Monocyte chemoattractant protein-1 is generated via TGF-beta by myofibroblasts in gastric intestinal metaplasia and carcinoma without H. pylori infection

Abstract

Helicobacter pylori (H. pylori) stimulates secretion of monocyte chemoattractant protein 1 (MCP-1) from gastric mucosa. Monocyte chemoattractant protein-1 (MCP-1) expression and macrophage infiltration are recognized in human gastric carcinoma. We have previously generated Cdx2-transgenic mice as model mice for intestinal metaplasia. Both chronic H. pylori-associated gastritis and Cdx2-transgenic mouse stomach develop intestinal metaplasia and finally gastric carcinoma. In this study we have directed our attention to MCP-1 expression in the intestinal metaplastic mucosa and the gastric carcinoma of Cdx2-transgenic mouse stomach. Quantitative real-time PCR was performed to determine MCP-1 and transforming growth factor-beta1 (TGF-beta1) mRNA expression levels and single- or double-label immunohistochemistry was used to evaluate the localization of MCP-1, TGF-beta type I receptor, and alpha-smooth muscle actin (alphaSMA). We determined that MCP-1 mRNA dramatically increased in the intestinal metaplastic mucosa and the gastric carcinoma of Cdx2-transgenic mouse stomach, compared with normal mouse stomach. Both MCP-1 and TGF-beta type I receptor were co-expressed in the alphaSMA-positive myofibroblasts of intestinal metaplastic mucosa and gastric carcinoma. Exogenous application of TGF-beta1 increased MCP-1 mRNA expression levels in the intestinal metaplastic tissue. Furthermore, TGF-beta1 was overexpressed and macrophage was strongly infiltrated in the gastric carcinoma. In conclusion, MCP-1 expression, which was stimulated by TGF-beta1, was recognized in the TGF-beta type I receptor-expressing myofibroblasts of the intestinal metaplastic mucosa and the gastric carcinoma of Cdx2-transgenic mouse stomach. The present results suggest that intestinal metaplasia and gastric carcinoma themselves induce MCP-1 expression independently of H. pylori infection.

Figure 1

Quantitative real‐time PCR analysis of monocyte chemoattractant protein‐1 (MCP‐1) and vascular endothelial growth factor (VEGF) expression. Hematoxylin–eosin staining of the normal mouse stomach (a), intestinal metaplastic mucosa (b), and gastric carcinoma (c) of Cdx2‐transgenic mouse stomach. Scale bar: 100 μm. Monocyte chemoattractant protein‐1 (MCP‐1) gene expression characterised by quantitative real‐time PCR (d). Monocyte chemoattractant protein‐1 (MCP‐1) mRNA levels were compared among the normal mouse stomach and the intestinal metaplasia and gastric carcinoma of Cdx2‐transgenic mouse stomach. Monocyte chemoattractant protein‐1 (MCP‐1) exhibited a six‐fold increase in intestinal metaplastic mucosa and a 37‐fold increase in gastric carcinoma compared with normal mouse stomach. Each column indicates mean ± SE of six tissue samples. *P < 0.05 and **P < 0.01 versus control values. Vascular endothelial growth factor (VEGF) gene expression characterised by quantitative real‐time PCR (e). Vascular endothelial growth factor (VEGF) mRNA levels were compared among the normal mouse stomach and the intestinal metaplasia and gastric carcinoma of Cdx2‐transgenic mouse stomach.

Figure 2

Colocalization of α‐smooth muscle actin (αSMA) and monocyte chemoattractant protein‐1 (MCP‐1) in intestinal metaplastic mucosa and gastric carcinoma by dual labeling immunofluorescence. By immunohistochemistry, we examined the expression of MCP‐1 in normal mouse stomach (a), intestinal metaplastic mucosa (b), and gastric carcinoma (c) of Cdx2‐transgenic mouse stomach. Intestinal metaplastic mucosa (d) and gastric carcinoma (e) of Cdx2‐transgenic mouse stomach were used for analyzing the expression of Cdx2 (Cy3) and MCP‐1 (Alexa 488) by dual immunofluorescence staining. Intestinal metaplastic mucosa (f,g,h) and gastric carcinoma (i,j,k) of Cdx2‐transgenic mouse stomach were used for analyzing the expression of αSMA (f,i) and MCP‐1 (g,j) by immunofluorescence staining (h,k; merged image). The same section was stained for αSMA (f,i: cy3) and MCP‐1 (g,j: Alexa 488) by dual immunofluorescence staining. α‐Smooth muscle actin (αSMA) and MCP‐1 were co‐localized. Panels (f–k) were counterstained with the nuclear dye 4′,6‐diamidino‐2‐phenylindole (DAPI). Scale bar: 100 μm.

