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Pathology International 2014; 64: 361–363
doi:10.1111/pin.12172
Letter to the Editor
Lymphocytic gastritis showing concomitant occurrence of both CD4 + and CD8 + T-cells among epithelial cells To the Editor: Lymphocytic gastritis (LG), first described by Haot et al.,1 is microscopically featured by marked increase of intraepithelial lymphocytes (IELs), more than 25 IELs per 100 epithelial cells, in addition to mucin depletion, nuclear stratification of the foveolar cells and chronic active inflammation in the lamina propria mucosae.2 Common etiologies of LG include celiac disease and Helicobacter pylori (Hp) infection: LG was found in 33% of patients with celiac disease and 4.1% of histopathologically defined Hp gastritis.3 The frequency of LG was highest in celiac disease without Hp infection (50%), followed by Hp-infected celiac disease (20%), Hp infection without celiac disease (8.3%), but none in the control group.4 Kushima and Borchard described relative infrequency of Hp colonization in LG when compared with conventional chronic gastritis.5 Varioliform gastritis, endoscopically showing nodular targetoid and eroded lesions along proximal rugae, represents a form of LG.6 Intraepithelial lymphocytes in LG belong to T-cells, express CD8+, and are mostly positive for granzyme B, being categorized in activated cytotoxic T-cells.7 Expression of CD4 on IELs is an exceptional event.8 We briefly report herein a case of unique LG showing concomitant occurrence of both CD4 + and CD8 + T-cells among the epithelia. A 53 year-old Japanese female felt upper abdominal discomfort, and endoscopic evaluation revealed mild mucosal redness and rugal swelling on the gastric body. Hp eradication therapy was given 5 years earlier, with urea breath test being negative. An ulcer scar at the supra-angular region was biopsied. A control case of LG was exampled from the antral mucosa of a 55-year-old male case. No features of celiac disease were found in either cases. The gastric biopsy of the present case and control case was performed at Takahashi Clinic and Komori Clinic, respectively. Both biopsy samples histologically revealed numerous small lymphocytes among surface epithelia. The foveolar cells were regenerative and the mucin content was decreased. Dense lymphocytic infiltration was seen in the lamina propria, but without Hp colonization and formation of lymphoid follicles or lymphoepithelial lesions. Two LG samples were evaluated by immunostaining with the amino acid polymer method (Simple Stain Max, Nichirei, Tokyo). Primary mouse monoclo-
nal antibodies targeted at CD3, CD4, CD8, granzyme B (Dako, Carpinteria, USA), perforin (Novocastra, Newcastle, UK), T-cell-restricted intercellular antigen-1 (TIA-1 = nucleolysin: Coulter Immunology, Tokyo, Japan), CD25 (Nichirei) and FoxP3 (Abcam, Cambridge, UK). Granzyme B, perforin and TIA-1 are representative cytotoxic substances in azurophilic granules of killer T-cells and natural killer cells.7 CD25 and FoxP3 are the markers of regulatory (suppressor) T-cells, expressed on the plasma membrane and in the nuclei of CD4+ T-cells.9 Pressure pan-assisted heat-induced epitope retrieval was performed in 10 mM citrate buffer, pH 6 or 7 or in 1 mM ethylenediamine tetraacetic acid. Diaminobenzidine gave a brown color reaction. Sections of the tonsil served as positive control. Using consecutive sections, the marker-positive IELs were counted on color prints after taking a total of 20 (present case) or 29 (control case) high-powered photomicrographs using a x40 objective lens. CD3+ IELs in the present and control cases summed up 82 ± 30 and 102 ± 49 per 100 foveolar epithelial cells, respectively. In the present case, both CD4+ and CD8+ IELs were distributed among the foveolar cells, while in the control case only CD8+ IELs were observed. Figure 1 illustrates representative features of expression of CD3, CD4 and CD8. CD3+ IELs consisted of both CD4+ T-cells and CD8+ T-cells (CD4: 33 ± 22 and CD8: 53 ± 27 per 100 epithelial cells). In the lamina propria, CD4+ T-cells predominated over CD8+ T-cells. Three killer substances in CD8+ T-cells were further evaluated. In the present case, TIA-1-positive cells predominated over granzyme B- and perforin-positive