Human neutrophils depend on extrinsic factors produced by monocytes for their survival response to TLR4 stimulation

LPS delays neutrophil apoptosis by a process generally assumed to involve cell-intrinsic TLR4 signaling. However, neutrophil survival responses to LPS have been reported to be monocyte-dependent, which would indicate more complexity than is currently appreciated. We compared the survival responses of conventionally purified vs highly purified neutrophils to confirm or refute the need for secondary cell-types and to identify the cellular or molecular mechanisms involved. Direct stimulation of TLR4 failed to extend the survival of highly purified neutrophils, but survival activity was retained in less pure neutrophil preparations containing low numbers of eosinophils, monocytes, platelets and CD3(+) lymphocytes. Sequential depletions identified monocytes as the only cell type required. Transfer of culture supernatants after lipid A-conditioning revealed that purified monocytes were sufficient for production of nearly all of the survival activity observed in mixed populations. The survival factors secreted upon TLR4 stimulation remain unidentified, but were not correlated with IL-1β, IL-6 or TNF-α nor could survival activity be inhibited by Ab blockade of IL-8 or of several other candidate factors other than endogenously produced GM-CSF, which was responsible for about one-tenth of the survival activity present in conditioned supernatants. These observations confirm that ex vivo neutrophil survival responses to TLR4 agonists are not cell intrinsic and involve potentially novel factors secreted by TLR4-stimulated monocytes.