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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 4 1445-1452
Copyright © 2003 by The Endocrine Society

Special Feature

Histidine Decarboxylase, a Pyridoxal Phosphate-Dependent Enzyme, Is an Autoantigen of Gastric Enterochromaffin-Like Cells

Filip Sköldberg, Guida M. Portela-Gomes, Lars Grimelius, Gunnar Nilsson, Jaakko Perheentupa, Corrado Betterle, Eystein S. Husebye, Jan Gustafsson, Anders Rönnblom, Fredrik Rorsman and Olle Kämpe

Departments of Medical Sciences (F.S., A.R., F.R., O.K.), Women’s and Children’s Health (J.G.), and Genetics and Pathology (G.M.P.-G., L.G., G.N.), Uppsala University, University Hospital, 751 85 Uppsala, Sweden; Hospital for Children and Adolescents (J.P.), University of Helsinki, 00029 Helsinki, Finland; Department of Medical and Surgical Sciences (C.B.), University of Padova, 35128 Padova, Italy; and Division of Endocrinology (E.S.H.), Institute of Medicine, Haukeland Hospital, 5021 Bergen, Norway

Address all correspondence and requests for reprints to: Dr. Filip Sköldberg, Department of Medical Sciences, Uppsala University, University Hospital, 751 85 Uppsala, Sweden. E-mail: Filip.Skoldberg{at}medsci.uu.se.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Patients with autoimmune polyendocrine syndrome type 1 often have autoantibodies against neurotransmitter synthesizing enzymes, including the pyridoxal phosphate-dependent enzymes glutamic acid decarboxylase and aromatic L-amino acid decarboxylase. Using a candidate approach, we have identified the histamine-synthesizing enzyme histidine decarboxylase, also pyridoxal phosphate dependent, as an autoantigen in this disorder. Anti-histidine decarboxylase antibodies reacting with in vitro translated antigen were found in 36/97 (37%) of autoimmune polyendocrine syndrome type 1 patients studied. The antibodies also reacted with the native enzyme in HMC-1 cell lysates and did not cross-react with the highly homologous aromatic L-amino acid decarboxylase. Anti-histidine decarboxylase antibodies were associated with a history of intestinal dysfunction (P = 0.017). Gastric and duodenal biopsies from a patient with anti-histidine decarboxylase antibodies were studied by immunohistochemistry. The oxyntic mucosa was found to lack the histamine producing enterochromaffin-like cells, suggestive of an autoimmune destruction. To our knowledge, this is the first report of autoantibodies against histidine decarboxylase and absence of gastric enterochromaffin-like cells.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
AUTOIMMUNE POLYENDOCRINE syndrome type 1 (APS1), also known as autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (OMIM 240300), is an autosomal recessive disorder caused by mutations of the AIRE (autoimmune regulator) gene encoded on chromosome 21 (1, 2). The classical features are chronic mucocutaneous candidiasis, hypoparathyroidism, and adrenal failure, two of which should be present for the clinical diagnosis of APS1 (3). Other manifestations include gonadal failure, alopecia, vitiligo, insulin-dependent diabetes mellitus (IDDM), pernicious anemia, intestinal dysfunction, and chronic active hepatitis (4). APS1 can be regarded as a model disorder for organ-specific autoimmunity and recently, targeted inactivation of the AIRE gene in mice has provided an animal model, which shares some features of the human syndrome (5, 6). Several manifestations have been linked to the presence of specific autoantibodies, often directed against cytochrome P450 enzymes or neurotransmitter synthesizing enzymes, with restricted tissue distribution. These autoantigens include the pyridoxal phosphate-dependent enzymes glutamic acid decarboxylase (GAD) and aromatic L-amino acid decarboxylase (AADC; Refs. 7, 8). Intestinal dysfunction, including steatorrhea, diarrhea, and constipation is reported to be present in 15–25% of APS1 patients (3, 4, 9, 10) and is associated with an autoimmune reaction against tryptophan hydroxylase (TPH) and an autoimmune destruction of the serotonin producing enterochromaffin (EC) cells of the intestine (11, 12, 18).

