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Lucigenin- and luminol-enhanced chemiluminescence in turkey monocytes
A Van Nerom1, M Desmidt, R Ducatelle
1Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, University of Ghent, Belgium.
Insights
Turkey monocytes show oxidative activity measurable by chemiluminescence (CL). Lucigenin-enhanced CL, reflecting oxidase activity, was more sensitive than luminol-enhanced CL, which relies on peroxidase activity absent in these cells.
Area of Science:
- Immunology
- Avian Biology
- Cellular Physiology
Background:
- Monocytes play a crucial role in host defense.
- Assessing oxidative activity in avian monocytes is important for understanding their immune function.
- Previous studies have focused on mammalian monocyte oxidative responses.
Purpose of the Study:
- To characterize the oxidative activity of turkey monocytes using chemiluminescence assays.
- To compare the efficacy of lucigenin and luminol as chemiluminigenic probes for turkey monocyte oxidative activity.
- To evaluate the response of turkey monocytes to various stimuli.
Main Methods:
- Isolation of monocytes from specific pathogen-free turkey peripheral blood.
- Measurement of chemiluminescence (CL) using luminometer with lucigenin and luminol enhancers.
- Stimulation of monocytes with phorbol myristate acetate (PMA), Zymosan A, calcium ionophore A23187, and N-formyl-methionyl-phenylalanine.
Main Results:
- Turkey monocytes exhibited distinct oxidative responses to PMA and Zymosan A, with weaker responses to calcium ionophore.
- No oxidative activity was induced by N-formyl-methionyl-phenylalanine.
- Lucigenin-enhanced CL showed higher sensitivity than luminol-enhanced CL, particularly for PMA and Zymosan A.
- No peroxidase activity was detected in turkey monocytes, explaining lower luminol-enhanced responses.
Conclusions:
- Turkey monocytes possess significant oxidase activity, measurable by lucigenin-enhanced chemiluminescence.
- The absence of peroxidase activity impacts luminol-based assays.
- Chemiluminescence assays provide a valuable tool for studying turkey monocyte function and host defense mechanisms.
Abstract:
Monocytes from 10 week-old specific pathogen-free turkeys were isolated from peripheral blood by density centrifugation and assayed for their oxidative activity by means of a luminometer. Chemiluminescence (CL) properties after stimulation with different soluble and particulate stimuli were compared in lucigenin- and luminol-enhanced assays. A distinct response could be measured with 12-phorbol 13-myristate acetate (PMA) and Zymosan A, but only a weak signal was obtained with calcium ionophore A23187. No oxidative activity could be induced with N-formyl-methionyl-phenylalanine. Peak maxima for both lucigenin- and luminol-enhanced CL were ranked: PMA > Zymosan A > calcium ionophore. The velocity of the lucigenin- and luminol-enhanced responses induced by calcium ionophore were of similar magnitude, but the lucigenin-enhanced responses of Zymosan A and PMA-stimulated monocytes were respectively about 5 and 10 times higher than those obtained in luminol-enhanced assays. No peroxidase activity could be detected in the purified turkey monocytes. As luminol-enhanced CL primarily results from the peroxidase activity, this lack of myeloperoxidase may explain the observed lower responses to the different stimuli, in the presence of a luminol. In contrast, lucigenin-enhanced CL is not related to peroxidase activity, but is a selective probe of oxidase activity. Irrespective of the myeloperoxidase deficiency, different soluble and particulate stimuli induced a significant and reproducible CL response in turkey monocytes, in the presence of both chemiluminigenic probes, lucigenin and luminol. The possibility of measuring the phagocyte oxygenation activity of turkey monocytes represents a useful tool for the study of monocyte mediated host defence in the turkey.