(a) Fresh neutrophils were freeze thawed and then ultracentrifuged to generate membrane-free necrotic neutrophil supernatants (NN). NN was titrated into cultures containing LPS activated HMDMs. This was compared with the ability of NCM at a final vol:vol of 25% released from apoptotic neutrophils to inhibit TNF-α secretion. TNF-α in the supernatants collected from these stimulated macrophages (after 18 hrs of culture) was quantified by ELISA.
(b) Multicytokine analysis of these supernatants to show that NN inhibited the secretion of a wide range of pro-inflammatory cytokines as well as IL-10 by activated macrophages stimulated with LPS or CD40L/IFN-γ (CI). *** p ≤0.0002, ** p ≤0.003, * p ≤0.03.error bars =SEM. Experiments representative of 1 from 5 using separate donors.