There have been no reports on histamine producing enterochromaffin-like (ECL) cells being affected in APS1. As opposed to EC cells, which are primarily located in the antrum and duodenum, ECL cells are located in the oxyntic mucosa of the stomach. They express histidine decarboxylase (HDC), the histamine-synthesizing enzyme, and are the main site of histamine synthesis in the gastric mucosa (13). HDC is a pyridoxal phosphate-dependent enzyme (14) expressed in the brain, mast cells, gastric mucosa, and fetal liver (discussed in Ref. 15), structurally related to AADC and GAD (16), which are 52% homologous over 476 amino acids and 27% homologous over 316 amino acids by BLASTP alignment, respectively. The aim of the present study was to investigate whether HDC can be targeted by autoantibodies in APS1, and the possible relation to alterations of ECL cells.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Patients

Serum samples were analyzed from 10 Swedish, 16 Norwegian, 57 Finnish, and 14 Italian patients with APS1. As controls, we analyzed 44 patients with Addison’s disease, 54 patients with IDDM, 30 patients with Hashimoto’s disease, 30 patients with vitiligo, and 108 healthy blood donors. The clinical characteristics of the APS1 patients have been described elsewhere (17, 18, 19). We have used the definition of intestinal dysfunction as periodic steatorrhea, diarrhea, or severe constipation (18). Data on antibody reactivity against AADC and TPH in the Nordic patients were available from previous studies (17, 18). The methods used were approved by the local ethics committee.

Plasmids and antisera

The plasmid pVL-HDC74, containing a cDNA encoding full-length human HDC and a rabbit antiserum against human HDC, were kindly provided by Dr. Kimio Yatsunami (20). The cDNA was subcloned into the BamHI site of pGEM3zf+ (Promega Corp., Madison, WI) and the integrity verified by sequencing of the entire insert. A full-length cDNA for human AADC in pBluescript was generously donated by Dr. Hiroshi Ichinose (21). The pMALc2 expression vector was kindly provided by Dr. Mark Peakman. Bases 83–1930 of the AADC cDNA (GenBank accession no. NM_000790) were subcloned into the EcoRI site of pMALc2, for the production of recombinant AADC as described below. Normal rabbit serum was purchased from DAKO Corp. (Glostrup, Denmark).

In vitro transcription/translation

Human full-length HDC (amino acids 1–662) and AADC were expressed by coupled in vitro transcription and translation of 35S-labeled antigen in the TnT system (Promega Corp.) as previously described. Translation products were analyzed by SDS-PAGE and measurement of 35S-methionine incorporation by trichloroacetic acid precipitation, followed by scintillation counting.

Immunoprecipitation of in vitro translated antigen

Immunoprecipitation of in vitro translated HDC was carried out in 96-well plates, and all serum samples were analyzed in duplicate. An anti-HDC reactivity index of each serum was calculated as follows: (cpm of unknown sample - cpm of negative control)/(cpm of positive control - cpm of negative control) as described (22). Approximately 20,000 cpm of radiolabeled HDC and 2.5 µl serum in a final volume of 50 µl was used for each reaction. For some experiments, immunoprecipitations of HDC and AADC were carried out in microcentrifuge tubes. Bound immune complexes were then washed six times with 1 ml of 20 mM Tris-HCl (pH 8), 150 mM NaCl, 0.02% sodium azide, 1% Tween 20, and bound proteins analyzed by SDS-PAGE, followed by PhosphorImager analysis (Molecular Dynamics, Inc., Sunnyvale, CA).

