Figure 3.
Inhibition of BCL-XL kills neutrophils from inflamed human and mouse joints and halts the progression of STIA. (A) STIA was induced in Rosa26CreERT2.Bcl-xfl/fl mice. Arthritic mice were treated for 4 days with 2 mg tamoxifen (+TM) or vehicle control (−TM) by oral gavage starting day 4 postserum injection to induce deletion of Bcl-x leading to loss of BCL-XL protein (as indicated by arrows). Following treatment (d8 postserum injection), PMN were enumerated in joint and blood by PI staining and flow cytometry. Data show pooled data from n = 2 experiments; 10-11 animals/group; mean ± SEM. (B) STIA was induced in B6 mice by IP injection of K/BxN serum. Arthritic mice were treated for 5 days with 100 mg/kg BCL-XL inhibitor or vehicle by oral gavage, starting d2 postserum injection (as indicated by arrows). Disease severity was measured using a standardized visual scoring system.74 Following inhibitor treatment (d7 postserum injection) PMN were enumerated in joint and blood by PI staining and flow cytometry. Data shown were pooled from 2 experiments (n = 10/group STIA); mean ± SEM. (C) Joint and blood PMN were sorted from arthritic mice and western blot analysis performed to assess MCL-1, BCL-XL, A1, and actin expression. BCL-XL expression is displayed relative to actin loading control. Data shown were pooled from 2 experiments (n = 10/group); mean ± SEM. (D) Cell aspirates were obtained from inflamed joints of inflammatory arthritis patients and enriched for neutrophils on a Ficoll gradient. Cells were preconditioned in culture in the presence or absence of GM-CSF or G-CSF at the indicated dose for 16 hours. Cells were then cultured in the presence or absence of 1 µM BCL-XL inhibitor (A-1331852), MCL-1 inhibitor (S63845), or BCL-2 inhibitor (ABT-199). After 16 hours, viable PMN were enumerated by flow cytometry. Data are shown as mean ± SD of PMN recovery per well. Data show a single representative experiment of n = 2 experiments. All data were analyzed using a 2-tailed Student t test. *P < .05, **P < .01, ***P < .001.

Inhibition of BCL-XL kills neutrophils from inflamed human and mouse joints and halts the progression of STIA. (A) STIA was induced in Rosa26CreERT2.Bcl-xfl/fl mice. Arthritic mice were treated for 4 days with 2 mg tamoxifen (+TM) or vehicle control (−TM) by oral gavage starting day 4 postserum injection to induce deletion of Bcl-x leading to loss of BCL-XL protein (as indicated by arrows). Following treatment (d8 postserum injection), PMN were enumerated in joint and blood by PI staining and flow cytometry. Data show pooled data from n = 2 experiments; 10-11 animals/group; mean ± SEM. (B) STIA was induced in B6 mice by IP injection of K/BxN serum. Arthritic mice were treated for 5 days with 100 mg/kg BCL-XL inhibitor or vehicle by oral gavage, starting d2 postserum injection (as indicated by arrows). Disease severity was measured using a standardized visual scoring system.74 Following inhibitor treatment (d7 postserum injection) PMN were enumerated in joint and blood by PI staining and flow cytometry. Data shown were pooled from 2 experiments (n = 10/group STIA); mean ± SEM. (C) Joint and blood PMN were sorted from arthritic mice and western blot analysis performed to assess MCL-1, BCL-XL, A1, and actin expression. BCL-XL expression is displayed relative to actin loading control. Data shown were pooled from 2 experiments (n = 10/group); mean ± SEM. (D) Cell aspirates were obtained from inflamed joints of inflammatory arthritis patients and enriched for neutrophils on a Ficoll gradient. Cells were preconditioned in culture in the presence or absence of GM-CSF or G-CSF at the indicated dose for 16 hours. Cells were then cultured in the presence or absence of 1 µM BCL-XL inhibitor (A-1331852), MCL-1 inhibitor (S63845), or BCL-2 inhibitor (ABT-199). After 16 hours, viable PMN were enumerated by flow cytometry. Data are shown as mean ± SD of PMN recovery per well. Data show a single representative experiment of n = 2 experiments. All data were analyzed using a 2-tailed Student t test. *P < .05, **P < .01, ***P < .001.

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