Modulation of fibroblast proliferation by postsurgical macrophages

Abstract

Macrophages and fibroblasts are major components of postsurgical peritoneal repair. In order to understand the interaction between these two cell types, we studied the effects of spent macrophage culture media on fibroblast proliferation. Rabbits underwent resection and reanastomosis of their small intestine. Peritoneal exudative cells (PEC) were then collected from these animals on postoperative Days 4, 7, and 28 and from nonsurgical controls. PEC (5 × 10 5 cells/ml) were cultured in M-199 with 3% fetal calf serum. After 48 hr the spent media from the cultured PEC were harvested, centrifuged (200g for 10 min), and stored (medium: M-D 0, D 4, D 7, D 28). A second group of rabbits underwent peritoneal wall abrasion followed by collection of fibroblasts directly from the site of injury on postoperative Days 1, 4, and 7. After Day 7 of culture, fibroblasts were resuspended and seeded into dishes (1 × 10 5 cells in 1 ml medium), to which was added 1 ml of spent PEC culture medium. After 24 hr of incubation, 1 μCi [ 3H]thymidine was added for an additional 18 hr. Fibroblasts were then collected and the amount of [ 3H]thymidine incorporated into trichloroacetic acid-precipitable material was quantitated. In one protocol, fresh M-199 with 3% fetal calf serum was used, and in another protocol, “U-medium” (which was previously incubated for 48 hr with fibroblasts) was used. The cytology of the PEC was determined by Wright's staining, nonspecific esterase activity, and phagocytosis. At least 80% of the peritoneal exudative cells were identified as macrophages. Postsurgical Day 7 fibroblasts demonstrated greater [ 3H]thymidine incorporation compared to fibroblasts from postoperative Days 1 and 4. Thymidine incorporation into fibroblasts cultured with spent medium from macrophage cultures (M-D 0, D 4, D 7, or D 28) was suppressed compared to the control group, but the M-D 4 and D 7 medium group showed greater [ 3H]thymidine incorporation compared to the M-D 0, D 28 group. This pattern was repeated when the assay was performed with U-medium. In addition, M-D media obtained from various concentrations of PEC was investigated to determine the effect of cell dose on the [ 3H]thymidine assay. Thymidine incorporation was gradually decreased with increasing concentrations of macrophages, but at each macrophage concentration M-D 4 and D 7 media demonstrated greater activities compared to M-D 0 medium. These data suggest that macrophages act as both positive and negative modulators of fibroblast proliferation. Postsurgical macrophages may therefore accelerate the reconstruction of injured tissue as well as moderate the activity of fibroblasts once tissue repair is complete.