Evaluation of steroid hormone receptor protein expression in intact cells using flow cytometry.

Several methods are currently employed to evaluate expression of steroid hormone receptors in tissues and cells, including real-time reverse-transcriptase polymerase chain reaction (real-time RT-PCR) and western blot assays. These methods require homogenization of cells, thereby preventing evaluation of individual cells or specific cell types in a given tissue sample. In addition, methods such as real-time RT-PCR assess mRNA levels, which may be subject to posttranslational modifications that prevent subsequent production of functional proteins. Flow cytometry is a fluorescence-based technique commonly used to evaluate expression of cell surface and intracellular proteins. This method is especially useful as it allows for single-cell analysis and can be utilized to determine the amount of receptor expressed by individual cells. Flow cytometry is commonly used to analyze immune cell activity and determine functionality based on changes in expression of cell surface molecules, as well as intracellular proteins (such as cytokines). Here, we describe a method to identify protein expression of steroid hormone receptors by rat leukocytes from different organs (spleen, liver and thymus) using flow cytometry. We examined expression of glucocorticoid receptor (GR), androgen receptor (AR) and progesterone receptor (PR) by cells at these sites and were able to demonstrate expression of receptors, as well as the intensity of expression of each receptor. This method is useful for rapid, high throughput measurement of steroid hormone receptors at the protein level in single, intact cells and would be valuable to determine which cells are more likely to respond to steroid hormone treatment.

Progesterone inhibits mature rat dendritic cells in a receptor-mediated fashion.

A variety of extraimmune system factors, including hormones, play a critical role in regulating immunity. Progesterone has been shown to affect immunity in rodents and humans, mainly at concentrations commensurate with pregnancy. These effects are primarily mediated via the progesterone receptor (PR), which acts as a transcription factor, although non-genomic effects of PR activation have been reported. In this study, we evaluated the effects of progesterone on rat dendritic cells (DCs) at ranges encompassing physiologic and pharmacologic concentrations to determine whether progesterone plays a role in modulating DC-mediated immune responses. DCs were derived by culturing rat bone marrow cells in granulocyte macrophage colony-stimulating factor and IL-4. Cells were analyzed for expression of PR using FACS analysis, real-time reverse transcriptase-PCR and fluorescent microscopy. Progesterone treatment of LPS-activated, mature bone marrow-derived dendritic cells (BMDCs) suppressed production of the pro-inflammatory response-promoting cytokines tumor necrosis factor-alpha and IL-1beta in a dose-dependent manner but did not affect production of the pro-inflammatory response-inhibiting cytokine IL-10. Treatment of cells with progesterone also resulted in down-regulation of co-stimulatory molecule CD80 and MHC class II molecule RT1B expression. In addition, progesterone inhibited DC-stimulated proliferation of T cells. Suppression of pro-inflammatory response-promoting cytokine production by progesterone was prevented using the PR antagonist RU486. There was no dose-dependent effect of progesterone treatment on immature DC capacity to take up antigenic peptide. These data indicate that progesterone directly inhibits mature rat BMDC capacity to drive pro-inflammatory responses. This mechanism could contribute to or account for some of the differential expression of autoimmune/inflammatory disease in females.