Figure 3

Immunohistochemical staining for transforming growth factor‐β (TGF‐β) type I receptor and monocyte chemoattractant protein‐1 (MCP‐1) expression in intestinal metaplastic mucosa and gastric carcinoma. With immunohistochemistry we examined the expression of TGF‐β type I receptor in normal mouse stomach (a), intestinal metaplastic mucosa (b), and gastric carcinoma (c) of Cdx2‐transgenic mouse stomach. Intestinal metaplastic mucosa (d) and gastric carcinoma (e) of Cdx2‐transgenic mouse stomach were used for analyzing the expression of Cdx2 (Cy3) and TGF‐β type I receptor (Alexa 488) by dual immunofluorescence staining. Intestinal metaplastic mucosa (f,g,h) and gastric carcinoma (i,j,k) of Cdx2‐transgenic mouse stomach were used for analyzing the expression of TGF‐β type I receptor and MCP‐1 by immunofluorescence staining (h,k; merged image). The same section was stained for TGF‐β type I receptor (f, i: cy3) and MCP‐1 (g,j: Alexa 488). Transforming growth factor‐β (TGF‐β) type I receptor and MCP‐1 were co‐localized. Panels (f–k) were counterstained with the nuclear dye DAPI. Scale bar: 100 μm.

Figure 4

Monocyte chemoattractant protein‐1 (MCP‐1) expression in intestinal metaplastic tissue was increased by the cultivation with transforming growth factor‐β1 (TGF‐β1) ex vitro. Monocyte chemoattractant protein‐1 (MCP‐1) mRNA in intestinal metaplastic tissue after 16 h of cultivation without TGF‐β1 (A); MCP‐1 mRNA in intestinal metaplastic tissue after 16 h of cultivation with TGF‐β1 (B); MCP‐1 mRNA in normal gastric tissue after 16 h of cultivation without TGF‐β1 (C); MCP‐1 mRNA in normal gastric tissue after 16 h of cultivation with TGF‐β1 (D). The expression levels of MCP‐1 mRNA in intestinal metaplasia significantly increased during 16 h of cultivation with TGF‐β1 while the MCP‐1 level in normal gastric mucosa was not changed during 16 h of cultivation with TGF‐β1 as determined by quantitative real‐time PCR. Each column indicates mean ± SE of five tissue samples. **P < 0.01 versus control values.

Figure 5

Transforming growth factor‐β1 (TGF‐β1) expression in normal gastric mucosa, intestinal metaplastic mucosa (IM), and gastric carcinoma of Cdx2‐transgenic mouse stomach. Transforming growth factor‐β1 (TGF‐β1) mRNA was compared among the normal gastric mucosa and the intestinal metaplastic mucosa and gastric carcinoma of Cdx2‐transgenic mouse stomach. The gastric carcinoma of Cdx2‐transgenic mouse stomach exhibited a 6.5‐fold increase in TGF‐β1 mRNA expression compared with the normal gastric mucosa. Each column indicates mean ± SE of six tissue samples. **P < 0.01 versus control values.

Figure 6

Immunofluorescence staining for macrophage marker F4/80. Macrophage accumulation, which is shown by the immunostaining for macrophage marker F4/80, was recognized in the normal mouse stomach (a) and the intestinal metaplasia (IM) (b) and the gastric carcinoma (c) of the Cdx2‐transgenic mouse stomach. Scale bar: 100 μm. The mean number of infiltrated macrophages per field is shown in panel (d). The number of tissue macrophages in the intestinal metaplastic mucosa and gastric carcinoma increased by two‐fold and six‐fold compared with the normal gastric mucosa, respectively. *P < 0.05 and **P < 0.01 versus control values.