's lymphoma was significantly increased in patients with dermatitis herpetiformis. The results also confirm that the patients with dermatitis herpetiformis treated mainly with a gluten free diet have no increased general mortality.     

HLA-DR expression, natural killer cells and IgE containing cells in the jejunal mucosa of coeliac children.

The expression of HLA-DR by surface and crypt epithelium and the numbers of cells of natural killer (NK) phenotype and of IgE containing cells were studied with monoclonal antisera using the peroxidase technique. We examined 48 jejunal biopsy specimens taken from 35 coeliac children before treatment (11), during gluten free diet (20) and after gluten challenge (17), and 13 control specimens. The luminal surface of the epithelial cells stained with HLA-DR antiserum in all specimens, but the cytoplasm of the surface epithelial cells took up the stain more frequently in the specimens from the controls (5/13) than those from the coeliacs (2/48) (p less than 0.01). In 21/28 specimens taken from coeliacs when on a gluten containing diet the crypt epithelium showed strong HLA-DR expression, while only 4/20 (p less than 0.01) specimens of coeliacs on a gluten free diet and 1/13 specimens of controls had similar staining. Among the intraepithelial lymphocytes no cells of NK phenotype were found in specimens from patients or controls. As compared with control specimens biopsy specimens from untreated coeliac patients showed smaller numbers of NK cells in the lamina propria. No difference was found in the numbers of IgE containing cells between the patients and controls. The strong expression of HLA-DR by the crypt epithelial cells in coeliac children on a normal diet suggest that these cells are involved in the presentation of the antigen.     

Activated eosinophils in adult coeliac disease: evidence for a local release of major basic protein.

The eosinophil population is increased in the jejunal mucosa of patients with coeliac disease. Eosinophils may participate in the mucosal damage by releasing their granule components that have cytotoxic properties such as eosinophil cationic protein (ECP) and major basic protein (MBP). This study aimed to assess the presence of ECP and MBP in the jejunal mucosa of 10 adult patients with active coeliac disease who presented with villous flattening. Endoscopic jejunal biopsy specimens were obtained from macroscopically flattened jejunal mucosa and were processed for ultrastructural study and immunogold labelling using anti-MBP, anti-ECP, and anti-IgA antibodies. Numerous eosinophils were found in the upper part of the lamina propria and showed two types of morphological change: some were lytic and others exhibited ultrastructural signs of activation, containing altered granules with fading of the central core. IgA plasma cells were intermingled with eosinophils and had dense deposits on the external side of their cytoplasmic membrane. MBP was detected in central cores of granules but also diffusely in their matrix and in tight association with dense extracellular deposits. Conversely, ECP was detected only in the matrix of eosinophil granules. This study showed that numerous eosinophils are in an activated state in the mucosa of patients with active coeliac disease and release cytotoxic proteins such as MBP, which could contribute to the mucosal damage. The observation that eosinophils and IgA plasmocytes were closely associated in the mucosa supports a role for IgA in eosinophil recruitment and activation in coeliac disease.