Selection of insulinoma cell lines with resistance to interleukin-1 beta- and gamma-interferon-induced cytotoxicity

Engineered insulinoma cell lines may represent an alternative to isolated i slets for transplantation therapy of type 1 diabetes. Success of this appro ach may require development of cell lines that can withstand cytokine-media ted damage. To this end, we have cultured INS-1 insulinoma cells in increas ing concentrations of interleukin-1 beta (IL-1 beta) + gamma-interferon (IF N-gamma), with approximate weekly iterations over an 8-week period. Based o n the C,N diphenyl-N'-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium br omide (MTT) viability assay the selected cells, termed INS-1(res), were 100 %, viable after 5 days of treatment with 10 ng/ml of IL-1 beta. These cells were also 78 +/- 1.2%, viable after 5 days of exposure to the combination of 10 ng/ml IL-1 beta and 100 U/ml IFN-gamma, whereas parental INS-1 cells treated in the same manner were only 0.3 +/- 0.03% viable. INS-1(res) cells were also resistant to treatment with supernatants from activated rat peri pheral blood mononuclear cells, whereas only 20% of parental INS-1 cells su rvived such treatment. The resistance to IL-1 beta conferred by this proced ure was stable, whereas the partial resistance to IFN-gamma was transient b ut reinducible by culture in the presence of cytokines. Stable transfection of INS-1(res) cells with a plasmid containing the human insulin cDNA and e xpansion of the transfected colonies in the absence of cytokines produced c ell lines that were on average more resistant to IL-1 beta + IFN-gamma (53 +/- 11%) than similarly transfected clones derived from parental INS-1 cell s (15 +/- 7%). Importantly, several INS-1(res)-derived clones retained the capacity to secrete insulin in response to glucose concentrations over the normal physiological range, With regard to the mechanism by which selection was conferred, we found normal levels of IFN-gamma receptor mRNA, but a 60 % reduction in expression of the IL-1 receptor type I (IL-1R1) in INS-1(res ) cells compared with parental INS-1 cells. IL-1 beta signaling through p38 MAP kinase was found to be normal in INS-1(res) cells, suggesting that the ir expression of IL-1R1 is sufficient to maintain cytokine action, However, normal IL-1 beta-mediated translocation of NF-kB and induction of inducibl e nitric oxide synthase expression and nitric oxide production was severely impaired in the INS-1(res) cell lines, suggesting a mechanism for the IL-1 beta resistance. In sum, this study defines a strategy for isolation of cy tokine-resistant beta-cell lines and provides a new system for studying the mechanisms by which such resistance can be achieved.