Related Experiment Videos
Endogenous PMN-derived reactive oxygen intermediates provide feedback regulation on respiratory burst signal
A Derevianko1, R D'Amico, T Graeber
1Brown University School of Medicine/Rhode Island Hospital, Department of Surgery, Providence 02903, USA.
Abstract:
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.
Insights
Reactive oxygen intermediates (ROI) regulate polymorphonuclear leukocyte (PMN) signaling. Antioxidants protect the IL-8 signal transduction pathway, suggesting ROI scavengers may mitigate infection responses.
Area of Science:
- Immunology
- Cellular Signaling
- Biochemistry
Background:
- Polymorphonuclear leukocytes (PMN) are crucial in combating systemic infections through reactive oxygen intermediates (ROI).
- ROI also function as signaling molecules, influencing cellular autoregulation via positive and negative feedback.
- Understanding ROI's role in PMN signal transduction is vital for infection control.
Purpose of the Study:
- To investigate the impact of endogenous ROI on the G-protein-phospholipase D (PLD) and G-protein-phospholipase C (PLC) signal transducing pathways.
- To determine how ROI affect the interleukin-8 (IL-8)-induced respiratory burst in human PMN.
- To evaluate the effects of various ROI scavengers/antioxidants on these pathways.
Main Methods:
- Human PMN were primed and stimulated with IL-8 in the presence or absence of ROI scavengers (DMSO, N(a)N3, L-alanine, catalase, superoxide dismutase).
- IL-8 receptor expression was measured using radioligand binding assays.
- PLD and PLC activation were assessed by quantifying specific metabolite formation (phosphatidyl ethanol, diacylglycerol).
- G alpha-inhibitory subunit expression was analyzed via SDS-PAGE and immunoblotting.
Main Results:
- Antioxidants generally stimulated components of the signal transducing complex.
- N(a)N3 and superoxide dismutase (SOD) showed the most widespread effects, with N(a)N3 up-regulating IL-8 receptor expression.
- Myeloperoxidase (MPO) significantly inhibited effector enzyme activity, even at low concentrations.
- Endogenous ROI were found to target specific sites within the signal-transducing complex.
Conclusions:
- Endogenous ROI play a significant role in modulating PMN signal transduction pathways.
- ROI scavengers/antioxidants demonstrate a protective effect on these pathways.
- Targeting ROI interactions could offer therapeutic strategies for managing infections involving PMN.