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Interferon gamma-resistant mutants are defective in the induction of indoleamine 2,3-dioxygenase
1Department of Biology, Indiana University, Bloomington 47405.
Abstract:
Several mutants of the human cell line ME-180 resistant to the cytotoxic effect of interferon gamma (IFN-gamma) were isolated after mutagenesis with nitrosoguanidine. Two of the mutant lines (ME-IR3b and ME-IR6g) examined had significantly lower induction levels of the L-tryptophan (L-Trp) degradative enzyme indoleamine 2,3-dioxygenase activity in response to IFN-gamma. Moreover, culture medium supplemented with low concentrations of L-Trp reversed the cytotoxic effect of IFN-gamma, whereas higher concentrations of L-Trp in the medium were extremely toxic to both parental and mutant cells. These mutants were still protected against herpes simplex virus infection by IFN-gamma and expressed the HLA-DR alpha gene normally in the presence of this lymphokine. Thus, the mutation in these cells is specific to indoleamine 2,3-dioxygenase and not a global effect of an IFN-gamma-induced gene. This genetic evidence indicates that the major pathway of IFN-gamma cytotoxicity in this cell line is mediated primarily by induction of indoleamine 2,3-dioxygenase and deprivation of L-Trp.
Insights
Interferon gamma (IFN-gamma) cytotoxicity in ME-180 cells is primarily mediated by the L-tryptophan (L-Trp) degrading enzyme, indoleamine 2,3-dioxygenase. Mutants resistant to IFN-gamma showed reduced enzyme activity, indicating this pathway
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Interferon gamma (IFN-gamma) is a cytokine with diverse biological effects, including cytotoxicity.
- The mechanisms underlying IFN-gamma-induced cytotoxicity in human cell lines are not fully elucidated.
- L-tryptophan (L-Trp) metabolism, particularly via indoleamine 2,3-dioxygenase, has been implicated in immune responses.
Purpose of the Study:
- To investigate the role of indoleamine 2,3-dioxygenase in IFN-gamma-induced cytotoxicity.
- To isolate and characterize ME-180 cell mutants resistant to IFN-gamma.
- To determine if the mutation affects other IFN-gamma-mediated responses.
Main Methods:
- Chemical mutagenesis of ME-180 cells using nitrosoguanidine.
- Isolation and characterization of IFN-gamma-resistant mutant cell lines (ME-IR3b, ME-IR6g).
- Assay of indoleamine 2,3-dioxygenase activity induction by IFN-gamma.
- Assessment of L-Trp concentration effects on cytotoxicity.
- Evaluation of IFN-gamma-mediated protection against herpes simplex virus and HLA-DR alpha gene expression.
Main Results:
- Two mutant cell lines (ME-IR3b and ME-IR6g) exhibited significantly reduced indoleamine 2,3-dioxygenase induction by IFN-gamma.
- Low L-Trp concentrations reversed IFN-gamma cytotoxicity, while high concentrations were toxic to all cells.
- Mutants retained IFN-gamma-mediated protection against herpes simplex virus and normal HLA-DR alpha gene expression.
Conclusions:
- The primary mechanism of IFN-gamma cytotoxicity in ME-180 cells involves the induction of indoleamine 2,3-dioxygenase and subsequent L-Trp deprivation.
- The identified mutations are specific to indoleamine 2,3-dioxygenase, not a general defect in IFN-gamma signaling.
- This study provides genetic evidence for the critical role of indoleamine 2,3-dioxygenase in IFN-gamma-induced cell death.