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Temperature‐sensitive polymer‐conjugated IFN‐γ induces the expression of IDO mRNA and activity by fibroblasts populated in collagen gel (FPCG)
Author(s) -
Sarkhosh Kourosh,
Tredget Edward E.,
Uludag Hasan,
Kilani Ruhangiz T.,
Karami Ali,
Li Yunyuan,
Iwashina Takashi,
Ghahary Aziz
Publication year - 2004
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.20043
Subject(s) - indoleamine 2,3 dioxygenase , peripheral blood mononuclear cell , northern blot , western blot , kynurenine , chemistry , microbiology and biotechnology , messenger rna , in vitro , foreskin , interferon gamma , blot , interferon , conjugated system , cell culture , immunology , biochemistry , tryptophan , biology , polymer , gene , genetics , organic chemistry , amino acid
Indoleamine 2,3‐dioxygenase (IDO) is an intracellular tryptophan‐catabolizing enzyme possessing various immunosuppressive properties. Here, we report the use of this enzyme to suppress the proliferation of peripheral blood mononuclear cells (PBMC) co‐cultured with IDO‐expressing fibroblasts of an allogeneic skin substitute in vitro. Fetal foreskin fibroblasts populated within collagen gel (FPCG) were treated with interferon‐gamma (IFN‐γ) conjugated with a temperature‐sensitive polymer to induce the expression of IDO mRNA and protein. SDS–PAGE showed successful conjugation of IFN‐γ with the temperature‐sensitive polymer. Expression of IDO mRNA was evaluated by Northern analysis. IDO enzyme activity was evaluated by the measurement of kynurenine levels. The results of Northern blot analysis showed an induction of IDO mRNA expression when treated with polymer‐conjugated IFN‐γ. Kynurenine levels, as a measure of IDO bioactivity, were significantly higher in IFN‐γ‐treated fibroblasts than in controls ( P < 0.001). In a lasting effect experiment, the expression of IDO mRNA in FPCG treated with polymer‐conjugated IFN‐γ was significantly longer than in those treated with free (non‐conjugated) IFN‐γ ( P < 0.001). IFN‐γ radiolabeling showed a prolonged retention of IFN‐γ within collagen gel in its polymer‐conjugated form, compared to its free form. Presence of IDO protein in FPCG was demonstrated by Western analysis even 16 days after removal of the conditioned medium (containing released IFN‐γ). To demonstrate the immunosuppressive effects of IDO on the proliferation of PBMC, IDO‐expressing FPCG treated with polymer‐conjugated IFN‐γ were co‐cultured with PBMC for a period of 5 days. The results showed a significant reduction in proliferation of PBMC co‐cultured with IFN‐γ‐treated IDO‐expressing fibroblasts, compared to those co‐cultured with non‐IDO‐expressing fibroblasts ( P < 0.001). The addition of an IDO inhibitor (1‐methyl‐ D ‐tryptophan) reversed the suppressive effects of IDO on PBMC proliferation. In conclusion, IDO expression in FPCG suppresses the proliferation of immune cells in vitro. The use of a temperature‐sensitive polymer further prolongs the effect of IFN‐γ on the expression of IDO. Therefore, modulating IDO levels in situ might be an alternative for prolonging the survival of skin allografts. J. Cell. Physiol. 201: 146–154, 2004. © 2004 Wiley‐Liss, Inc.