Determination of the eicosanoid response to inflammatory stimuli in whole blood and its pharmacological modulation ex vivo.
Recognition of pathogens by immune cells initiates the release of numerous signaling molecules, including cytokines and eicosanoids. Here, we describe a simple procedure by which eicosanoids such as prostaglandin E(2) (PGE(2)), leukotriene B(4) (LTB(4)) and thromboxane B(2) (TxB(2)) can be measured using commercial enzyme immunoassays (EIAs) in the supernatant of whole blood stimulated with inflammatory stimuli. This is illustrated for numerous stimuli. The kinetics by which lipopolysaccharide (LPS) induces cyclooxygenase (COX)-2 expression in this setup were determined by quantitative reverse transcription polymerase chain reaction (RT-PCR). The eicosanoid response of the blood of 160 healthy volunteers to 1 microg/ml LPS was measured. To determine whether the action of a drug in vivo is represented ex vivo in the eicosanoid response of blood, one volunteer took a standard dose of a number of commercially available cyclooxygenase inhibitors on different days and the eicosanoid response of his blood to LPS was determined before ingestion as well as 2 and 6 h afterwards. The efficacy of the different pharmaceuticals on cyclooxygenase but not lipoxygenase products or cytokines could be monitored ex vivo. Similarly, ex vivo eicosanoid release was measured in blood from 10 volunteers who had taken 50 mg flurbiprofen. The method described extends approaches for studying whole blood cytokine release to the lipid mediators formed from arachidonic acid. These important signaling molecules represent targets for pharmacological intervention, which can now be monitored in vitro, as well as ex vivo employing the same model. Furthermore, the assay could be used to characterize the immune status of patient groups or to monitor the course of disease.
von Aulock
Sonja
S
Biochemical Pharmacology, University of Konstanz, POB M655, 78457, Konstanz, Germany.
Hermann
Corinna
C
Hartung
Thomas
T
D000328:Adult; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000276:immunology / Q000494:pharmacology; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D015232:Dinoprostone / Q000097:blood* / Q000276:immunology; D006801:Humans; D007124:Immunoenzyme Techniques; D007249:Inflammation / Q000097:blood / Q000276:immunology; D007527:Isoenzymes / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D007700:Kinetics; D007975:Leukotriene B4 / Q000097:blood* / Q000276:immunology; D008070:Lipopolysaccharides / Q000276:immunology / Q000494:pharmacology; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology; D008297:Male; D008565:Membrane Proteins; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013929:Thromboxane B2 / Q000097:blood* / Q000276:immunology
D000894:Anti-Inflammatory Agents, Non-Steroidal; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D007527:Isoenzymes; D008070:Lipopolysaccharides; D016859:Lipoxygenase Inhibitors; D008565:Membrane Proteins; D012333:RNA, Messenger; D007975:Leukotriene B4; D013929:Thromboxane B2; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone