Figure 2. Suppression of inflammatory signaling in recombinant AAT-Fc-pretreated mouse BM cells, AAT-Fc competing with IL-32γ binding to PR3-Fc, and the dogma of AAT anti-inflammatory activity on IL-32-induced inflammation in BM transplantation. (A) The phosphorylation of NF-κB and p38MAPK by IL-32γ in BM cells from 6-week-old C57BL/6 mice was assessed using anti-phospho antibodies. (B) Similar experiments were performed by injecting AAT-Fc (2 mg/kg) into mice for 48 h prior to isolating BM cells. (C) Recombinant PR3-Fc was used for IL-32γ binding assay. IL-32γ was added at the concentration indicated on the bottom, and IL-32γ binds to PR3-Fc in a dose dependent manner. (D) The binding of IL-32γ was blocked with PR3-Fc in a dose dependent manner. Data in C are comparisons between control blank and IL-32γ-treated cells. Data in D are comparisons between control without AAT-Fc and with AAT-Fc. Mean±SEM; ^*p<0.05; ^**p<0.01; ^#p<0.001 from three replicates. Representative data from 1 of 4 independent experiments are shown. (C) Schematic representation of AAT-Fc suppression on IL-32-mediated inflammation in BM transplantation. Allogeneic BM transplantation induces different pro-inflammatory cytokines, such as IL-32, TNFα, IL-1α, and IL-6, resulting in immune reaction causing GvHD and death, while the specific IL-32 blockade AAT-Fc reduces immune reaction increasing survival.