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Blood, Vol. 110, Issue 6, 1748-1755, September 15, 2007
Apoptosis is a natural stimulus of IL6R shedding and contributes to the proinflammatory trans-signaling function of neutrophils
Blood Chalaris et al.
110: 1748
Supplemental materials for Chalaris et al, Vol. 110, Issue 6, 1748-1755
Files in this Data Supplement:
- Figure S1. Doxorubicin-induced apoptosis of the murine pre-B-cell line Ba/F3 (PDF, 81.7 KB) -
(A) Ba/F3-gp130/IL6R cells were stimulated with 500 ng/mL doxorubicin for 0 hours, 2 hours, 4 hours and 8 hours, and apoptosis examined by annexin V-FITC and propidium iodide staining. (B) Ba/F3-gp130/IL6R cells were treated with 500 ng/mL doxorubicin for 0 hours, 3 hours, 6 hours and 8 hours. Cell lysates were prepared and the cleaved form of PKC (c-PKC) analyzed by Western blot. (C) Ba/F3-gp130/IL6R cells were treated for 8 hours with 500 ng/mL doxorubicin. The metalloprotease inhibitor marimastat (10 µM) was added as indicated 30 minutes prior to stimulation. Cell-associated IL-6R and immunoprecipitated sIL6R were analyzed by Western blot with anti-IL6R 14-18 antibody.
- Figure S2. Apoptosis of primary human neutrophils (PDF, 12.4 KB) -
Peripheral blood neutrophils were isolated as described in “Materials and methods,” and were incubated with (A) 500 ng/mL agonistic anti-Fas antibody CH-11 or were UV-treated (200,000 µj/cm2) and incubated for 6 hours. To monitor apoptosis, the cells were stained with annexin V-FITC. Results are representative of three independent experiments from separate blood donors. (B) Neutrophils were stimulated with 500 ng/mL agonistic anti-Fas antibody CH-11 and incubated for 7 hours in RPMI-medium. Recombinant human IL6 (10 ng/mL) and recombinant human Hyper-IL6 (fusion protein of IL6/sIL6R, 10 ng/mL) were added to the culture medium together with the  Fas antibody as indicated. To monitor apoptosis the cells were stained with annexin V-FITC and analyzed by flow cytometry.
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