GM-CSF-induced granulation tissue formation: relationships between macrophage and myofibroblast accumulation

We have studied the formation of granulation tissue around osmotic minipumps delivering granulocyte macrophage-colony stimulating factor (GM-CSF) chronologically in the rat using electron microscopy and immunohistochemistry at the light and electron microscopic levels, with specific antibodies against alpha-smooth muscle (SM) actin and rat macrophages. At 2 and 3 days after pump implantation, GM-CSF application produced an extensive inflammatory reaction characterized by edema and the accumulation of polymorphonuclear cells and macrophages. Gradually, polymorphonuclear cells decreased in number and macrophages became arranged in large clusters. The expression of alpha-SM actin in fibroblastic cells of the granulation tissue started from the 4th day after pump implantation and progressed up to the 7th day. Double immunofluorescence staining showed macrophage clusters in relation to alpha-SM actin-rich fibroblastic cells. Electron microscopic examination confirmed that the fibroblasts containing alpha-SM actin-positive stress fibers were found initially in close proximity to clustered macrophages. The delivery of platelet-derived growth factor (PDGF) and tumor necrosis factor-alpha (TNF-alpha) by the osmotic minipump induced an accumulation of macrophages, but in a much smaller number compared with those seen after GM-CSF application; these macrophages were never assembled in clusters and, furthermore, TNF-alpha and PDGF did not stimulate alpha-SM actin expression in fibroblastic cells. Our results suggest that after GM-CSF administration, the cluster-like accumulation of macrophages plays an important role in stimulating alpha-SM actin expression in myofibroblasts. Our results may be relevant to the understanding of the processes leading to granulation tissue formation in this and other experimental models.