cells (TIA-1 20 ± 14, granzyme B 15 ± 8, and perforin 6 ± 8). On average, 38% (20/53) of CD8+ IELs expressed TIA-1. In the control case, more IELs were labeled for granzyme B and perforin than TIA-1 (TIA-1 38 ± 25, granzyme B 68 ± 38, and perforin 55 ± 26), and CD3+ IELs expressed CD8 alone, with granzyme B and perforin seen in 66% (68/103) and 53% (55/103) of the IELs, respectively. Representative microscopic features are shown in Fig. 2. Supplementary Figure S1 summarizes the results in a column chart: the data of the present case are shown in red, and the control data in blue. For evaluating intraepithelial CD4+ cells in the present case as regulatory T-cells, CD25 and FoxP3 were immunostained. The results were negative. In both cases, a few lymphoid cells in the lamina propria were labeled for CD25 on the plasma membrane and for FoxP3 in the nuclei. A novel phenotype of IELs in LG was documented in the present case, devoid of celiac disease or Hp infection. In
© 2014 The Authors Pathology International © 2014 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd
362
Letter to the Editor
a
b
c
d Figure 1 Representative immunoperoxidase features of LG in the present case. (a) hematoxylin and eosin; (b) CD3; (c) CD4; (d) CD8. There are numbers of small lymphocytes among the foveolar cells showing decreased mucin content. CD3+ IELs are composed of both CD4+ and CD8+ T-cells. In the lamina propria mucosae, CD4+ T-cells predominate over CD8+ T-cells.
a
b
c
d
Figure 2 Expression of cytotoxic T-cell substances in LG (upper panels, the present case; lower panels, the control case; (a,c) granzyme B; (b,d) perforin). The expression of granzyme B and perforin is suppressed when compared with the control case showing activated killer substance expression.
contrast to the conventional CD8+ IELs (as seen in our control male case),7,8 IELs in our female LG case were composed of both CD8+ T-cells and CD4+ T-cells, with a ratio of 1.6:1. Han et al. recently reported the relative frequency of CD4+ IELs in Hp-infected LG. In Hp-negative LG, the ratio of CD8+ IELs to CD4+ IELs was 15:1, while the ratio in Hp-infected LG increased to 6:1.10 Expression of granzyme B and perforin in CD8+ IELs was suppressed in the present case. In the control case, granzyme B and perforin were actively expressed in the
CD8+ IELs. Suppressed expression of granzyme B in Hp-infected LG was also described.10 TIA-1 is expressed in both resting and activated cytotoxic T-cells, whereas granzyme B is a marker of activated cytotoxic T-cells.7 Intraepithelial CD4+ IELs and CD8+ resting cytotoxic T-cells characterized LG in the present case. Regulatory T-cells commonly express CD4, CD25 and FoxP3.9 It is intriguing to suppose that the CD4+ IELs functioned as regulatory T-cells, to induce the resting state of adjacent CD8+
© 2014 The Authors Pathology International © 2014 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd
Letter to the Editor
IELs. However, immunostaining for CD25 and FoxP3 gave negativity. Further accumulation of similar cases is needed to elucidate functions of CD4+ IELs in the gastrointestinal mucosa. Hideyuki Fujiwara,1,2 Himuro Fujiwara,1,2 Isao Akatsuka,1,2 Toshiaki Takahashi,3 Yasuo Komori,4 Kazuya Shiogama2 and Yutaka Tsutsumi2 1
Medical Student of Fujita Health University School of Medicine, 2Department of Pathology, Fujita Health University School of Medicine, Toyoake, 3 Takahashi Clinic of Internal Medicine, Hamamatsu, and 4 Komori Clinic of Internal Medicine, Okazaki, Japan
REFERENCES 1 Haot J, Jouret-Mourin A, Wallez Z et al. Anatomoclinical study of a series of chronic gastritis characterized by intraepithelial lymphocytic infiltration. Acta Endosc 1986; 16: 69–74. 2 Ribeiro VL, Barbosa AJ. Lymphocytic gastritis: A study of its frequency and review of the literature. Arq Gastroenterol 1998; 35: 26–31. 3 Feeley KM, Heneghan MA, Stevens FM, McCarthy CF. Lymphocytic gastritis and coeliac disease: Evidence of a positive association. J Clin Pathol 1998; 51: 207–10. 4 Wu TT, Hamilton SR. Lymphocytic gastritis: Association with etiology and topology. Am J Surg Pathol 1999; 23: 153–8. 5 Kushima R, Borchard F. Lymphocytic gastritis: Autoimmune disease or variant of Helicobacter gastritis? Verh Dtsch Ges Pathol 1996; 80: 208–11. (in German).