Immunoprecipitation and immunoblotting of HMC-1 cell lysates

The human mast cell line HMC-1, which expresses HDC (23) was maintained as described (24). Cells were labeled for 5 h with 35S-methionine (125 µCi/ml) in DMEM with 10% dialyzed FCS, washed in PBS, and stored at -70 C. Cell pellets (~2 x 107 cells) were either lysed on ice for 1 h in 1 ml nondenaturing lysis buffer (20 mM Tris-HCl, pH 8; 150 mM NaCl; 0.02% sodium azide; 1% Triton X-100) or resuspended in 50 µl PBS, followed by addition of 50 µl 2x denaturing lysis buffer (20 mM Tris-HCl, pH 8; 150 mM NaCl; 0.02% sodium azide; and 2% sodium dodecyl sulfate) and denatured at 80 C for 10 min. Denatured lysates were diluted with 10 vol nondenaturing lysis buffer and incubated on ice for 30 min. Insoluble material was removed by centrifugation at 16,000 x g for 30 min. In immunoprecipitations, nondenatured lysates were used for human sera, whereas denatured lysates were used for rabbit sera. Immunoprecipitation, SDS-PAGE, and transfer to nitrocellulose membranes were performed essentially as described (25), except that antibodies were bound to Fast Flow Protein A-Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden) before adding the cell lysates. Bound proteins on the same nitrocellulose membrane were analyzed both by phosphorimaging and by probing with a specific anti-HDC antiserum (dilution 1:5000; Ref. 20). Antibody binding was detected using a horseradish peroxidase conjugated rat antirabbit antibody (Amersham Pharmacia Biotech) diluted 1:5000 and Western Blotting Luminol Reagent chemiluminescent substrate (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Competition experiments

To investigate possible cross-reactivity between anti-HDC antibodies and AADC, competition experiments were performed essentially as described (26). The pMALc2 bacterial expression vector was used to express amino acids 4–480 of human AADC as a fusion protein with maltose binding protein (MBP) in the XL1-Blue MRF’ Escherichia coli strain grown in Terrific Broth. Protein expression was induced with 0.5 mM isopropyl-ß-D-thiogalactopyranoside at 18 C. Cells were harvested after 22 h and lysed with lysozyme and Triton X-100 as described (27). Competition was conducted by preincubating the sera with E. coli extract containing approximately 10 µg MBP-AADC fusion protein, or control E. coli extract, for 1 h at 4 C before adding 35S-labeled in vitro translated HDC or AADC and conducting an immunoprecipitation as described above. Immune complexes were analyzed by SDS-PAGE, followed by phosphorimaging.

Immunohistochemistry

Archival, paraffin-embedded biopsies were available from one single APS1 patient who had undergone gastroscopy because of epigastrial pains. This was a 26-yr-old woman with a history of mucocutaneous candidiasis, hypoparathyroidism, adrenal failure, hypogonadism, alopecia, and vitiligo. At gastroscopy, no macroscopic abnormalities were found. As controls, paraffin sections of histologically normal mucosa from patients who underwent surgery for gastric tumors were immunostained.

Biopsies from gastric corpus, antrum, and distal duodenum of the present patient (n = 3 each) and controls were fixed in 10% buffered neutral formalin and routinely processed to paraffin. The sections, 4 µm thick, were stained with hematoxylin-eosin, according to van Gieson or immunostained using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA) with mouse monoclonal antibodies to human chromogranin A (CgA, clone LK2H10, Roche Molecular Biochemicals, Mannheim, Germany; dilution 1:1000), human serotonin (clone 5HT-H209, code no. M0758, DAKO Corp., Glostrup, Denmark; dilution 1:50) human N-terminal gastrin (clone 4C7A1, code no. 1537, Immunotech, Marseille, France; dilution 1:500), or human mast cell tryptase (code no 444905, Calbiochem-Novabiochem, San Diego, CA; dilution 1:3000) and a rabbit polyclonal antiserum to human C-terminal vesicular monoamine transporter-2 (VMAT-2; code no. H-V003, Phoenix Pharmaceuticals, Inc., Belmont, CA; dilution 1:2000), or human somatostatin (code no. A 568, DAKO Corp.; dilution 1:500).

All immunostainings were performed without microwave pretreatment unless indicated. Furthermore, immunostaining for CgA was performed on paraffin sections pretreated for enhanced sensitivity in a microwave oven (Whirlpool Nordic AB, Stockholm, Sweden) for 2 x 5 min at 750 W, using a citrate buffer (pH 6.0) as retrieval solution.

