Engineered
insulinoma cell lines may represent an alternative to isolated islets for
transplantation therapy of
type 1 diabetes. Success of this approach may require development of cell lines that can withstand
cytokine-mediated damage. To this end, we have cultured INS-1
insulinoma cells in increasing concentrations of
interleukin-1beta (IL-1beta) +
gamma-interferon (IFN-gamma), with approximate weekly iterations over an 8-week period. Based on the C,N
diphenyl-N'-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium+ ++
bromide (MTT) viability assay, the selected cells, termed INS-1res, were 100% viable after 5 days of treatment with 10 ng/ml of IL-1beta. These cells were also 78 +/- 1.2% viable after 5 days of exposure to the combination of 10 ng/ml IL-1beta and 100 U/ml IFN-gamma, whereas parental INS-1 cells treated in the same manner were only 0.3 +/- 0.03% viable. INS-1res cells were also resistant to treatment with supernatants from activated rat peripheral blood mononuclear cells, whereas only 20% of parental INS-1 cells survived such treatment. The resistance to IL-1beta conferred by this procedure was stable, whereas the partial resistance to IFN-gamma was transient but reinducible by culture in the presence of
cytokines. Stable transfection of INS-1res cells with a plasmid containing the human
insulin cDNA and expansion of the transfected colonies in the absence of
cytokines produced cell lines that were on average more resistant to IL-1beta + IFN-gamma (53 +/- 11%) than similarly transfected clones derived from parental INS-1 cells (15 +/- 7%). Importantly, several INS-1res-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-1RI) in INS-1res cells compared with parental INS-1 cells. IL-1beta signaling through
p38 MAP kinase was found to be normal in INS-1res cells, suggesting that their expression of IL-1RI is sufficient to maintain
cytokine action. However, normal IL-1beta-mediated translocation of
NF-kappaB and induction of
inducible nitric oxide synthase expression and
nitric oxide production was severely impaired in the INS-1res cell lines, suggesting a mechanism for the IL-1beta resistance. In sum, this study defines a strategy for isolation of
cytokine-resistant beta-cell lines and provides a new system for studying the mechanisms by which such resistance can be achieved.