Induction of heat shock protein gp96 by immune cytokines

Abstract

Cytokines play a major role in regulating both humoral and cell-mediated immune responses. Recent advances in our understanding of cell-mediated immune responses have focused on the antigen presentation machinery and the proteins in the endoplasmic reticulum (ER). These proteins help the formation and stabilization of the major histocompatibility complex (MHC)-peptide interaction. A 96-kDa, ER-resident glycoprotein (gp96) is being evaluated as a therapeutic agent in cancer because of its ability to associate with a vast number of cellular peptides irrespective of size or sequence. Because the antigen presentation complex is assembled in the ER and a number of ER-resident proteins are modulated by cytokines, it is important to examine the regulation of gp96 in response to immune cytokines interferon gamma (IFN-gamma), and interleukin 2 (IL-2). Defects in signaling pathway in either of the cytokines can result in suboptimal immune response. We examined the effect of the cytokines IFN-gamma and IL-2 on the induction of gp96 in different cancer cell lines and examined the induction of DNA-binding proteins that recognize gamma interferon-activating sequence (GAS), present in the promoter region of gp96. The induction of GAS binding protein correlated with the induction of STAT 1 protein, a transcriptional regulator and mediator of IFN-gamma-mediated gene expression. The use of cytokines in inducing gp96 levels may have significance in maintaining high levels of gp96 for a sustained immune response.

Fig. 1.

(A) Detection of gp96 in LG-2 cells by Western blot. Cells were either untreated (lane 1) or treated with 2 and 4 ng/mL interferon gamma (IFN-γ) for 12 hours (lanes 2, 3) and 2.5 and 5 Units/mL interleukin 2 (IL-2) for 12 hours (lanes 4, 5), and lysates were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane for Western blot analysis. Gp96 was detected using anti-grp94 monoclonal antibody (Neomarkers) and developed using enhanced chemiluminescence. (B) Detection of gp96 in T-2 cells by Western blot. Cells were either untreated (lane 1) or treated with 2 and 4 ng/mL IFN-γ for 12 hours (lanes 2, 3) and 5 and 2.5 Units/mL IL-2 for 12 hours (lanes 4, 5) and gp96 detected in lysates as above

Fig. 2.

Detection of gp96 in MCF-7 cells by Western blot. (A) Cells were either untreated (lane 1) or treated with interleukin 2 (IL-2) (5 Units/mL) for 3, 6, and 12 hours (lanes 2–4) and with interferon gamma (IFN-γ) (4 ng/mL) for 3, 6, and 12 hours (lanes 5–7), and lysates were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane for Western blot analysis. Gp96 was detected using anti-grp94 monoclonal antibody (Neomarkers) and developed using enhanced chemiluminescence. Actin was used as protein-loading control, and gp96 induction levels were normalized to actin levels. (B) Cells were either untreated (lane 1) or treated with 1, 2, and 4 ng/mL IFN-γ (lanes 2–4) for 12 hours and gp96 detected in lysates as above

Fig. 3.

Detection of gp96 in MCF-7 cells by Western blot. Cells were either untreated (lane 1) or treated with interferon gamma (IFN-γ) (4 ng/mL) for 12 hours (lane 2), IFN-γ and actinomycin D (1 μg/mL) for 12 hours (lane 3), IFN-γ and actinomycin D for 6 hours (lane 4), IFN-γ and actinomycin D for 3 hours (lane 5), or actinomycin D alone for 12 hours (lane 6), and lysates were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane for Western blot analysis. Gp96 was detected using anti-grp94 monoclonal antibody (Neomarkers) and developed using enhanced chemiluminescence

Fig. 4.

Detection of STAT 1 in MCF-7 cells by Western blot. Cells were either untreated (lane 1) or treated with interferon gamma (4 ng/mL) for 3, 6, and 12 hours (lanes 2–4), and lysates were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane for Western blot analysis. STAT 1 was detected using anti-STAT 1 polyclonal antibody (Santa Cruz Biotechnology) and developed using enhanced chemiluminescence

Fig. 5.

Detection of gamma interferon–activating sequence (GAS)–binding proteins by gel shift assay. MCF-7 cells were either untreated (lane 1) or treated with interferon gamma (4 ng/mL) for 3, 6, and 12 hours (lanes 2–4) and nuclear extracts made. The extracts were incubated with ³²P-labeled GAS deoxyribonucleic acid element for 20 minutes at room temperature and run on 4% polyacrylamide gel electrophoresis. The bands were detected by autoradiography