363
6 Haot J, Jouret A, Willette M, Gossuin A, Mainguet P. Lymphocytic gastritis: Prospective study of its relationship with varioliform gastritis. Gut 1990; 31: 282–5. 7 Oberhuber G, Bodingbauer M, Mosberger I, Stolte M, Vogelsang H. High proportion of granzyme B-positive (activated) intraepithelial and lamina propria lymphocytes in lymphocytic gastritis. Am J Surg Pathol 1998; 22: 450–8. 8 Drut R, Drut RM. Lymphocytic gastritis in pediatric celiac disease. Immunohistochemical study of the intraepithelial lymphocytic component. Med Sci Monit 2004; 10: CR38–42. 9 Roncador G, Brown PJ, Maestre L et al. Analysis of FOXP3 protein expression in human CD4+CD25+ regulatory T cells at the single-cell level. Eur J Immunol 2005; 35: 1681–91. 10 Han S-H, Joo M, Kim K-M. High proportion of granzyme B+ intraepithelial lymphocytes contributes to epithelial apoptosis in Helicobacter pylori-associated lymphocytic gastritis. Helicobacter 2013; 18: 290–8.
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1 Summary of the expression of CD3, CD4, CD8, TIA-1, granzyme B and perforin in the present (red columns) and control cases (blue columns). The number represents positive cells per 100 epithelial cells. The CD3+ IELs in the present case equal CD 4 + cells plus CD8+ cells, while CD8+ cells are the main IEL component in the control case. The expression of granzyme B and perforin is relatively suppressed in the present case.
© 2014 The Authors Pathology International © 2014 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd
Pathology International 2014; 64: 361–363
doi:10.1111/pin.12172
Letter to the Editor
Lymphocytic gastritis showing concomitant occurrence of both CD4 + and CD8 + T-cells among epithelial cells To the Editor: Lymphocytic gastritis (LG), first described by Haot et al.,1 is microscopically featured by marked increase of intraepithelial lymphocytes (IELs), more than 25 IELs per 100 epithelial cells, in addition to mucin depletion, nuclear stratification of the foveolar cells and chronic active inflammation in the lamina propria mucosae.2 Common etiologies of LG include celiac disease and Helicobacter pylori (Hp) infection: LG was found in 33% of patients with celiac disease and 4.1% of histopathologically defined Hp gastritis.3 The frequency of LG was highest in celiac disease without Hp infection (50%), followed by Hp-infected celiac disease (20%), Hp infection without celiac disease (8.3%), but none in the control group.4 Kushima and Borchard described relative infrequency of Hp colonization in LG when compared with conventional chronic gastritis.5 Varioliform gastritis, endoscopically showing nodular targetoid and eroded lesions along proximal rugae, represents a form of LG.6 Intraepithelial lymphocytes in LG belong to T-cells, express CD8+, and are mostly positive for granzyme B, being categorized in activated cytotoxic T-cells.7 Expression of CD4 on IELs is an exceptional event.8 We briefly report herein a case of unique LG showing concomitant occurrence of both CD4 + and CD8 + T-cells among the epithelia. A 53 year-old Japanese female felt upper abdominal discomfort, and endoscopic evaluation revealed mild mucosal redness and rugal swelling on the gastric body. Hp eradication therapy was given 5 years earlier, with urea breath test being negative. An ulcer scar at the supra-angular region was biopsied. A control case of LG was exampled from the antral mucosa of a 55-year-old male case. No features of celiac disease were found in either cases. The gastric biopsy of the present case and control case was performed at Takahashi Clinic and Komori Clinic, respectively. Both biopsy samples histologically revealed numerous small lymphocytes among surface epithelia. The foveolar cells were regenerative and the mucin content was decreased. Dense lymphocytic infiltration was seen in the lamina propria, but without Hp colonization and formation of lymphoid follicles or lymphoepithelial lesions. Two LG samples were evaluated by immunostaining with the amino acid polymer method (Simple Stain Max, Nichirei, Tokyo). Primary mouse monoclo-
