Production of soluble HLA-class-I molecules by IFN-gamma-induced colon-adenocarcinoma cells

High levels of soluble HLA-class-I molecules (sHLA) were found to be produced in a time- and dose-dependent manner by colon-adenocarcinoma Colo205 cells in response to IFN-gamma stimulation. Among other cytokines tested, only IL-6, TNF, IFN-alpha and IFN-beta showed weak inducibility. IFN-gamma-induced production of sHLA was synergistically enhanced by IL-1 alpha, IL-6 or TNF, and combined treatment with TNF and IL-6 exhibited an additive to synergistic induction. Expression of sHLA is unlikely to result from IFN-gamma-induced enhancement of overall HLA-class-I expression, as at low concentrations IFN-alpha, IFN-beta and IFN-gamma stimulated increased expression of cell membrane HLA-class-I molecules in Colo205 cells with almost equal efficiency, whereas only IFN-gamma induced high level production of sHLA. Immunoblotting with a monoclonal antibody recognizing beta 2-microglobulin-free HLA-class-I heavy chain revealed 3 major forms of sHLA heavy chain, i.e., 45/43-, 37- and 33-kDa molecules, in the culture supernatants of IFN-gamma-induced Colo205 cells. The 45/43-kDa proteins can be partitioned into Triton X-114, representing intact HLA-class-I heavy chains shed from the cell membrane. The hydrophilic 37- and 33-kDa heavy chain, which remained almost exclusively in the aqueous phase after extraction with Triton X-114, could well be due to alternative RNA splicing, with deletion of exon 5 encoding the hydrophobic transmembrane region of membrane-anchored HLA-class-I heavy chain.