112. The pig (Sus scrofa) as a suitable non-rodent model system: Enabling the quantification of number and affinity of glucocorticoid receptors in porcine leukocytes

Abstract

Glucocorticoids, being one central link between the neuroendocrine and the immune system, act on leukocytes directly via endogenous glucocorticoid receptors (GR). Consequently, the number and affinity of these receptor sites in immune cells is crucial for the sensitivity towards glucocorticoids and thus their immune-modulating effects. Although the pig is considered as a suitable model organism for studying the immune system, there is no research concerning GR in porcine leukocytes so far and methods allowing their quantification are currently missing. Therefore, this study aimed at the establishment of a quantification method of GR number and affinity in porcine leukocytes. A 3 H-dexamethasone-based receptor-binding assay adapted for processing in 96-well microtiter plates allowing quantitative analysis of GR in porcine leukocytes was accomplished. A detection limit of 1.5 × 10 8 total receptor sites and a quantification limit of 7.6e8 total receptor sites was found. Evaluation of the intra-assay-precision for quantification of the GR number and affinity showed a coefficient of variation of 2.5% and 3.0%, respectively. A range of 1500–2900 GR/cell showing affinities of K d = 1.4–3.1 nM were found for porcine PBMC, thereby closely corresponding to results for human PBMC. Comparison of PBMC and granulocytes revealed similar numbers of GR/cell on both leukocyte types but lower GR affinities in granulocytes, which resembles findings in goats. In summary, the established binding assay allows reliable quantification of GR number and affinity in porcine leukocytes.