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

High levels of soluble HLA-class-l molecules (sHLA) were found to be produced in a time- and dose-dependent manner by colon-adenocarcinoma Colo20S cells in response to IFN-γ stimulation. Among other cytokines tested, only IL-6, TNF, IFN-α and IFN-β showed weak inducibility. IFN-γ-induced production of sHLA was synergistically enhanced by IL-lα, 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-γ-induced enhancement of overall HLA-class-l expression, as at low concentrations IFN-α, IFN-β and IFN-γ stimulated increased expression of cell membrane HLA-class-l molecules in Colo205 cells with almost equal efficiency, whereas only IFN-γ induced high level production of sHLA. Immunoblot-ting with a monoclonal antibody recognizing, β2-microglobulin-free HLA-class-l 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-γ-induced Colo20S cells. The 45/43-kDa proteins can be partitioned into Triton X-114, representing intact HLA-class-l 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-l 14, could well be due to alternative RNA splicing, with deletion of exon 5 encoding the hydrophobic transmembrane region of membrane-anchored HLA-class-l heavy chain.