Figure 1
Figure 1. PSGL-1 is highly expressed on MM cells and regulates interaction with P-selectins. (A) Expression of PSGL-1 detected in BM biopsies from MM patients (n = 17) and healthy individuals (n = 3) using immunohistochemistry. Images are showing 20× magnification and inserts are showing 100× magnification. All MM patients presented with a higher expression of PSGL-1 compared with healthy individuals. (B) Expression of PSGL-1 evaluated on MM cell lines (MM.1S, OPM1, OPM2, LR7, RPMI.8226, U266, and H929) using flow cytometry and expressed as a ratio between the MFI of PSGL-1 and the MFI of the isotype control. (C) Gene-expression profiling of PSGL-1 from available dataset series number GSE 6477. The expression level of PSGL-1 significantly increases with MM tumor progression from MGUS to smoldering MM to newly diagnosed MM. Significant differences are observed between healthy subjects and MM patients (both smoldering MM and newly diagnosed MM) (P < .01). (D) CD138+ cells isolated from either normal BM (n = 3) or MM BM (n = 6) and MM cell lines (MM.1S, OPM1, OPM2, RPMI.8226, U266, LR7, and H929) incubated with free human Fc-chain (isotype control) or with chimeras of human-Fc chain and recombinant human E-, L-, or P-selectin (10 μg/mL) for 1 hour, followed by FITC-conjugated mouse anti–human Fc. The interaction of the selectins with MM cells was analyzed by flow cytometry and quantified as the ratio of the MFI of each selectin to the MFI of the isotype control. P- and L-selectins (but not E-selectin) interacted with MM primary cells and cell lines, whereas none of the selectins interacted with the normal plasma cells. (E) MM1s cells were transfected with either PSGL-1 siRNA or scramble siRNA. Cells were exposed to recombinant human E-, L-, and P-selectin (10 μg/mL) for 1 hour. The interaction of selectins with MM cells was analyzed by flow cytometry and quantified as ratio of MFI of each selectin to the MFI of the isotype control. Down-regulation of PSGL-1 reduced the interaction of both L- and P-selectins with MM cells. (F) MM.1S cells were treated with increasing concentrations of GMI-1070 (0, 250, and 500μM) for 1 hour and then exposed to recombinant E-, L-, and P-selectin (10 μg/mL, for 1 hour). The interaction of selectins with MM cells was analyzed by flow cytometry and quantified as a ratio of MFI of each selectin to the MFI of the isotype control. Dose-dependent inhibition of the interaction of L- and P-selectins was observed. All data represent means ± SD of triplicate experiments.

PSGL-1 is highly expressed on MM cells and regulates interaction with P-selectins. (A) Expression of PSGL-1 detected in BM biopsies from MM patients (n = 17) and healthy individuals (n = 3) using immunohistochemistry. Images are showing 20× magnification and inserts are showing 100× magnification. All MM patients presented with a higher expression of PSGL-1 compared with healthy individuals. (B) Expression of PSGL-1 evaluated on MM cell lines (MM.1S, OPM1, OPM2, LR7, RPMI.8226, U266, and H929) using flow cytometry and expressed as a ratio between the MFI of PSGL-1 and the MFI of the isotype control. (C) Gene-expression profiling of PSGL-1 from available dataset series number GSE 6477. The expression level of PSGL-1 significantly increases with MM tumor progression from MGUS to smoldering MM to newly diagnosed MM. Significant differences are observed between healthy subjects and MM patients (both smoldering MM and newly diagnosed MM) (P < .01). (D) CD138+ cells isolated from either normal BM (n = 3) or MM BM (n = 6) and MM cell lines (MM.1S, OPM1, OPM2, RPMI.8226, U266, LR7, and H929) incubated with free human Fc-chain (isotype control) or with chimeras of human-Fc chain and recombinant human E-, L-, or P-selectin (10 μg/mL) for 1 hour, followed by FITC-conjugated mouse anti–human Fc. The interaction of the selectins with MM cells was analyzed by flow cytometry and quantified as the ratio of the MFI of each selectin to the MFI of the isotype control. P- and L-selectins (but not E-selectin) interacted with MM primary cells and cell lines, whereas none of the selectins interacted with the normal plasma cells. (E) MM1s cells were transfected with either PSGL-1 siRNA or scramble siRNA. Cells were exposed to recombinant human E-, L-, and P-selectin (10 μg/mL) for 1 hour. The interaction of selectins with MM cells was analyzed by flow cytometry and quantified as ratio of MFI of each selectin to the MFI of the isotype control. Down-regulation of PSGL-1 reduced the interaction of both L- and P-selectins with MM cells. (F) MM.1S cells were treated with increasing concentrations of GMI-1070 (0, 250, and 500μM) for 1 hour and then exposed to recombinant E-, L-, and P-selectin (10 μg/mL, for 1 hour). The interaction of selectins with MM cells was analyzed by flow cytometry and quantified as a ratio of MFI of each selectin to the MFI of the isotype control. Dose-dependent inhibition of the interaction of L- and P-selectins was observed. All data represent means ± SD of triplicate experiments.

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