Endogenous PMN-derived reactive oxygen intermediates provide feedback regulation on respiratory burst signal transduction

The role of polymorphonuclear leukocytes (PMN) in stemming systemic infection is executed mainly by the utilization of molecular O-2 leading to the production of reactive oxygen intermediates (ROI). PMN-derived ROI also serve as intra- and extracellular second messengers providing both positive and negative feedback on cellular autoregulation. We investigated the effect of endogenous ROI on two signal transducing pathways: the receptor (R)-G-protein-phospholipase D (PLD) and receptor (R)-G-protein-phospholipase C pathways responsible for the subsequent interleukin-8 (IL-8)-induced PMN respiratory burst. Purified human PMN were printed with LPS adhered to plastic surfaces and stimulated with IL-8 with or without the presence of each of five different selective ROI scavengers/antioxidants: DMSO, NaN3, L-alanine, catalase, or superoxide dismutase. Total IL-8 surface receptor expression was assessed by I-125-IL-8 and I-125-labeled mAbs against IL-8R type A and B binding assays; PLD activation was assessed by measuring formation of phosphatidyl ethanol (PEt) in the presence of ethanol; PLC activation was measured by quantitative conversion of [P-32]ATP-labeled phosphatidic acid (PA) into diacylglycerol (DAG); expression of G alpha-inhibitory subunit was assessed by SDS-PAGE and immunoblotting with polyclonal Abs against this subunit. Production of O-2(-), H2O2, HClO, and myeloperoxidase (MPO) in the experimental model was confirmed in a separate set of experiments. The overall impact of antioxidants on each component of the transducing tripartite complex was stimulatory; however, NaN3 and SOD exhibited the most ubiquitous effect with consistent up-regulation by NaN3 of IL-8R expression, whereas even trace amounts of externally added authentic MPO significantly down-regulated the functional activity of both effector enzymes. These results demonstrate a multiple site-specific targeting of the signal-transducing complex by endogenous PMN-derived ROI and an overall protective effect of ROI scavengers/antioxidants.