Statistical analyses

Frequencies of intestinal dysfunction and other manifestations in APS1 patients with and without anti-HDC antibodies were compared by the use of Fisher’s exact test. P value less than 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Immunoprecipitation of in vitro translated HDC with patient sera

Coupled in vitro transcription and translation of the full-length HDC and AADC cDNA yielded major products of approximately 64 kDa and 50 kDa, respectively, as estimated by SDS-PAGE. The in vitro translated HDC was recognized specifically by the anti-HDC antiserum in immunoblotting (data not shown). In vitro translated HDC was used in a 96-well immunoprecipitation assay, and the antibody reactivity was expressed as an anti-HDC reactivity index. The results from the 96-well immunoprecipitation assay are shown in Fig. 1Go. Using a cut-off value at 0.188, at 4 SD above the mean index value of the blood donors, 36 of 97 APS1 patients (37%) were positive, whereas all of the control sera tested were negative. We have not been able to detect any antibody reactivity directed against the C-terminal approximately 20-kDa fragment (amino acids 478–680) expressed in vitro (data not shown).



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  		Figure 1. Scattergram showing the anti-HDC antibody reactivity of serum samples from patients with APS1 (n = 97), Addison’s disease (n = 44), IDDM (n = 54), Hashimoto’s thyroiditis (n = 30), vitiligo (n = 30), and blood donors (n = 108). Dashed line indicates the cut-off value of positive results (mean value of blood donors + 4 SD).

 
Immunoprecipitation and immunoblotting of HMC-1 cell lysates

To determine whether the antibodies reacting with in vitro translated HDC were also able to immunoprecipitate the native protein from lysates of cells expressing endogenous HDC, immunoprecipitation experiments with 35S-methionine-labeled HMC-1 cell lysates were performed. Patient sera previously found to be positive in the anti-HDC assay reacted with a protein migrating at approximately 64 kDa, which comigrated with the major band immunoprecipitated with a specific rabbit anti-HDC serum (Fig. 2AGo). Sera of anti-HDC-negative patients and healthy blood donors did not react with this protein. Immunoblotting of the same nitrocellulose membrane with the rabbit anti-HDC serum showed that HDC was present in these lanes and had an apparently identical molecular mass, strongly suggesting that the radioactive 64-kDa band represents HDC (Fig. 2BGo).



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  		Figure 2. Immunoprecipitates of 35S-methionine labeled HMC-1 cell lysates subjected to SDS-PAGE and transfer to nitrocellulose, followed by analysis by phosphorimaging (A), and immunoblotting with a specific anti-HDC rabbit antiserum (B), respectively. Lane 1, Anti-HDC rabbit antiserum; lane 2, normal rabbit serum; lanes 3–5, anti-HDC positive APS1 patient sera; lane 6, anti-HDC negative APS1 patient serum; lanes 7 and 8, healthy blood donor sera; lane 9, unlabeled HMC-1 cell lysate; lane 10, prestained molecular weight standard (not visible); and lane 11, in vitro translated HDC. B, Composite of two exposure times (lanes 1 and 2 and 3–11, respectively) due to high background in the first two lanes.

 
Competition experiments

Considering the sequence similarity between AADC, a well known autoantigen in APS1, and HDC we investigated whether anti-HDC reactivity could be reduced by preincubation with excess AADC. As shown in Fig. 3Go, it was found that the amount of HDC immunoprecipitated by different sera was only marginally decreased by preincubation with AADC. This was true for sera, which had both anti-HDC and anti-AADC reactivity, as well as a serum, which was only anti-HDC positive. On the other hand, binding of radiolabeled AADC was almost abolished when preincubating sera with unlabeled AADC.



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  		Figure 3. Immunoprecipitation of 35S-methionine labeled in vitro translated HDC (first and second rows), and AADC (third and fourth rows), respectively, followed by SDS-PAGE and phosphorimaging. Serum samples were preincubated with extracts of bacteria expressing MBP alone (first and third rows) or MBP-AADC fusion protein (second and fourth rows) before adding the respective radiolabeled antigen. Lanes 1 and 2, Two different APS1 patient sera positive for anti-HDC and anti-AADC antibodies; lane 3, APS1 patient serum positive for anti-HDC and negative for anti-AADC antibodies; lane 4, APS1 patient serum negative for anti-HDC and positive for anti-AADC antibodies; and lane 5, serum from a healthy blood donor.

