Quantifying the number and affinity of glucocorticoid receptors in porcine peripheral blood mononuclear cells – Establishment of a ligand binding assay

Abstract

Glucocorticoids display a central link between the neuroendocrine and the immune system. As they act on leukocytes directly via endogenous glucocorticoid receptors (GR), both the number and affinity of these receptor sites are crucial for the immune-modulating effects of glucocorticoids. Although the pig is considered as suitable model organism for studying the immune system, there is no research concerning GR in porcine leukocytes so far. Moreover, methods allowing their quantification are currently missing. Therefore, this study aimed at the establishment of a method for the quantification of GR number and affinity in porcine peripheral blood mononuclear cells (PBMC). A 3H-dexamethasone-based receptor-binding assay adapted for processing in 96-well microtiter plates allowing quantitative analysis of GR in porcine PBMC was established. By calculating the signal to noise ratio using 106 PBMC/well a detection limit of 1.5 × 108 total receptor sites and a quantification limit of 7.6 × 108 total receptor sites were found. Evaluation of the intra-assay-precision for quantification of 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 Kd = 1.4–3.1 nM were found for porcine PBMC (n = 15), thereby closely corresponding to results for human PBMC. Comparing fresh and frozen PBMC probes revealed that PBMC stored at −80 °C showed a 13.8% increase in GR number/cell, most probably resulting from a concomitant shift in monocyte to lymphocyte ratios in the frozen probes due to different cell viabilities. In summary, the established binding assay reliably allows quantification of GR number and affinity in porcine PBMC.