Prior to the reduction in myofibroblasts 7 days after BMM delivery, there were increases in the numbers of cells producing MMP-13 and -9 protein (P < 0.01 and < 0.05, respectively, Fig. 5A,B). These MMP-expressing cells were predominantly located in the hepatic scar. Within 1 day of BMM therapy, whole liver gene expression of MMP-9 was markedly elevated (P < 0.05) alongside trends toward increases in MMP-13 (P = 0.21), MMP-8 (neutrophil collagenase, P = 0.17), and MMP-12 (macrophage metalloelastase, P = 0.08) (Fig. 5C). Serial section
analysis indicated that a subset of predominantly scar associated macrophages (SAMs) produced MMP-13 (Fig. 5D). We have previously shown that SAMs are an important cellular source of MMP-13 contributing to scar resolution after liver injury.6 Dual immunostaining revealed the MMP-9 producing cells to be neither donor nor endogenous macrophages (Fig. 5Ei) but endogenous see more Ly-6G+ neutrophils (Fig. 5Eii). Therefore, the initial donor BMMs caused an increase in the numbers of MMP-producing leukocytes in the hepatic scar. Within 1 day of BMM infusion, Daporinad there was a marked change in the cellular composition of the fibrotic liver.
F4/80 immunostaining demonstrated a 44% increase in macrophages (P < 0.05, Fig. 6A,B). The absolute increase in macrophage number in BMM-treated mice (from 53 to 76, i.e., an additional 23 per ×200 field) is greater than the number of donor BMMs (mean <7) in the same area of tissue, indicating that the majority of these macrophages were recruited. Ly-6G selleck products immunostaining revealed a 242% increase in hepatic neutrophils
(P < 0.01, Fig. 6A,B). Analysis of whole liver protein from this timepoint revealed that BMM recipients had significantly higher levels of several chemokines expressed by the donor BMMs (Figs. 1E, 6C). The macrophage chemoattractant MCP-1 (CCL2) was increased to 160% (P < 0.001), whereas MIP-1α (CCL3) was 137% of control (P < 0.05). The neutrophil chemoattractants KC (CXCL1) and MIP-2 (CXCL2) were also strongly up-regulated (242%, P < 0.001 and 842%, P < 0.01, respectively). Whole liver protein levels of the antiinflammatory cytokine IL-10 were elevated to 346% in BMM recipients (P < 0.05), whereas proinflammatory mediators such as IL-6 and TNF-α were unchanged (Fig. 7F). Four weeks after BMM delivery, serum ALT levels were not significantly reduced in recipient mice (399.2 ± 120.7) compared to controls (505.7 ± 91.7 u/l, P = 0.5). Therefore, BMM therapy switches the hepatic milieu towards an antiinflammatory cytokine environment while recruiting host macrophages and neutrophils into this altered setting. Serum albumin was increased in BMM recipients 4 weeks after cell delivery (46.0 ± 2.6 g/l versus 39.9 ± 0.9 g/l, P = 0.05, Fig. 7A). The elevated serum albumin was confirmed in mice receiving GFP+ BMMs (43.3 ± 0.6 g/l versus 40.4 ± 1.0 g/l, P < 0.05, Fig. 7A), suggesting improved regeneration.