Expression of the integrin alpha8beta1 during pulmonary and hepatic fibrosis

Abstract

The fibrotic response after diverse forms of injury is characterized by the accumulation of extracellular matrix proteins, proliferation of myofibroblast-like cells, and organ contraction. Myofibroblasts are key effector cells in the development of the fibrotic response. They contribute to fibrosis through both increased cell number (proliferation) and enhanced matrix synthesis. Integrins, a class of cell adhesion molecules, are mediators of cell-extracellular matrix protein interactions that are important in the proliferative and migratory response of cells to matrix proteins. We have previously cloned the human integrin subunit alpha8, documented its high expression in lung tissue, and established it as a receptor for the matrix proteins fibronectin, vitronectin, and tenascin. We now demonstrate that alveolar interstitial cells are the primary cell type expressing alpha8beta1 in the lung parenchyma. Expression of alpha8beta1 is concentrated primarily along the thinned extensions of cells and at the tips of filopodia. Because of its unique distribution in alveolar interstitial cells, we hypothesized that it may play a role in the fibrotic response after injury. In bleomycin-induced pulmonary fibrosis, there is increased expression of alpha8beta1 by interstitial fibroblasts, the majority of which coexpress alpha smooth muscle actin, a marker of tissue myofibroblasts. To establish a more general role for alpha8beta1 during organ fibrosis, we further examined its expression in two rat models of liver fibrosis. During hepatic injury due to either carbon tetrachloride injury or bile duct ligation, we demonstrate de novo expression of alpha8beta1 in activated hepatic stellate cells, the myofibroblast equivalent in liver. Taken together, the data localize alpha8beta1 to myofibroblast-like cells during wound healing and suggest that signal transduction through the alpha8beta1 integrin may contribute to the fibrotic response of organs to injury.

Figure 1.

Electron microscopy of normal rat lung demonstrating α8-immunoreactivity in contractile interstitial cells. A–D: α8 immunoreactivity is visualized at the tips and along the membrane (arrows) of contractile interstitial cells (CIC). A: Actin filaments (*) are noted in an α8-expressing CIC. C: Higher magnification of CIC illustrating α8-reactivity (arrow) along the filopodia as it protrudes into the basement membrane. D: Lipid droplet (arrowhead) within an α8-expressing CIC. Immunoperoxidase staining. Scale bars, 0.5 μm in A, B, and D; 0.2 μm in C.

Figure 2.

A: Western blot of α8β1 in primary lung fibroblasts. Fibroblasts isolated from rat lung were lysed, and aliquots of lysates were analyzed under nonreducing conditions. The samples were separated on SDS-PAGE gel and immunoblotted with anti-α8 antiserum. Lanes 1–3 represent three different rat fibroblast preparations. The arrow indicates the position of α8. Positions of molecular size markers in kd are shown to the left. B: Immunofluorescence of α8β1 in primary lung fibroblasts. Primary rat lung fibroblasts were grown on fibronectin-coated plates and then fixed, permeabilized, and labeled with anti-α8 antibody. α8-containing focal contacts are indicated by arrows.

Figure 3.

Immunohistochemistry of α8β1 in normal lung and pulmonary fibrosis. Immunohistochemical staining with anti-α8 antibody of mouse lung after saline (A) or bleomycin (B–J) treatment. Frozen sections of lung were stained with hematoxylin and affinity-purified anti-α8 antibodies. In the control (A) 4 weeks after saline treatment, normal lung architecture is preserved, and α8-immunoreactive cells are seen in the interstitium. In the bleomycin-treated animals, alveolar wall thickening, increased cellularity, and obliteration of air spaces are seen at early time points (B–F). The majority of cells within alveolar wall thickening are α8β1-positive. At higher magnification, some areas of α8β1-negative inflammatory cells are observed (asterisk) (D). Vascular smooth muscle and airway smooth muscle are α8β1-positive (arrowheads) (C). By 4 weeks (G and H), areas of dense cellular infiltration with α8β1-positive cells are seen with complete loss of normal alveolar architecture. I: Nine weeks. Higher magnification of a less involved area shows α8β1-positive cells within thickened alveolar septa. Control specimens incubated with preimmune rabbit serum displayed no significant staining (data not shown). Immunoperoxidase staining. Original magnifications: A–C and E–G, ×10; D and H, ×20; I, ×40.

Figure 4.

α8β1 and α-smooth muscle actin expression during pulmonary fibrosis. Mouse lung sections obtained 4 days after bleomycin treatment were stained for α8β1 (FITC) and α-smooth muscle actin (Cy3). Superimposition of the images reveals that most cells that express α-smooth muscle actin also express α8β1 (yellow). However, some cells express α8β1 but not α-smooth muscle actin (white arrows). Control specimens incubated with preimmune serum displayed no significant staining (data not shown). Original magnification, ×40.

Figure 5.

Immunohistochemistry of α8β1 in normal and fibrotic liver. In normal rat liver, α8-positive cells are visualized in smooth muscle surrounding vessels in portal veins and hepatic arteries within portal triads. Parenchyma is negative for α8β1. Vascular smooth muscle cells are α8β1-positive (arrowheads) (A and D). Two weeks after bile duct ligation, α8β1 is detected in cells surrounding proliferating ductules. B and E: After 4 weeks of CCl₄ administration, bridging fibrosis is present within lobules (arrow). Cells within fibrotic bands show strong immunoreactivity for α8β1 (arrow) (C and F). Immunoperoxidase staining. Original magnifications: A–C, ×2.5; D–F, ×10.

Figure 6.

Immunoprecipitation of α8β1 from immortalized rat hepatic stellate cells. HSC T6 cells were surface-labeled with biotin, lysed, and then immunoprecipitated with anti-α8 antibody. Proteins were analyzed by SDS-PAGE under nonreducing conditions. The positions of α8 and β1 subunits are indicated by arrows on the right; positions of molecular mass markers (kd) are indicated on the left.