Research ArticleCytokines

Soluble gp130 prevents interleukin-6 and interleukin-11 cluster signaling but not intracellular autocrine responses

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Science Signaling  02 Oct 2018:
Vol. 11, Issue 550, eaar7388
DOI: 10.1126/scisignal.aar7388
  • Fig. 1 sgp130Fc binds to membrane-bound IL-6:IL-6R complexes.

    (A) Simple model of how sgp130 (white) may bind to IL-6 (light gray):IL-6R (dark gray) complexes on the cell surface of Ba/F3 cells. (B) Flow cytometric analysis of cell surface IL-6R abundance on control (gray-filled) or IL-6R stably transduced (line) Ba/F3 cells. Histograms are representative of three independent experiments. Normalized mean fluorescence intensity (MFI) data are means ± SEM from all experiments. ** P = 0.01. (C) Flow cytometric analysis of recombinant IL-6 binding to control (gray-filled) or IL-6R transduced (line) or Ba/F3 cells. Histograms are representative of three independent experiments. Normalized MFI data are means ± SEM from all experiments. ns, not significant. (D) Flow cytometric analysis of recombinant sgp130Fc binding to membrane-bound IL-6:IL-6R complexes on control (gray-filled) or IL-6R transduced (line) Ba/F3 cells. Histograms are representative of three independent experiments. Normalized MFI data are means ± SEM from all experiments. (E) Flow cytometric analysis of recombinant sgp130Fc binding to membrane-bound IL-6R on control (gray-filled) or IL-6R transduced (line) Ba/F3 cells. Histograms are representative of three independent experiments. Normalized MFI data are means ± SEM from all experiments.

  • Fig. 2 sgp130Fc binds to mbHIL-6 complexes.

    (A and B) Molecular model of mbHIL-6 (A) and nmbHIL-6 (B) that indicates IL-6 (yellow), D2 and D3 of the IL-6R (green), the linker between IL-6 and IL-6R (red), and the stalk region, transmembrane (TM), and intracellular domain (ICD) of IL-6R (gray). (C) Simple model of how sgp130 (white) may bind to mbHIL-6 (gray) on the cell surface of Ba/F3 cells. (D) Flow cytometric analysis of IL-6R abundance on control (gray-filled) and mbHIL-6 stably transduced (line) Ba/F3 cells. Histograms are representative of three independent experiments. Normalized MFI data are means ± SEM from all experiments. *P ≤ 0.05. (E) Flow cytometric analysis of recombinant sgp130Fc binding to control (gray-filled) or mbHIL-6 transduced (line) Ba/F3 cells. Histograms are representative of three independent experiments. Normalized MFI data are means ± SEM from all experiments. (F) Simple model of how sgp130 (white) binds to nmbHIL-6 (gray) on the cell surface of Ba/F3 cells. (G) Flow cytometric analysis of nmbHIL-6 abundance on control (gray-filled) and nmbHIL-6 transduced (line) Ba/F3 cells. Histograms are representative of three independent experiments. Normalized MFI data are means ± SEM from all experiments. ***P = 0.001. (H) Flow cytometric analysis of recombinant sgp130Fc binding to nmbHIL-6 on Ba/F3 cells. Histograms are representative of three independent experiments. Normalized MFI data are means ± SEM from all experiments. **P = 0.01.

  • Fig. 3 Transactivation by IL-6:IL-6R complexes is inhibited by sgp130Fc.

    (A) Simple model of IL-6 transactivation, where IL-6 (light gray):IL-6R (dark gray) complexes on transmitter cells activate gp130 (white) on receiver cells. (B) CellTiter-Blue analysis of the proliferation of Ba/F3-gp130 cells, Ba/F3–IL-6R cells, or cocultures incubated with the indicated cytokines or recombinant proteins for 3 days. Data are means ± SD representative of three independent experiments. RFU, relative fluorescence unit. (C and D) Western blot analysis of pSTAT3 in lysates of Ba/F3–IL-6R or Ba/F3-gp130 cells (C) or cocultured Ba/F3–IL-6R and Ba/F3-gp130 cells stimulated with cytokines or recombinant proteins, as indicated. Blots are representative of three independent experiments. Normalized band intensity data are means ± SEM from all experiments. (E) Western blot analysis of pSTAT3 in lysates of cocultured Ba/F3–IL-6R and Ba/F3-gp130 cells incubated with IL-6 and sgp130Fc, tocilizumab, or GW280264X (GW) as indicated. Blots are representative of three independent experiments. Normalized band intensity data are means ± SEM from all experiments. **P = 0.01. (F) Western blot analysis of pSTAT3 in lysates of Ba/F3-gp130 cells stimulated by 10% cellular supernatants collected after cocultivation experiments in (E). Blots are representative of three independent experiments. Normalized band intensity data are means ± SEM from all experiments. *P ≤ 0.05 and ***P ≤ 0.001 by analysis of variance (ANOVA) test with Dunnett’s correction.

