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 O2 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 primed 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, N(a)N3, L-alanine, catalase, or superoxide dismutase. Total IL-8 surface receptor expression was assessed by 125I-IL-8 and 125I-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 [32P]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 O2-, 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, N(a)N3 and SOD exhibited the most ubiquitous effect with consistent up-regulation by N(a)N3 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.
Derevianko
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A
Brown University School of Medicine/Rhode Island Hospital, Department of Surgery, Providence 02903, USA.
D'Amico
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R
Graeber
T
T
Keeping
H
H
Simms
H H
HH
D000409:Alanine / Q000494:pharmacology; D015703:Antigens, CD / Q000378:metabolism; D000975:Antioxidants / Q000494:pharmacology; D001386:Azides / Q000494:pharmacology; D002374:Catalase / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005246:Feedback; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D006801:Humans; D016209:Interleukin-8 / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D009504:Neutrophils / Q000502:physiology*; D009195:Peroxidase / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism; D017382:Reactive Oxygen Species; D018123:Receptors, Interleukin / Q000378:metabolism; D023062:Receptors, Interleukin-8A; D016897:Respiratory Burst; D015290:Second Messenger Systems; D015398:Signal Transduction; D013482:Superoxide Dismutase / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism
D015703:Antigens, CD; D000975:Antioxidants; D001386:Azides; D016209:Interleukin-8; D008070:Lipopolysaccharides; D017382:Reactive Oxygen Species; D018123:Receptors, Interleukin; D023062:Receptors, Interleukin-8A; D002374:Catalase; D009195:Peroxidase; D013482:Superoxide Dismutase; D010738:Type C Phospholipases; D010739:Phospholipase D; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000409:Alanine