nal antibodies targeted at CD3, CD4, CD8, granzyme B (Dako, Carpinteria, USA), perforin (Novocastra, Newcastle, UK), T-cell-restricted intercellular antigen-1 (TIA-1 = nucleolysin: Coulter Immunology, Tokyo, Japan), CD25 (Nichirei) and FoxP3 (Abcam, Cambridge, UK). Granzyme B, perforin and TIA-1 are representative cytotoxic substances in azurophilic granules of killer T-cells and natural killer cells.7 CD25 and FoxP3 are the markers of regulatory (suppressor) T-cells, expressed on the plasma membrane and in the nuclei of CD4+ T-cells.9 Pressure pan-assisted heat-induced epitope retrieval was performed in 10 mM citrate buffer, pH 6 or 7 or in 1 mM ethylenediamine tetraacetic acid. Diaminobenzidine gave a brown color reaction. Sections of the tonsil served as positive control. Using consecutive sections, the marker-positive IELs were counted on color prints after taking a total of 20 (present case) or 29 (control case) high-powered photomicrographs using a x40 objective lens. CD3+ IELs in the present and control cases summed up 82 ± 30 and 102 ± 49 per 100 foveolar epithelial cells, respectively. In the present case, both CD4+ and CD8+ IELs were distributed among the foveolar cells, while in the control case only CD8+ IELs were observed. Figure 1 illustrates representative features of expression of CD3, CD4 and CD8. CD3+ IELs consisted of both CD4+ T-cells and CD8+ T-cells (CD4: 33 ± 22 and CD8: 53 ± 27 per 100 epithelial cells). In the lamina propria, CD4+ T-cells predominated over CD8+ T-cells. Three killer substances in CD8+ T-cells were further evaluated. In the present case, TIA-1-positive cells predominated over granzyme B- and perforin-positive cells (TIA-1 20 ± 14, granzyme B 15 ± 8, and perforin 6 ± 8). On average, 38% (20/53) of CD8+ IELs expressed TIA-1. In the control case, more IELs were labeled for granzyme B and perforin than TIA-1 (TIA-1 38 ± 25, granzyme B 68 ± 38, and perforin 55 ± 26), and CD3+ IELs expressed CD8 alone, with granzyme B and perforin seen in 66% (68/103) and 53% (55/103) of the IELs, respectively. Representative microscopic features are shown in Fig. 2. Supplementary Figure S1 summarizes the results in a column chart: the data of the present case are shown in red, and the control data in blue. For evaluating intraepithelial CD4+ cells in the present case as regulatory T-cells, CD25 and FoxP3 were immunostained. The results were negative. In both cases, a few lymphoid cells in the lamina propria were labeled for CD25 on the plasma membrane and for FoxP3 in the nuclei. A novel phenotype of IELs in LG was documented in the present case, devoid of celiac disease or Hp infection. In
© 2014 The Authors Pathology International © 2014 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd
362
Letter to the Editor
a
b
c
d Figure 1 Representative immunoperoxidase features of LG in the present case. (a) hematoxylin and eosin; (b) CD3; (c) CD4; (d) CD8. There are numbers of small lymphocytes among the foveolar cells showing decreased mucin content. CD3+ IELs are composed of both CD4+ and CD8+ T-cells. In the lamina propria mucosae, CD4+ T-cells predominate over CD8+ T-cells.
a
b
c
d
Figure 2 Expression of cytotoxic T-cell substances in LG (upper panels, the present case; lower panels, the control case; (a,c) granzyme B; (b,d) perforin). The expression of granzyme B and perforin is suppressed when compared with the control case showing activated killer substance expression.
