Differential susceptibility to hepatic inflammation and proliferation in AXB recombinant inbred mice chronically infected with Helicobacter hepaticus - PubMed (original) (raw)

Differential susceptibility to hepatic inflammation and proliferation in AXB recombinant inbred mice chronically infected with Helicobacter hepaticus

M Ihrig et al. Am J Pathol. 1999 Aug.

Abstract

Helicobacter hepaticus is a naturally occurring pathogen of mice and has been used to develop models of chronic hepatitis, liver cancer, and, more recently, inflammatory bowel disease, in selected mouse strains. A/JCr mice are particularly susceptible to H. hepaticus-induced hepatitis and subsequent development of liver neoplasms, whereas C57BL/6 mice are resistant. In this study, we inoculated nine AXB recombinant inbred (RI) mouse strains, derived from A/J and C57BL/6 mice, with H. hepaticus to determine the genetic basis of resistance to Helicobacter-induced liver disease. Mice were surveyed 14 months after inoculation by culture and PCR for H. hepaticus colonization of the liver and cecum, and microscopic morphometric evaluations of the liver were performed to quantify and correlate the severity of inflammation, apoptosis, and proliferation. Analysis of variance of hepatic inflammation demonstrated significant variation among the RI strains (P < 0.0001), and the strain distribution pattern suggested a multigenic basis of disease resistance. Quantitative trait analysis using linear regression suggested possible linkage to loci on mouse chromosome 19. Hepatocellular and biliary epithelial apoptosis and proliferation indices, including proliferation of oval cells, were markedly increased and correlated with severity of inflammation. Prevalence of hepatic neoplasia was also increased in susceptible RI strains. These findings demonstrate a genetic basis for susceptibility to Helicobacter-induced disease and provide insight into its pathogenesis.

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Figures

Figure 1.

Figure 1.

Representative photomicrographs of AXB RI strains demonstrating differential severity, distribution, and morphology of chronic inflammation 14 months after inoculation with H. hepaticus. A and B: Characteristic lesions of mild hepatitis with involvement of portal areas in relatively resistant RI strains 4 and 5, respectively. H&E, ×57 and ×114. C and D: Progressive chronic inflammation centered around veins and bile ductules in RI strains 2 and 10, respectively. Inflammation is chiefly lymphocytic, including numerous plasma cells but also contains scattered neutrophils and eosinophils and involves areas of parenchyma (arrows). H&E, ×57 and ×114. E and F: Severe lesions from RI strains 8 and 1, respectively. Inflammation has spread through the liver and is often associated with biliary epithelial hyperplasia and dysplasia. H&E, ×57 and ×95, respectively. G, Left: A RI strain 1 animal with severe hepatitis and high PCNA proliferation index also had pronounced oval cell proliferation, shown by immunostaining for A6 antibody. AEC, hematoxylin counterstain, ×95. G, Right: Hepatic parenchymal inflammation centered around apoptotic cells from AXB RI strain 8. H&E, ×370 (top), and TUNEL immunohistochemistry with hematoxylin counterstain, ×370 (bottom). H: Warthin-Starry stained liver section from RI strain 8, showing intercellular H. hepaticus organisms (arrows).

Figure 2.

Figure 2.

A: Photomicrograph of AXB RI strain 1 male with hepatocellular adenoma that has compressed adjacent liver parenchyma (open arrows); variable degrees of cytoplasmic vacuolization and residual multifocal inflammation (solid arrows) are present within the neoplasm. H&E, ×31. The inset shows an area of lymphocytic inflammation surrounding poorly organized biliary epithelium within neoplasm (lower arrow). H&E, ×165. B: Hepatocellular carcinoma with irregular cords and clusters of large, pleomorphic hepatocytes and foci of lymphocytic inflammation from AXB RI strain 6 male. H&E, ×100. C: AXB RI strain 12 female with B-cell lymphosarcoma that has diffusely infiltrated an area of large intestine from the muscularis mucosa (mm) to the superficial epithelium (left). H&E, ×78. The same animal had lymphosarcoma within the liver (right). A remaining bile ductule is present with the neoplasm (white arrow). H&E, ×160. D: AXB RI strain 1 female with severe hepatitis and development of poorly differentiated hemangiosarcoma lining sinusoids and vascular channels. H&E, ×160.

Figure 3.

Figure 3.

Geometric means and standard errors for the morphometric scores of hepatic inflammation in the nine AXB recombinant inbred mouse strains. The surface area affected by inflammation was determined for each mouse by examination of three liver sections prepared from samples collected in a standardized manner. The number of 50 μm boxes in an ocular grid filled with inflammatory cells was counted, and the area of those counted boxes was calculated. The number of portal areas contained within the three liver sections was used as an estimate of total surface area of the three liver sections. The area calculated from the boxes filled with inflammatory cells (surface area of inflammation) was divided by the number of portal areas counted (total surface area estimate) to give the area affected by inflammation per portal area in square microns.

Figure 4.

Figure 4.

The apoptotic index for the four AXB recombinant inbred strains assessed for apoptosis. The TUNEL procedure was performed on liver sections from four RI strains with the most extreme inflammation phenotypes. The positively stained cells in liver sections were counted and categorized, the total surface area of the liver sections was determined with image analysis software, and the number of positive cells was divided by the total surface area. The data were then log transformed to obtain the apoptotic index. The graphs represent the mean apoptotic indices for hepatocytes (A), bile duct epithelial cells (B), Kupffer cells (C), lymphocytes (D), and all cell types combined (E).

Figure 5.

Figure 5.

The proliferation index for the four AXB recombinant inbred strains assessed for proliferation. Liver sections from four RI strains with the most extreme inflammation phenotypes were immunostained with PCNA antibody. The positively stained cells in liver sections were counted and categorized, the total surface area of the liver sections was determined with image analysis software, and the number of positive cells was divided by the total surface area. The data were then log transformed to obtain the proliferation index. The graphs represent the mean proliferation indices for hepatocytes (A), bile duct epithelial cells (B), Kupffer cells (C), lymphocytes (D), and all cell types combined (E).

Figure 6.

Figure 6.

The oval cell index for the four AXB recombinant inbred strains assessed for oval cell hyperplasia. Liver sections from four RI strains with the most extreme inflammation phenotypes were immunostained with A6 antibody, which is specific for oval cells. The positively stained cells in liver sections were counted, the surface area of the liver sections was determined with image analysis software, and the number of positive cells was divided by the total surface area. The data were then log transformed to obtain the oval cell index. The graphs represent the mean oval cell indices for the four RI strains.

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