Activation of pancreatic stellate cells in human and experimental pancreatic fibrosis

Abstract

The mechanisms of pancreatic fibrosis are poorly understood. In the liver, stellate cells play an important role in fibrogenesis. Similar cells have recently been isolated from the pancreas and are termed pancreatic stellate cells. The aim of this study was to determine whether pancreatic stellate cell activation occurs during experimental and human pancreatic fibrosis. Pancreatic fibrosis was induced in rats (n = 24) by infusion of trinitrobenzene sulfonic acid (TNBS) into the pancreatic duct. Surgical specimens were obtained from patients with chronic pancreatitis (n = 6). Pancreatic fibrosis was assessed using the Sirius Red stain and immunohistochemistry for collagen type I. Pancreatic stellate cell activation was assessed by staining for alpha-smooth muscle actin (alphaSMA), desmin, and platelet-derived growth factor receptor type beta (PDGFRbeta). The relationship of fibrosis to stellate cell activation was studied by staining of serial sections for alphaSMA, desmin, PDGFRbeta, and collagen, and by dual-staining for alphaSMA plus either Sirius Red or in situ hybridization for procollagen alpha(1) (I) mRNA. The cellular source of TGFbeta was examined by immunohistochemistry. The histological appearances in the TNBS model resembled those found in human chronic pancreatitis. Areas of pancreatic fibrosis stained positively for Sirius Red and collagen type I. Sirius Red staining was associated with alphaSMA-positive cells. alphaSMA staining colocalized with procollagen alpha(1) (I) mRNA expression. In the rat model, desmin staining was associated with PDGFRbeta in areas of fibrosis. TGFbeta was maximal in acinar cells adjacent to areas of fibrosis and spindle cells within fibrotic bands. Pancreatic stellate cell activation is associated with fibrosis in both human pancreas and in an animal model. These cells appear to play an important role in pancreatic fibrogenesis.

Figure 1.

Histological appearance of experimental rat pancreatic fibrosis. A: Hematoxylin and eosin stain demonstrating focal periductal fibrosis with inflammatory infiltration. An area of lobular atrophy is seen at the right edge of the field. Original magnification, ×40. B: Higher power view of an area of focal fibrosis showing periductal changes that extend into the lobule with associated acinar atrophy. Original magnification, ×200. C: Sirius Red collagen stain demonstrating marked periductal fibrosis with focal extension of collagen fibrils into peri-acinar regions of adjacent parenchyma. Original magnification, ×100. D: Serial section of the fibrotic area shown in C stained for αSMA (brown). There is co-localization of αSMA positive cells with areas of fibrosis. Original magnification, ×100.

Figure 2.

Histological appearance of human chronic pancreatitis. A: Hematoxylin and eosin stain demonstrating periductal fibrosis similar to the rat model (see Figure 1A ▶ ). Original magnification, ×100. B: Sirius Red stain shows collagen deposition in the peri-acinar areas. Original magnification, ×400. C: Immunohistochemistry demonstrating positive staining (pink) for collagen type I in bands of fibrosis. Original magnification, ×100. D: αSMA positive stellate cells are found in peri-acinar areas. Original magnification, ×100. E: Co-localization of collagen (red) and αSMA (brown) in cells. Original magnification, ×400. F: TGFβ expression (brown) in acinar cells adjacent to a fibrotic band; some weakly positive spindle-shaped cells are also observed within the fibrotic band (arrows). Original magnification, ×400.

Figure 3.

Identification of activated PSCs as the predominant cellular source of collagen production in pancreatic fibrosis. These images show colocalization of αSMA (brown) and procollagen α₁ (I) mRNA expression (blue) A: Rat pancreatic fibrosis. Original magnification, ×200. B. Human chronic pancreatitis showing a band of fibrosis with adjacent acinar tissue. Original magnification, ×100. C: Human chronic pancreatitis at higher magnification. Original magnification, ×400.

Figure 4.

The relationship between areas of rat pancreatic fibrosis, desmin, and PDGFRβ using serial sections. A: Sirius Red collagen stain. Original magnification, ×40. B: Desmin stain of the same area showing accumulation of desmin-positive cells in fibrotic area. Original magnification, ×40. C: The fibrotic area is also PDGFRβ-positive. Original magnification, ×40.