Activation of Stat Proteins Following Ischemia Reperfusion Injury Demonstrates a Distinct IL-6 and G-CSF Mediated Profile C. Hierholzer, J.C. Kalff, T.R. Billiar, A. Bauer, and D.J. Tweardy I SCHEMIA/REPERFUSION injury of the gut results in cytokine production and a delayed impairment of mus- cle contractility with subsequent decreased gut motility and stasis. The inflammatory response includes the local pro- duction of cytokines in the gut. We have previously shown that IL-6 and G-CSF are produced in the gut following hemorrhagic shock. 1 G-CSF and IL-6 induce activation of STATs, proteins that serve the dual functions of signal transducers and activators of transcription, but each acti- vates a distinct STAT protein profile: IL-6 activates Stat3 (p92), while extracts of G-CSF-stimulated PMN contain only Stat3 (p72). We examined the hypothesis that the systemic response to ischemia/reperfusion injury includes G-CSF and IL-6 mRNA production and the activation of STAT proteins. We also examined the profile of the activated STAT proteins and determined whether both phases of ischemia and reperfusion were required for activation of these signaling mediators. MATERIALS AND METHODS The gut of Sprague Dawley rats were subjected to 75 minutes of ischemia by clamping the superior mesenteric artery followed by reperfusion and sacrifice at 0 min, 30 min, and 24 hours. The gut was harvested, and the mucosa and muscularis from midjejunum were isolated using surgical techniques. Semiquantitative reverse transcription polymerase chain reaction was performed to measure levels of G-CSF and IL-6 mRNA in the isolates. Electrophoretic mobility shift assay was performed using protein extracts of mucosa and muscularis isolates and radiolabeled high-affinity serum-induc- ible element duplex oligonucleotide. Supershift analysis was per- formed using a specific antibody against Stat3. Where indicated, mucosa and muscularis externa were isolated from the midjejunum of normal rats and were incubated with G-CSF (100 ng/L) and IL-6 (100 ng/L) at 37° for 30 minutes. RESULTS Levels of G-CSF and IL-6 mRNA were elevated in the mucosa and the muscularis following ischemia and reper- fusion of 30 minutes and 24 hours compared to mRNA levels following ischemia alone. Electrophoretic mobility shift assay of mucosa and muscularis protein extracts in animals subjected to 75 minutes of ischemia alone demon- strated low levels of Stat3 activation that were not distinct from levels in normal control animals. In contrast, STAT protein activation increased 8.4-fold in mucosa (P = .0037) and 5.8-fold in muscularis (P = .0001) following ischemia and 30 min of reperfusion and was still detectable at 24 hours. Supershift analysis demonstrated that in the muscu- laris the major portion of the Stat3 complex was composed of Stat3 (p92) (76  1.6%), consistent with the action of IL-6 at this site. In the mucosa only a small portion of the complex consisted of Stat3 (8.7  4.8%), while the remainder was composed of either Stat3 (p83) or Stat3 (p72). The latter is a distinct isoform of Stat3 known to be activated by G-CSF in PMN. Protein extracts of normal mucosa and muscularis externa incubated with G-CSF (100 ng/L) or IL-6 (100 ng/L) demonstrated activation of Stat3 and Stat1 proteins. CONCLUSIONS G-CSF and IL-6 production and Stat3 activation require both the ischemia and reperfusion phases. 2 The profile of Stat3 activation observed in the gut is characteristic for G-CSF and IL-6 mediated activation. The profile of Stat3 activation in the gut reflects that observed in two important effector cells, PMN and monocytes. These findings suggest that there may be two distinct mechanisms of cell activation and cell-mediated impairment of gut motility, 3 one medi- ated by PMN and the other by monocytes. REFERENCES 1. Hierholzer C, Kalff JC, Bauer A, et al: Langenbecks Arch Chir (suppl 1):579, 1997 2. Hierholzer C, Kelly E, Billiar TR, et al: Arch Orthop Trauma Surg 116:173, 1997 3. Hierholzer C, Kalff JC, Billiar TR, et al: Gastroenterology 112:A747, 1997 From the Departments of Surgery, Medicine, Molecular Ge- netics and Biochemistry, University of Pittsburgh School of Medicine and the University of Pittsburgh Cancer Institute, University of Pittsburgh Medical Center, Pittsburgh, Pennsylva- nia. Supported, in part, by NIH grant GM 53789 and by the Deutsche Forschungsgemeinschaft (DFG) HI 614/1-1. Address reprint requests to David J. Tweardy, MD, W1052 Biomedical Science Tower, University of Pittsburgh Cancer Institute, 200 Lothrop Street, Pittsburgh, PA 15213. © 1998 by Elsevier Science Inc. 0041-1345/98/$19.00 655 Avenue of the Americas, New York, NY 10010 PII S0041-1345(98)00771-4 Transplantation Proceedings, 30, 2647 (1998) 2647