Release kinetics of tumor necrosis factor-α and interleukin-1 receptor antagonist in the equine whole blood.
BACKGROUND
Horses are much predisposed and susceptible to excessive and acute inflammatory responses that cause the recruitment and stimulation of polymorphnuclear granulocytes (PMN) together with peripheral blood mononuclear cells (PBMC) and the release of cytokines. The aim of the study is to develop easy, quick, cheap and reproducible methods for measuring tumor necrosis factor alpha (TNF-α) and interleukin-1 receptor antagonist (IL-1Ra) in the equine whole blood cultures ex-vivo time- and concentration-dependently.


RESULTS
Horse whole blood diluted to 10, 20 and 50 % was stimulated with lipopolysaccharide (LPS), PCPwL (a combination of phytohemagglutinin E, concanavalin A and pokeweed mitogen) or equine recombinant TNF-α (erTNF-α). TNF-α and IL-1Ra were analyzed in culture supernatants, which were collected at different time points using specific enzyme-linked immunosorbent assays (ELISA). Both cytokines could be detected optimal in stimulated 20 % whole blood cultures. TNF-α and IL-1Ra releases were time-dependent but the kinetic was different between them. PCPwL-induced TNF-α and IL-1Ra release was enhanced continuously over 24-48 h, respectively. Similarly, LPS-stimulated TNF-α was at maximum at time points between 8-12 h and started to decrease thereafter, whereas IL-1Ra peaked later between 12-24 h and rather continued to accumulate over 48 h. The equine recombinant TNF-α could induce also the IL-1Ra release.


CONCLUSIONS
Our results demonstrate that similar to PCPwL, LPS stimulated TNF-α and IL-1Ra production time-dependently in whole blood cultures, suggesting the suitability of whole blood cultures to assess the release of a variety of cytokines in health and diseases of horse.
Institute of Pharmacology, Pharmacy and Toxicology, Faculty of Veterinary Medicine, Leipzig University, An den Tierkliniken 15, 04103, Leipzig, Germany.
Simon
S
Rütten
Department of Large Animal Medicine, Faculty of Veterinary Medicine, Leipzig University, An den Tierkliniken 11, 04103, Leipzig, Germany.
Gerald F
GF
Schusser
Institute of Pharmacology, Pharmacy and Toxicology, Faculty of Veterinary Medicine, Leipzig University, An den Tierkliniken 15, 04103, Leipzig, Germany. gabraham@rz.uni-leipzig.de.
Getu
G
Abraham
Institute of Bacteriology and Mycology, Faculty of Veterinary Medicine, Leipzig University, An den Tierkliniken 29, 04103, Leipzig, Germany.
Wieland
W
Schrödl
D000818:Animals; D001774:Blood Chemical Analysis / Q000662:veterinary*; D004797:Enzyme-Linked Immunosorbent Assay; D006736:Horses; D066298:In Vitro Techniques; D007249:Inflammation / Q000662:veterinary; D053590:Interleukin 1 Receptor Antagonist Protein / Q000097:blood* / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000187:drug effects; D008070:Lipopolysaccharides / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000097:blood* / Q000378:metabolism*
D053590:Interleukin 1 Receptor Antagonist Protein; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha
ELISA; Horse; IL-1receptor antagonist; Inflammation; TNF-α; Whole blood culture