Neutrophil cannibalism triggers transforming growth factor 1 production and self regulation of neutrophil inflammatory function in monosodium urate monohydrate crystalinduced inflammation in mice

Objective To identify macrophage-independent sources of transforming growth factor 1 (TGF1) production during monosodium urate monohydrate (MSU) crystalinduced inflammation and to determine how TGF1 alters MSU crystalrecruited neutrophil functions. Methods C57BL/6J mice were injected intraperitoneally with MSU crystals with or without TGF1-neutralizing antibody. MSU crystalrecruited peritoneal and blood neutrophils were purified and cultured ex vivo. Peritoneal neutrophils were treated with the caspase inhibitor Q-VD-OPh, anti-TGF1 antibody, or fluorochrome-labeled apoptotic neutrophils. Neutrophils were analyzed for expression of annexin V, caspase 3, and TGF1 by flow cytometry or fluorescence microscopy, for superoxide production using the redox-sensitive dye water-soluble tetrazolium 1, and for TGF1 and interleukin-1 (IL-1) production by enzyme-linked immunosorbent assay. Results Eighteen hours after MSU crystal administration in vivo, TGF1 levels were elevated in peritoneal lavage fluids, and a significant number of peritoneal neutrophils were TGF1+. Purified blood or peritoneal neutrophils cultured ex vivo showed TGF1+ neutrophils coexpressing the apoptosis marker caspase 3 and increased TGF1 production, both of which dropped following inhibition of apoptosis. Live neutrophils that had phagocytosed apoptotic neutrophils showed greatest TGF1 expression. Superoxide production by purified MSU crystalrecruited neutrophils ex vivo was enhanced by anti-TGF1 antibody treatment. Neutrophils purified from the peritoneum of MSU crystalchallenged mice treated with anti-TGF1 antibody produced elevated levels of superoxide, but neutrophil IL-1 production was unaffected. Conclusion Neutrophil cannibalism and TGF1 production have the potential to make a significant contribution to the controlled resolution of neutrophil-driven inflammatory diseases such as gout.