![Figure 1. Coculture of mouse macrophages with fresh blood or blood degradation products. RBCs (4 × 109/mL) (1 hour, 38 ± 38 pg/mL; 2 hours, 146 ± 25 pg/mL) (A) and blood extract from frozen and thawed blood (1 hour, 104 ± 21 pg/mL; 2 hours, 167 ± 23 pg/mL) (B) promoted significantly increased TNF-α shedding from RAW 264.7 cells compared with their controls. (C) Incubation of RAW 264.7 cells with 20 µM hemin also stimulated the release of TNF-α (45 ± 5 pg/mL compared with dimethyl sulfoxide–treated cells, 1 ± 0.8 pg/mL). Incubation with RBC for 4 hours (4 × 109/mL) (D) or with 20 µM hemin for 2 hours (E) stimulated the shedding of TNF-α from WT BMDMs (RBCs, 185 ±56 pg/mL; hemin, 80 ± 8 pg/mL), but not from BMDMs lacking iRhom2 (iRhom2−/−; RBCs, 21 ± 7 pg/mL; hemin, 30 ± 6 pg/mL). (F) Activation of different TLRs on RAW 264.7 cells for 2 hours also triggered the production of TNF-α from macrophages (1424 ± 445 pg/mL for TLR1/2-Pam3CSK4 [0.1 μg/mL]; 1008 ± 191 pg/mL for TLR2, keyhole limpet hemocyanin (108 cells per mL); 1787 ± 671 pg/mL for TLR4, lipopolysaccharide from E. coli (10 ng/mL); 714 ± 278 pg/mL for TLR6/2, FLS-1 (0.5 μg/mL); 506 ± 23 pg/mL for TLR7-ssRNA40 (2.5 μg/mL); 2601 ± 1185 pg/mL for TLR9, ODN1826 [2.5 μg/mL]). The mean ± SEM of 3 independent experiments in triplicate are shown. *P > .05 vs untreated control (A,D), vs EDTA-treated control (B), or vs DMSO-treated control (C,E).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/132/10/10.1182_blood-2017-12-820571/4/blood820571f1.jpeg?Expires=1730227467&Signature=Nj~78k2oV8w7gasD77a-d9HM5cNqouBkr~VmKhjzovFgc9IePznguFi8aYLWvnPVKvu1i9MYeGl5~ivXZRzkz85Jk4LFh0neaN9-BBB7GdQnc0uNTvPbjhamVUAiCM2MbvsG-5i8cMm3I-Q9ryJ5TEwufz8k-NUNQ~s~RC4t9DUHRiBTMmyhG8uWe1Qe~~Umk3xxI0Myw7QjoeWAwdxBL-zxAvFIGnWy9yEaDS2eJhdv0AZxC32~ZxBv-y34JW43sD30GEwDVQVMvGp-peyVx9K466ANDEYgffJp-aePVb-ThgfS-i9ZnEzpuyoUeE2Z6E26Oo66WH5yAamCys~S-w__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Coculture of mouse macrophages with fresh blood or blood degradation products. RBCs (4 × 109/mL) (1 hour, 38 ± 38 pg/mL; 2 hours, 146 ± 25 pg/mL) (A) and blood extract from frozen and thawed blood (1 hour, 104 ± 21 pg/mL; 2 hours, 167 ± 23 pg/mL) (B) promoted significantly increased TNF-α shedding from RAW 264.7 cells compared with their controls. (C) Incubation of RAW 264.7 cells with 20 µM hemin also stimulated the release of TNF-α (45 ± 5 pg/mL compared with dimethyl sulfoxide–treated cells, 1 ± 0.8 pg/mL). Incubation with RBC for 4 hours (4 × 109/mL) (D) or with 20 µM hemin for 2 hours (E) stimulated the shedding of TNF-α from WT BMDMs (RBCs, 185 ±56 pg/mL; hemin, 80 ± 8 pg/mL), but not from BMDMs lacking iRhom2 (iRhom2−/−; RBCs, 21 ± 7 pg/mL; hemin, 30 ± 6 pg/mL). (F) Activation of different TLRs on RAW 264.7 cells for 2 hours also triggered the production of TNF-α from macrophages (1424 ± 445 pg/mL for TLR1/2-Pam3CSK4 [0.1 μg/mL]; 1008 ± 191 pg/mL for TLR2, keyhole limpet hemocyanin (108 cells per mL); 1787 ± 671 pg/mL for TLR4, lipopolysaccharide from E. coli (10 ng/mL); 714 ± 278 pg/mL for TLR6/2, FLS-1 (0.5 μg/mL); 506 ± 23 pg/mL for TLR7-ssRNA40 (2.5 μg/mL); 2601 ± 1185 pg/mL for TLR9, ODN1826 [2.5 μg/mL]). The mean ± SEM of 3 independent experiments in triplicate are shown. *P > .05 vs untreated control (A,D), vs EDTA-treated control (B), or vs DMSO-treated control (C,E).
