N. Uyama1, S. Wu1, M. Sudo1, E. Hatano1, H. Tsutsui1, J. Fujimoto1 1Hyogo College of Medicine,Surgery,Nishinomiya, HYOGO, Japan
Introduction: Postsurgical fibrous adhesions often result in severe complications of intestinal obstruction, female infertility, and chronic abdominal pain. We revealed that IFN-γ and PAI-1 played a pivotal role in adhesion formation by regulating the coagulation fibrinolysis system in mouse abdominal adhesion formation model (Nature Medicine 2008). In the present study, we investigated another molecular mechanism of adhesion formations by microarray analysis.
Methods: Cecum cauterization model was used in the present study. Intraabdominal adhesions were formed at 7 days after cecum cauterization by bipolar forceps. Immunohistochemistry for Ly6G (neutrophil) and collagen Type I (COL(I)) and triple immunostaining for podoplanin (PDPN), WT-1 (mesothelial cell markers) and αSMA (a myofibroblast marker) were performed to characterize cell type in adhesion areas. To determine the key molecules and signaling pathways for adhesion formations, microarray analysis was performed with damaged ceca at 0h, 3h, 12h, and 72h after operation. The kinetics of expression of IL-6, TNF-α, CXCL-2, TGF-β, COL(I) in damaged ceca was investigated by qRT-PCR. In in vitro experiments, effect of IL-6, TNF-α and TGF-β on expressions of TNF-α, CXCL-2, TGF-β and COL(I) by Met-5A cells (a human mesothelial cell line) and isolated human neutrophils was investigated by qRT-PCR.
Results:Neutrophil accumulation in damaged ceca was found at 3h after operation. At day 7, COL(I) deposition, neutrophil accumulation and PDPN+WT-1+αSMA+ cells (activated mesothelial cells) were found in adhesion areas. As for microarray analysis, IL-6-, TNFα- and TGFβ-signalings were found to be elevated in adhesion formations. mRNA expression levels of IL-6, TNF-α, CXCL-2, TGF-β and COL(I) in damaged ceca were upregulated to about 450-fold, 18-fold, 18000-fold, 3-fold and 40-fold of control during 7days after surgery, respectively. In in vitro experiments, IL-6 significantly induced mRNA expression of TNF-α (2 fold) and CXCL2 (1.5 fold) in Met-5A cells. TNF-α significantly induced mRNA expression of TNF-α (8.2 fold) and TGF-β (5.9 fold) by neutrophils. TGF-β significantly upregulates mRNA expression of TGF-β (1.9 fold) and COL(I) (2.3 fold) by Met5A cells. Collectively, we assume that IL-6 may induce neutrophil accumulation through the production of CXCL2 by mesothelial cells. IL-6 may also stimulate TNFα production by mesothelial cells, which induce TGF-β production by neutrophils. In turn, TGF-β may stimulate production of TGF-β and COL(I) by mesothelial cells.
Conclusion:Mesothelial cells and neutrophils may be key players of adhesion formations. On adhesiogenesis, IL-6 may induce neutrophil accumulation, eventually leading to collagen production presumably via activating cross-talk between neutrophils and mesothelial cells.