  • Fig. 4 Transactivation by mbHIL-6 or nmbHIL-6 complexes is inhibited by sgp130Fc.

    (A) Simple model of IL-6 transactivation, where IL-6 (light gray):IL-6R (dark gray) complexes encoded in mbHIL-6 and nmbHIL-6 on transmitter cells activate gp130 (white) on receiver cells. (B) CellTiter-Blue analysis of the proliferation by Ba/F3-gp130 cells, Ba/F3–IL-6R cells, or cocultures incubated with the indicated cytokines or recombinant sgp130Fc for 3 days. Data are means ± SD representative of three independent experiments. (C) Western blot analysis of pSTAT3 in lysates of Ba/F3-gp130, Ba/F3–mbHIL-6, or Ba/F3–nmbHIL-6 cells stimulated with HIL-6 or IL-3 for 30 min. Blots are representative of three independent experiments. Normalized band intensity data are means ± SEM from all experiments. (D and E) Western blot analysis of pSTAT3 in lysates of Ba/F3-gp130 cells cocultured with Ba/F3–mbHIL-6 cells (D) or Ba/F3–nmbHIL-6 cells (E) and incubated with sgp130Fc or tocilizumab and GW280264X, as indicated. Blots are representative of three independent experiments. Normalized band intensity data are shown in fig. S1 (A and B). (F and G) Western blot analysis of pSTAT3 in lysates of Ba/F3-gp130 cells stimulated with 10% supernatants collected from the coculture experiments in panel (D) (F) or panel (E) (G). Blots are representative of three independent experiments. Normalized band intensity data are shown in fig. S1 (C and D). *P ≤ 0.05 and ***P ≤ 0.001 by ANOVA test with Bonferroni correction (B) or ANOVA test with Dunnett’s correction (C).

  • Fig. 5 Transactivation by membrane-bound IL-11:IL-11R or mbHIL-11 complexes is inhibited by sgp130Fc.

    (A) Simple model of IL-11 transactivation, where IL-11 (light gray): IL-11R (dark gray) or mbHIL-11 on transmitter cells activates gp130 (white) on receiver cells. (B) Flow cytometric analysis of human IL-11R cell surface abundance on control (gray-filled) and IL-11R, mbHIL-11, or mbHIL-11R355E stably transduced Ba/F3 cells. Histograms are representative of three independent experiments. Normalized MFI data from all experiments are shown in fig. S2A. (C) Flow cytometric analysis of recombinant sgp130Fc binding to membrane-bound IL-11:IL-11R complexes, mbHIL-11, or mbHIL-11R355E expressed on Ba/F3 cells. Histograms are representative of three independent experiments. Normalized MFI data from all experiments are shown in fig. S2C. (D) CellTiter-Blue analysis of cellular proliferation by Ba/F3–IL-11R, Ba/F3–mbHIL-11, or Ba/F3–mbHIL-11R355E cells co-incubated with Ba/F3-gp130 cells in the presence of the indicated cytokines and recombinant sgp130Fc for 3 days. Data are means ± SD representative of three independent experiments. (E) Western blot analysis of pSTAT3 in lysates of Ba/F3-gp130 cells cocultured with Ba/F3–IL-11R, Ba/F3–mbHIL-11, or Ba/F3–mbHIL-11R355E cells and treated with IL-11, sgp130Fc, or GW as indicated. Blots are representative of two independent experiments. Normalized band intensity data from each experiment are shown in fig. S2D. (F) Western blot analysis of pSTAT3 in Ba/F3-gp130 cells stimulated with 10% supernatants collected from cocultivation in experiments in (E). Blots are representative of two independent experiments. Normalized band intensity data from each experiment are shown in fig. S2E. ***P ≤ 0.001 by ANOVA test with Bonferroni correction.

  • Fig. 6 Autocrine classic and trans-signaling by nmbHIL-6 or HIL-6 is not inhibited by extracellular inhibitors.

