Can antibodies reacting with Auerbach's plexuses be demonstrated in achalasia?
W.B. Storch (Heidelberg)
Achalasia is a motor disorder of the esophagus characterized by decrease in ganglion cell density in Auerbach's plexus. Aetiology and pathogenesis of this disease are unknown. We described recently the occurrence of autoimmune phenomena in patients with achalasia, in particular circulating antibodies against Auerbach's plexus [1, 2]. The present study, therefore, was designed to extend our previous studies.
Patients and methods
Studies were performed on sera of 67 patients with primary achalasia of the esophagus after manometric, radiographic and endoscopic diagnosis. Patients ranged from 17 to 88 years in age with a mean of 48 years. The patients exhibited different stages of the disease [I-IV]. The duration of the disease ranged from 1 to 20 years. Sera from 54 healthy subjects (age range: 17 to 83 years, mean age 50.2 years) as well as from 12 patients with congenital megacolon (Hirschsprung's disease, aganglionosis coli), 12 patients with myasthenia gravis, 12 patients with cancer of esophagus, and 11 patients with peptic esophagitis served as controls.
Antibodies against Auerbach's plexus were determined by standard indirect immunofluorescence [3, 4]. Briefly, 4 mm cryotome sections of a block consisting of rat stomach, liver and kidney were used as antigen substrate. Antihuman-IgG serum, marked with fluoresceinisothiocyanate and further substituted with Evans blue (Mardx, USA, and Viramed, München, Germany) served as the conjugate. For the evaluation of antigen antibody complexes we used fluorescence microscope (Axiophot, Oberkochen, Germany, Leica DM, Wetzlar, Germany, and Olympus BX 60, Olympus Opt. Corp. Europa, Hamburg, Germany) as well as confocal laser scanning microscopy (Leica TCS, Wetzlar, Germany, LSM 4, Zeiss, Oberkochen, Germany). Co-localization studies to show neurone structure were performed by double labelling using mouse monoclonal antibodies against the neurofilament protein kDa 68 (code 814326, Boehringer, Mannheim, Germany) and a Cy 3-marked anti-mouse-immunoglobulin (Jackson Immuno Research Laboratories, Dianova, Hamburg, Germany). A positive reaction was defined as a strong cytoplasmic immunofluorescent labelling of Auerbach's plexus cells and nerve fibres in serum diluted 1:20. We measured Auerbach's plexus antibody titres by doubling dilution of sera in phosphate-buffered saline.
The data were tested for statistical significant differences using Fisher's exact test. The level of significance was set to p < 0.05.
Results
A majority of patients with achalasia exhibited positive test results (Table I). This occurred only in few of the controls. Most of the reactions were seen at low titres (1:20), this was the case in all healthy controls. Only 2 patients with achalasia exhibited fluorescence when the sera were diluted 1:160 and 1:320, respectively. Positive test results did not correlate with either the duration or the severity of the disease.
All sera from patients with congenital megacolon and cancer of esophagus gave negative results. Only one of 11 patients with peptic esophagitis as well as one of 13 patients with myasthenia gravis had a positive result (titre 1:20).
Discussion
This study showed that about two thirds of patients with achalasia of the esophagus exhibit autoantibodies against Auerbach's plexus, while this phenomenon is rarely observed in
healthy subjects. These newly described antibodies against Auerbach's plexus were occasionally observed also in diseases which are unrelated to achalasia and not characterized by
ganglion cell degeneration [3, 5]. The question, therefore, arises whether these antibodies are epiphenomena or factors that are related to the cause of the disease. The antibodies exhibited a considerable heterogeneity. Most recently, it was shown that patients with achalasia exhibit antibodies against nerve fibres of the small bowel [6]. Whether these are related or identical to the currently described antibodies against Auerbach's plexus remains to be determined.
We were unable to detect any correlation between the presence or absence of the antibodies with the stage of the disease. Further subgroups of patients are being studied.
This demonstration of antibodies against Auerbach's plexus should stimulate further studies investigating the role of autoimmune phenomena in the aetiology of achalasia. Research is needed to test whether the antibody status is predictive of disease susceptibility and to identify the antigenic structures of the antibodies as well as to clarify the heterogeneity of the fluorescence patterns.
References
3. Storch W. Antikröper gegen Auerbach'schen Plexus und Glomerula. Lab Med 1993;17:343-346.