**2. Histopathological analysis of esophagus in MCTD patients**

#### **2.1. Comparison between changes in the upper, middle, and lower portion of the esophagus.**

To date, there have been studies demonstrating a high frequency of esophageal symptoms in patients with MCTD [1-7,13] (Table 1). In our study, evidence of histological changes was found in 25 of the 27 cases examined (91%). The differences may be due to differences in the method of measurement. Esophageal dysmotility in MCTD patients is sometimes associated with the dilatation of the distal esophagus (Figure 1). The main sites of esophageal change were generally different between CTDs [8]. In patients with SSc and MCTD, the lower portion of the esophagus changes histologically. Therefore, we examined 3 different regions of the

esophagus, which we defined as follows: 1) upper, at the height of the ring around the cartilage of the trachea; 2) middle, at the height of the bifurcation of the trachea; and 3) lower, just above the esophago-cardiac junction. We compared histological changes for each portion. Of 12 cases examined, 9 showed slight to severe changes in the lower portion, 3 showed slight to severe changes in the middle portion, and none showed histopathological changes in the upper portion. According to these results, the lower portion was involved in many cases of MCTD.

**Figure 1.** X-ray photograph of esophagus in MCTD patients.



**Table 1.** The frequency of esophageal involvement in patients with mixed connective tissue disease.

#### **2.2. Comparison between changes in the inner circular muscular layer and outer longitudinal muscular layer of the esophagus**

As regards histological changes in the muscular layers, the inner circular muscular layer (IM) exhibited more severe changes than the outer longitudinal muscular layer (OM) in 17 of 27 cases (63%). Eight cases (30%) showed similar changes. Two cases (7%) showed no pathological changes in either IM or OM, and no cases (0%) showed more severe involvement of OM than IM. Muscular dynamisms of IM and OM in esophageal motility are different. The IM is fairly active and subject to greater stress than the OM [14]. Furthermore, esophageal regurgitation often occurs and exerts direct effects on the IM, particularly in the lower esophagus. Thus the IM in the lower portion may carry a larger physical stress than the OM. Therefore, more severe histological changes may occur in the IM of the lower portion than in the OM.

#### **2.3. Cellular and tissue change**

esophagus, which we defined as follows: 1) upper, at the height of the ring around the cartilage of the trachea; 2) middle, at the height of the bifurcation of the trachea; and 3) lower, just above the esophago-cardiac junction. We compared histological changes for each portion. Of 12 cases examined, 9 showed slight to severe changes in the lower portion, 3 showed slight to severe changes in the middle portion, and none showed histopathological changes in the upper portion. According to these results, the lower portion was involved in many cases of MCTD.

**Figure 1.** X-ray photograph of esophagus in MCTD patients.

230 Seminars in Dysphagia

• Heartburn 10/17 (58.8%) • Regurgitation 6/17 (35.3%) • Dysphagia 1/17 (5.9%) **Abnormal esophageal motility** 14/17 (82.4%)

• Dysphagia 6/12 (50%) **Abnormal esophageal motility** 6/12 (50%)

• Heartburn or regurgitation 29/61 (47.5%) • Dysphagia 23/61 (37.7%) **Abnormal esophageal motility** 21/35 (60.0%)

• Heartburn 5/21 (23.8%)

**Symptoms and dysmotility Actual number (Frequency) Reference Abnormal esophageal motility** 8/17 (47.1%) Bennett (1980) [1] **Esophageal symptoms** 11/17 (64.7 %) Gutierrez (1982) [2]

**Esophageal symptoms** Dantas (1985) [3]

**Esophageal symptoms** Marshall (1990) [4]

**Esophageal symptoms** 14/21 (66.6%) Doria (1991) [5]

In our study, the most striking change of the esophagus in MCTD was severe atrophy and occasional disappearance of muscular fibers followed by fibrosis in muscular layer (Figure 2). In contrast to smooth muscle, however, striated muscle of the upper esophageal portion exhibited no marked changes. Similar histopathological changes occur in SSc [15,16]. In SSc patients, histological features are also characterized by degeneration and disappearance of smooth muscle cells with fibrosis, especially in the IM of the lower portion [17]. In our study, ganglionic cells had not decreased in number and were not particularly atrophic except in severely fibrotic areas. Vascular changes were also not overly severe in non-fibrotic regions, although slight intimal thickening of small vessels was sporadically found in the fibrotic area. The vein wall was injured and smooth muscle cell disruption and inflammatory cell invasion were observed (Figure 3).

#### **2.4. Pathogenesis of esophageal lesions**

The factors that seem to be associated with esophageal dysfunction have been reported in some studies, and include extracellular matrix degradation, disorder of blood circulation, and

**Figure 2.** Esophageal muscle degeneration and fibrosis in MCTD patients.

**Figure 3.** Vascular changes in the esophagus of MCTD patients.

autoantibodies [10,18-20]. Our hypothesis was that autoantibodies are associated with the pathogenesis of esophageal lesions. In immunohistochemical studies, anti-human IgG and anti-C3 antibodies reacted positively with muscle tissues showing a myolytic appearance accompanied by edema and inflammatory cell infiltration in MCTD autopsy case (Figures 4). No IgM deposition was found (Figure 4). The reactivity of IgG extracted from sera of MCTD patients against normal esophageal tissues was then assessed. Esophageal tissues used here were non-cancerous parts taken intraoperatively from esophageal cancer patients without specific immunological disorder. The IgG reacted with smooth muscle cells in the muscularis mucosa, muscular layer and venous wall, the ganglion cells in Auerbach's plexus, and squamous epithelium of the esophagus (Figure 5), but did not react with striated muscle in upper portion (Figure 5 A,B). IgG from MCTD patients also reacted with primary-cultured smooth muscle cells prepared from surgical specimens of esophagus (unpublished data) (Figure 6). These results suggested that antibodies in the serum of patients with MCTD attack smooth muscle tissues as well as other tissues of the esophagus.

**Figure 4.** Immunoglobulin and complement deposition in the muscular layer of the esophagus from MCTD patients. Deposition of IgG (A), IgM (B), and complement C3 (C).

autoantibodies [10,18-20]. Our hypothesis was that autoantibodies are associated with the pathogenesis of esophageal lesions. In immunohistochemical studies, anti-human IgG and anti-C3 antibodies reacted positively with muscle tissues showing a myolytic appearance accompanied by edema and inflammatory cell infiltration in MCTD autopsy case (Figures 4). No IgM deposition was found (Figure 4). The reactivity of IgG extracted from sera of MCTD patients against normal esophageal tissues was then assessed. Esophageal tissues used here were non-cancerous parts taken intraoperatively from esophageal cancer patients without specific immunological disorder. The IgG reacted with smooth muscle cells in the muscularis mucosa, muscular layer and venous wall, the ganglion cells in Auerbach's plexus, and squamous epithelium of the esophagus (Figure 5), but did not react with striated muscle in upper portion (Figure 5 A,B). IgG from MCTD patients also reacted with primary-cultured

**Figure 2.** Esophageal muscle degeneration and fibrosis in MCTD patients.

232 Seminars in Dysphagia

**Figure 3.** Vascular changes in the esophagus of MCTD patients.

**Figure 5.** Reaction of IgG from MCTD patients with smooth muscles and other cells composing the esophagus. (A) Esophageal smooth muscle tissue, (B) Higher magnification of esophageal smooth muscle tissue, (C) Medial smooth muscle of the venous wall, (D) Ganglionic cell in Auerbach's plexus, (E) Squamous epithelium of esophagus

**Figure 6.** Reaction of IgG from MCTD patients with primary-cultured smooth muscle cells from esophagus.