 
Clinical characteristics of anti-HDC positive APS1 patients

Anti-TPH antibodies and the specific loss of TPH expressing EC cells of the gastrointestinal tract have been found in APS1 patients with intestinal dysfunction (18, 28). As HDC is present in a distinct endocrine cell type of the gastrointestinal tract, namely the ECL cells, we investigated whether anti-HDC antibodies may also be linked to intestinal dysfunction. APS1 patients with and without anti-HDC antibodies were compared regarding the frequency of intestinal dysfunction and other supposedly autoimmune manifestations of APS1. Fifteen of twenty-six (58%) of patients with a history of intestinal dysfunction were anti-HDC positive, compared with 20 of 71 (28%) patients without intestinal dysfunction (P < 0.02). Anti-HDC antibodies were also found more frequently among patients with a history of signs of chronic active hepatitis (11 of 16, 69%) than those without (25 of 81, 31%; P < 0.01). No other associations between anti-HDC antibodies and different manifestations of APS1 were found (Table 1Go). Data on AADC and TPH immunoreactivity was available in 83 patients, 21 of which had a history of intestinal dysfunction (17, 18). No patients with intestinal dysfunction were identified which were anti-HDC positive and anti-TPH negative, suggesting that anti-HDC antibodies do not add to the sensitivity in identifying patients with intestinal dysfunction. Notably, 13 of 21 (62%) patients with intestinal dysfunction were positive for all three antibodies, whereas this was the case in only 12 of 62 (19%) patients without intestinal dysfunction (P = 0.0006).


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  		Table 1. Clinical disorders and anti-HDC antibodies in 97 patients with APS1

 
Immunohistochemical findings

To investigate whether anti-HDC antibodies could be linked to loss of HDC-expressing cells (ECL cells and mast cells), sections of biopsies from the corpus, antrum, and duodenum of an HDC-positive APS1 patient were studied. The biopsies from the corpus showed some atrophy of the mucosa and those from the antrum a slight foveolar hyperplasia. The duodenal mucosa showed a normal histology. None of the biopsies displayed any signs of inflammation. Parietal cells and chief cells could be identified by hematoxylin-eosin staining (data not shown). The histopathological findings are summarized in Table 2Go.


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  		Table 2. Summary of histological findings in the gastric and duodenal mucosa of the APS1 patient studied

 
Sections of the mucosal biopsies were immunostained for CgA, which is expressed in several endocrine cells (29). In the corpus, only very few CgA-positive cells could be observed (Fig. 4AGo) compared with the normal control section (Fig. 4BGo). In the APS1 patient biopsies, these cells could only be detected after microwave pretreatment of the sections to enhance sensitivity (Fig. 4AGo), whereas this was not required for the control sections (Fig. 4BGo).



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  		Figure 4. Immunohistochemical stainings of biopsies from the gastric corpus (A–H), antrum (I–L), and duodenum (M and N) obtained from an anti-HDC-positive APS1 patient (A, C, E, G, I, K, and M) and control patients (B, D, F, H, J, L, and N), respectively. A, Corpus mucosa of the APS1 patient stained for CgA. The mucosa displays slight atrophy and marked reduction of CgA positive cells (microwave pretreated section). Two CgA-positive cells can be seen (indicated by the square). Inset, Higher magnification of the immunoreactive cells. Bar, 100 µm; inset bar, 35 µm. B, Normal corpus mucosa stained for CgA showing numerous immunoreactive cells (no microwave pretreatment). Bar, 100 µm. C, Corpus mucosa of the APS1 patient stained for VMAT-2. A few immunoreactive cells, presumably mast cells, are present in the stroma (indicated by arrows). Bar, 42 µm. D, Normal corpus mucosa stained for VMAT-2. Numerous positive cells are present, both in the glandular epithelium (ECL cells) and in the stroma. Bar, 100 µm. E, Corpus mucosa of the APS1 patient stained for somatostatin, displaying only a single immunoreactive cell (indicated by the arrow). Bar, 42 µm. F, Normal corpus mucosa stained for somatostatin. Bar, 42 µm. G, Corpus mucosa of the APS1 patient stained for mast cell tryptase. Immunoreactive mast cells are present in the stroma. Bar, 42 µm. H, Normal corpus mucosa stained for mast cell tryptase. Bar, 50 µm. I, Antral mucosa of the APS1 patient stained for CgA, displaying a normal frequency of immunoreactive cells. Bar, 63 µm. J, Normal antral mucosa stained for CgA. Bar, 63 µm. K, Antral mucosa of the APS1 patient stained for gastrin, displaying a normal frequency of immunoreactive cells. Bar, 63 µm. L, Normal antral mucosa stained for gastrin. Bar, 63 µm. M, Duodenal mucosa of the APS1 patient stained for CgA, displaying a normal frequency of immunoreactive cells. Bar, 63 µm. N, Normal duodenal mucosa stained for CgA. Bar, 63 µm.

 
The histamine producing ECL cells are the major CgA positive cell type in the oxyntic mucosa (30), and to confirm that ECL cells were lacking, we did immunostainings of the corpus biopsies with a VMAT-2 antiserum (31). The corpus mucosa completely lacked ECL cells detected by VMAT-2 staining (Fig. 4CGo). Some cells were present in the stroma, presumably mast cells, expressing VMAT-2 (Fig. 4CGo, indicated by arrows). The normal control section contained numerous ECL cells in the middle portion of the crypts (Fig. 4DGo). Somatostatin cells, which do not express CgA in the corpus (29), were almost completely absent from the corpus mucosa. In three sections from separate biopsies from the APS1 patient, one single somatostatin-positive cell could be found (Fig. 4EGo). In the control section, a small number of somatostatin-positive cells were seen scattered in the mucosa (Fig. 4FGo). The presence of mast cells could be detected in both the APS1 patient biopsies and the control sections by staining for mast cell tryptase (Fig. 4Go, G and H). In the antrum, the number of CgA-positive and gastrin-positive cells was comparable to that in normal biopsies (Fig. 4Go, I–L). The abundance of CgA-positive cells in the duodenal biopsies from the APS1 patient and control patient was also similar (Fig. 4Go, M and N). Serotonin-positive EC cells could be detected in the duodenal biopsies of both the APS1 patient and the control patient (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
Using a candidate approach based on structural similarity, we have identified HDC as a novel pyridoxal-phosphate dependent autoantigen in APS1. The anti-HDC antibodies were found to react with in vitro translated HDC as well as the native enzyme present in HMC-1 cells and showed little cross-reactivity with recombinant AADC, indicating that unique epitopes are targeted. Purified HDC has been found to have a lower molecular weight than predicted from the cDNA sequence (32, 33, 34). This difference is believed to be due to posttranslational cleavage of the C-terminus, which does not have a homolog in the AADC amino acid sequence. In rat gastric mucosa, this has been proposed to represent proteolytic activation of the HDC (35). We have not been able to detect antibody reactivity directed against the C-terminal approximately 20-kDa fragment, indicating that the antibodies are directed against the part of the protein that is similar to AADC. Knowledge of the existence of specific non-cross-reactive autoantibodies against these distinct proteins with structural similarities may be useful for future mapping of conformational epitopes using chimeric hybrid molecules and also for studies of possible intermolecular epitope spreading (36, 37).

In the APS1 patient material studied, anti-HDC antibodies were associated with intestinal dysfunction and chronic active hepatitis. HDC is expressed in a subset of endocrine cells of the stomach, the ECL cells, and an immune reaction to these cells may possibly be linked to gastrointestinal symptoms. In adult liver on the other hand, HDC is not normally detectable at significant levels, suggesting that the association with chronic active hepatitis may rather be of indirect nature.

Gastric and duodenal biopsies were available from one patient with anti-HDC antibodies for immunohistochemical studies. It was found that the oxyntic mucosa almost completely lacked endocrine cells, including ECL cells, suggestive of an autoimmune destruction. Högenauer et al. (28) reported an APS1 patient with gastrointestinal symptoms, in which a subset of duodenal endocrine cells were absent, and subsequently reappeared in conjunction with clinical improvement. This suggests that enteroendocrine cells are not lacking, e.g. due to a congenital aplasia caused by the defect AIRE gene, and supports the hypothesis of an autoimmune pathogenesis. To our knowledge, this is the first report of absence of ECL cells in human and provides a novel basis for studies of ECL cell function in the human gastric mucosa. In contrast, mucosal mast cells, which also express HDC, could be detected at all levels. The patient studied also had antibodies against AADC and TPH (8, 18). AADC activity has been demonstrated in ECL cells (38) and both AADC and TPH has been detected in serotonin-containing EC cells (18, 39). This highlights that the connection between autoantibody reactivity, antigen distribution, and histopathological findings in autoimmune disorders is yet to be fully understood. Similarly, the widely expressed E2 component of the pyruvate dehydrogenase enzyme complex is the main autoantigen in primary biliary cirrhosis, where biliary epithelial cells are specifically targeted by the immune system (discussed in Ref. 40). Possibly, these apparent discrepancies could be related to differences in proteasome mediated processing and presentation of autoantigenic epitopes (41).

Autoantibodies against intracellular proteins are generally not believed to be pathogenic but may reflect activation of autoreactive T cells, which could mediate target cell destruction. The identification of HDC as an autoantigen illustrates the intriguing propensity of intracellular enzymes to be the targets of B cell responses in autoimmune disorders (42). These enzymes may have conserved structural features that can provoke immune responses or may not be presented to T cells in the thymus or periphery to a sufficient extent to achieve immunological tolerance. The gene mutated in APS1, AIRE, and its murine ortholog, have been identified (1, 2, 43) and targeted inactivation of this gene in mice may provide a tool for addressing this issue (5, 6).

In conclusion, we have found that HDC, the histamine-synthesizing enzyme, is a B cell autoantigen in APS1 and report an APS1 patient with anti-HDC antibodies who was found to lack ECL cells in the gastric oxyntic mucosa. These findings extend our knowledge of autoantibody specificity and alterations of endocrine cells in this disorder. They also provide a basis for studies of ECL cell physiology in human and a candidate antigen for the development of immunotherapy of ECL-derived tumors. Notably, the vitiligo-associated APS1 autoantigen SOX10 (19) was recently found to be targeted by tumor infiltrating lymphocytes in a patient with malignant melanoma who responded to immunotherapy (44).


   Acknowledgments
 
We thank Dr. Mona Landin-Olsson for sera from diabetes patients, Dr. Anthony P. Weetman for sera from vitiligo patients, Dr. Kimio Yatsunami for the pVL-HDC74 plasmid and the anti-HDC antiserum, Dr. Hiroshi Ichinose for the human AADC cDNA, and Dr. Mark Peakman for the pMALc2 plasmid. We are also indebted to Dr. Rolf Håkanson and Niclas Olsson for valuable discussions and technical advice, and to Lars Berglund (Uppsala Clinical Research Center) for expert statistical advice.


   Footnotes
 
This work was supported in part by the Medical Research Council, the Torsten and Ragnar Söderberg Foundation, the Petrus and Augusta Hedlund Foundation, the Swedish Medical Society, the Claes Groschinsky Memorial Foundation, the Agnes and Mac Rudberg Foundation, the Tore and Wera Cornell Foundation, and the Professor Nanna Svartz’ Foundation.

Abbreviations: AADC, Aromatic L-amino acid decarboxylase; AIRE, AIRE autoimmune regulator; APS1, autoimmune polyendocrine syndrome type 1; CgA, chromogranin A; EC, enterochromaffin; ECL, enterochromaffin-like; GAD, glutamic acid decarboxylase; HDC, histidine decarboxylase; IDDM, insulin-dependent diabetes mellitus; MBP, maltose binding protein; TPH, tryptophan hydroxylase; VMAT-2, vesicular monoamine transporter-2.

Received November 12, 2002.

Accepted January 2, 2003.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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