contrast to the conventional CD8+ IELs (as seen in our control male case),7,8 IELs in our female LG case were composed of both CD8+ T-cells and CD4+ T-cells, with a ratio of 1.6:1. Han et al. recently reported the relative frequency of CD4+ IELs in Hp-infected LG. In Hp-negative LG, the ratio of CD8+ IELs to CD4+ IELs was 15:1, while the ratio in Hp-infected LG increased to 6:1.10 Expression of granzyme B and perforin in CD8+ IELs was suppressed in the present case. In the control case, granzyme B and perforin were actively expressed in the
CD8+ IELs. Suppressed expression of granzyme B in Hp-infected LG was also described.10 TIA-1 is expressed in both resting and activated cytotoxic T-cells, whereas granzyme B is a marker of activated cytotoxic T-cells.7 Intraepithelial CD4+ IELs and CD8+ resting cytotoxic T-cells characterized LG in the present case. Regulatory T-cells commonly express CD4, CD25 and FoxP3.9 It is intriguing to suppose that the CD4+ IELs functioned as regulatory T-cells, to induce the resting state of adjacent CD8+
© 2014 The Authors Pathology International © 2014 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd
Letter to the Editor
IELs. However, immunostaining for CD25 and FoxP3 gave negativity. Further accumulation of similar cases is needed to elucidate functions of CD4+ IELs in the gastrointestinal mucosa. Hideyuki Fujiwara,1,2 Himuro Fujiwara,1,2 Isao Akatsuka,1,2 Toshiaki Takahashi,3 Yasuo Komori,4 Kazuya Shiogama2 and Yutaka Tsutsumi2 1
Medical Student of Fujita Health University School of Medicine, 2Department of Pathology, Fujita Health University School of Medicine, Toyoake, 3 Takahashi Clinic of Internal Medicine, Hamamatsu, and 4 Komori Clinic of Internal Medicine, Okazaki, Japan
REFERENCES 1 Haot J, Jouret-Mourin A, Wallez Z et al. Anatomoclinical study of a series of chronic gastritis characterized by intraepithelial lymphocytic infiltration. Acta Endosc 1986; 16: 69–74. 2 Ribeiro VL, Barbosa AJ. Lymphocytic gastritis: A study of its frequency and review of the literature. Arq Gastroenterol 1998; 35: 26–31. 3 Feeley KM, Heneghan MA, Stevens FM, McCarthy CF. Lymphocytic gastritis and coeliac disease: Evidence of a positive association. J Clin Pathol 1998; 51: 207–10. 4 Wu TT, Hamilton SR. Lymphocytic gastritis: Association with etiology and topology. Am J Surg Pathol 1999; 23: 153–8. 5 Kushima R, Borchard F. Lymphocytic gastritis: Autoimmune disease or variant of Helicobacter gastritis? Verh Dtsch Ges Pathol 1996; 80: 208–11. (in German).
363
6 Haot J, Jouret A, Willette M, Gossuin A, Mainguet P. Lymphocytic gastritis: Prospective study of its relationship with varioliform gastritis. Gut 1990; 31: 282–5. 7 Oberhuber G, Bodingbauer M, Mosberger I, Stolte M, Vogelsang H. High proportion of granzyme B-positive (activated) intraepithelial and lamina propria lymphocytes in lymphocytic gastritis. Am J Surg Pathol 1998; 22: 450–8. 8 Drut R, Drut RM. Lymphocytic gastritis in pediatric celiac disease. Immunohistochemical study of the intraepithelial lymphocytic component. Med Sci Monit 2004; 10: CR38–42. 9 Roncador G, Brown PJ, Maestre L et al. Analysis of FOXP3 protein expression in human CD4+CD25+ regulatory T cells at the single-cell level. Eur J Immunol 2005; 35: 1681–91. 10 Han S-H, Joo M, Kim K-M. High proportion of granzyme B+ intraepithelial lymphocytes contributes to epithelial apoptosis in Helicobacter pylori-associated lymphocytic gastritis. Helicobacter 2013; 18: 290–8.
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1 Summary of the expression of CD3, CD4, CD8, TIA-1, granzyme B and perforin in the present (red columns) and control cases (blue columns). The number represents positive cells per 100 epithelial cells. The CD3+ IELs in the present case equal CD 4 + cells plus CD8+ cells, while CD8+ cells are the main IEL component in the control case. The expression of granzyme B and perforin is relatively suppressed in the present case.
© 2014 The Authors Pathology International © 2014 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd
