Journal of Cellular Biochemistry 90:206–217 (2003) Immune Cell Proliferation Is Suppressed by the Interferon-g-Induced Indoleamine 2,3-Dioxygenase Expression of Fibroblasts Populated in Collagen Gel (FPCG) Kourosh Sarkhosh, 1 Edward E. Tredget, 2 Ali Karami, 1 Hasan Uludag, 3 Takashi Iwashina, 1 Ruhangiz T. Kilani, 1 and Aziz Ghahary 1 * 1 Department of Surgery, Wound Healing Research Group, University of Alberta, Edmonton, Alberta T6G 2E1, Canada 2 Division of Plastic and Reconstructive Surgery, Division of Critical Care, University of Alberta, Edmonton, Alberta T6G 2E1, Canada 3 Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta T6G 2E1, Canada Abstract Indoleamine 2,3-dioxygenase (IDO), a tryptophan-catabolizing enzyme, is an intracellular enzyme possessing various immunosuppressive properties. Here, we reportthe possible use ofthis enzyme to suppress proliferation of immune cells cocultured with IDO-expressing fibroblasts of an allogenic skin substitute. Fetalskin fibroblasts embedded within bovine collagen were treated with cytokine interferon-g (IFN-g) to induce expression of ID mRNA and protein. Expression of IDO mRNA was evaluated by Northern analysis. IDO enzyme activity was evaluated b measurement of kynurenine and tryptophan levels in the IFN-g untreated and treated fibroblasts. The results of North analysis showed a dose-dependent increase in expression of IDO mRNA in response to various concentrations of IFN-g used. The levels of kynurenine and tryptophan measured, as the bioactivity of IDO, were significantly different in the I treated fibroblasts, compared to those of controls (P < 0.001). In a lasting effect experiment, the expression of IDO m was gradually reduced to an undetectable level within 32 h ofIFN-g removal. The results of Western blot analysis, however, revealed a significantly longer (192 h) lasting effect of IFN-g on IDO protein level, relative to that of mRNA expression. To demonstrate immunosuppressive effects of IDO on proliferation ofimmune cells,IDO-expressing fibroblasts were cocultured with peripheral blood mononuclear cells (PBMC) for a period of 5 days. The results of 3 H- thymidine incorporation showed a significant reduction in proliferation of PBMC when cocultured with IDO-expressing fibroblasts, compared to those cocultured with non-IDO-expressing fibroblasts (P < 0.001). Furthermore, addition of ID inhibitor (1-methyl- D-tryptophan) reversed the suppressive effects of IDO on PBMC proliferation in a dose-dependant fashion. To test the viability of immune cells cocultured with IDO-expressing fibroblasts, FACS analysis of the PI staine PBMC was conducted and no significant difference was found between these cells and the controls. In another set of experiments, we showed that migration rate and subsequent proliferation of IDO-expressing fibroblasts are also the s as those of control cells. In conclusion, IDO-expressing allogenic fibroblasts embedded within collagen gel suppress the proliferation of allogenic immune cells, while they still remain viable in this IDO-induced tryptophan-deficient cultu environment. J. Cell. Biochem. 90: 206–217, 2003. ß 2003 Wiley-Liss, Inc. Key words:indoleamine 2,3-dioxygenase (IDO); interferon-g; fibroblasts; skin substitute Extensive skin loss from a variety of condi- tions such as severe thermal injury is associated with significant functional morbidity and mor- tality [Cairns et al., 1993]. In recent years, however,the overall mortality rate has been improved for patients suffering from burns due in part to the significant biotechnological advancements in skin replacement for wound closure [Cairns et al., 1993]. In vitro cultivation of keratinocytes with the support ofa feeder ß 2003 Wiley-Liss, Inc. *Correspondence to: Aziz Ghahary, PhD, Wound Healing Research Group, Department of Surgery, 161 HMRC, University of Alberta, Edmonton, Canada T6G 2E1. E-mail: aghahary@ualberta.ca Received 25 March 2003; Accepted 14 May 2003 DOI 10.1002/jcb.10593