    (A) Simple model of autocrine classic signaling through gp130 (white) by IL-6 (light gray):IL-6R (dark gray) encoded in nmbHIL-6 and trans-signaling by HIL-6. (B) Flow cytometric analysis of cell surface IL-6R abundance on control (gray-filled) and nmbHIL-6 stably transduced (line) Ba/F3-gp130 cells. Histograms are representative of three independent experiments. Normalized MFI data from all experiments are shown in fig. S3A. (C) Western blot (WB) analysis of HIL-6 abundance in cellular lysates (L) and cell culture supernatants (SN) of Ba/F3-gp130 control or HIL-6 stably transduced cells. Blots are representative of three independent experiments. (D) CellTiter-Blue analysis of cellular proliferation by Ba/F3-gp130, Ba/F3–gp130–nmbHIL-6, or Ba/F3–gp130–HIL-6 cells treated with HIL-6 or sgp130Fc, as indicated for 3 days. Data are means ± SD representative of three independent experiments. (E and F) Western blot analysis of pSTAT3 in lysates of Ba/F3-gp130 (E) or Ba/F3–gp130–nmbHIL-6, Ba/3–gp130–HIL-6, and Ba/F3–gp130–HIL-6R–IL-6 (F) cells treated with HIL-6, sgp130Fc, tocilizumab, P6, or B-R3, as indicated. Blots are representative of three independent experiments. Normalized band intensity data are means ± SD from all experiments (E) or are in fig. S3B (F). (G) Analysis of pSTAT3 in HEK 293 cells transfected with HIL-6Fc or sgp130Fc or treated with HIL-6Fc or sgp130Fc, as indicated. Blots are representative of three independent experiments. Normalized band intensity data from all experiments are shown in fig. S3C. *P ≤ 0.05 and ***P ≤ 0.001 by ANOVA test with Bonferroni correction (D) or ANOVA test with Dunnett’s correction (E).

  • Fig. 7 Autocrine classic and trans-signaling by mbHIL-11 is not inhibited by extracellular inhibitors.

    (A) Simple model of autocrine classic signaling through gp130 (white) by IL-11 (light gray):IL-11R (dark gray) complexes encoded in mbHIL-11. (B) Flow cytometric analysis of cell surface IL-11R abundance on control (gray-filled) and IL-11R or mbHIL-11 stably transduced (line) Ba/F3-gp130 cells. Histograms are representative of three independent experiments. Normalized MFI data are means ± SD from all experiments. (C) CellTiter-Blue analysis of cellular proliferation by Ba/F3-gp130 or Ba/F3–gp130–mbHIL-11 cells treated with HIL-6 and sgp130Fc, as indicated for 3 days. Data are means ± SD representative of three independent experiments. (D and E) Western blot analysis of pSTAT3 in lysates of Ba/F3–gp130–IL-11R (D) or Ba/F3–gp130–mbHIL-11 (E) cells treated with IL-11, sgp130Fc, tocilizumab, P6, or B-R3, as indicated. Blots are representative of at least two independent experiments. Normalized band intensity data are from each experiment (D) or are means ± SD from three experiments (E). *P ≤ 0.05 and ***P ≤ 0.001 by ANOVA test with Bonferroni correction (C) or ANOVA test with Dunnett’s correction (E).

  • Fig. 8 Schematic overview of sgp130 effects on IL-6 and IL-11 signaling modes.

    (A) sgp130Fc did not inhibit IL-6/IL-11 classic signaling. (B) sgp130Fc binds IL-6:sIL-6R and IL-11:sIL-11R complexes and inhibits paracrine trans-signaling. (C) sgp130Fc did not inhibit autocrine IL-6 trans-signaling. PM, plasma membrane. (D) sgp130Fc binds IL-6:IL-6R and IL-11:IL-11R complexes on transmitter cells and inhibits transactivation on receiver cells. White, gp130; light gray, IL-6/IL-11; dark gray, IL-6R/IL-11R.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/550/eaar7388/DC1

    Fig. S1. Statistical analysis of membrane-bound HIL-6–mediated IL-6 cluster signaling.

    Fig. S2. Statistical analysis of membrane-bound HIL-11–mediated IL-11 cluster signaling.

    Fig. S3. Autocrine classic and trans-signaling by nmbHIL-6 or HIL-6 is not inhibited by extracellular inhibitors.

  • This PDF file includes:

    • Fig. S1. Statistical analysis of membrane-bound HIL-6–mediated IL-6 cluster signaling.
    • Fig. S2. Statistical analysis of membrane-bound HIL-11–mediated IL-11 cluster signaling.
    • Fig. S3. Autocrine classic and trans-signaling by nmbHIL-6 or HIL-6 is not inhibited by extracellular inhibitors.

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