Cherie L. Butts

The role of glucocorticoids and progestins in inflammatory, autoimmune, and infectious disease

A bidirectional communication exists between the CNS and the immune system. The autonomic nervous system, through neurotransmitters and neuropeptides, works in parallel with the hypothalamic‐pituitary‐adrenal axis through the actions of glucocorticoids to modulate inflammatory events. The immune system, through the action of cytokines and other factors, in turn, activates the CNS to orchestrate negative‐feedback mechanisms that keep the immune response in check. Disruption of these interactions has been associated with a number of syndromes including inflammatory, autoimmune, and cardiovascular diseases, metabolic and psychiatric disorders, and the development of shock. The hypothalamic‐pituitary‐gonadal axis also plays an important part in regulating immunity through the secretion of sex hormones. Although numerous studies have established a role for immunomodulation by estrogen and testosterone, the role of progesterone is less well understood. Progesterone is crucial for reproductive organ development and maintenance of pregnancy, and more recent studies have clearly shown its role as an important immune regulator. The main focus of this review will be about the role of steroid hormones, specifically glucocorticoids and progesterone, in inflammatory responses and infectious diseases and how dysregulation of their actions may contribute to development of autoimmune and inflammatory disease.

Human dendritic cells require multiple activation signals for the efficient generation of tumor antigen‐specific T lymphocytes

Dendritic cells (DC) are specialized cells of the immune system responsible for the initiation and regulation of both cellular and humoral responses. DC function is highly dependent on their level of maturation. In this study, we postulated that full DC maturation would require a combination of activating signals. When cultured monocyte‐derived DC received stimulation with CD40 ligand (CD40L) and lipopolysaccharide (LPS) together, the IL‐12 secretion increased 5–60‐fold and the IL‐10 secretion increased 5–15‐fold when compared with either stimulation alone. In addition, poly I·C, a double‐stranded RNA analog that mimics viral infection, also synergized with CD40L to stimulate DC to secrete high levels of IL‐12 and IL‐10. Flow cytometry revealed an up‐regulation in the expression of CD80, CD86 and CD83 following activation with a soluble trimeric form of CD40L (CD40Ls) or LPS. However, no further up‐regulation was observed when both CD40Ls and LPS were used together compared with a single stimulatory signal, suggesting that there was no correlation between the expression of these markers and the level of IL‐12/IL‐10 secretion. Finally, specific cytotoxic T lymphocytes (CTL) were generated using DC pulsed with a modified HLA‐A2‐restricted peptide epitope derived from the melanoma antigen MART‐1. DC activated with a combination of CD40Ls and LPS were more efficient in eliciting MART‐specific reactivity compared to DC activated with CD40Ls or LPS alone. These results demonstrate that multiple maturational signals have a positive impact on the ability of DC to secrete IL‐12 and IL‐10 and more importantly, to generate antigen‐specific T lymphocytes.

Inhibitory Effects of Progesterone Differ in Dendritic Cells from Female and Male Rodents

BACKGROUND Steroid hormones, such as progesterone, are known to have immunomodulatory effects. Our research group previously reported direct effects of progesterone on dendritic cells (DCs) from female rodents. Primarily affecting mature DC function, progesterone effects included inhibition of proinflammatory cytokine secretion, downregulation of cell surface marker (major histocompatibility complex class II, CD80) expression, and decreased T-cell proliferative capacity, and were likely mediated through progesterone receptor (PR) because the PR antagonist RU486 reversed these effects. OBJECTIVE The goal of this study was to assess differences in response to progesterone by DCs from female and male rodents. METHODS Using real-time reverse-transcriptase polymerase chain reaction, transcriptional expression of steroid hormone receptors was measured in immature bone marrow-derived DCs (BMDCs) from male and female rats. Expression of steroid hormone receptor protein was also assessed in these cells using flow cytometry and fluorescence microscopy. To evaluate functional differences between BMDCs from female and male rats in response to the steroid hormone progesterone, levels of secreted cytokines were measured using enzyme-linked immunosorbent assay. RESULTS Higher numbers of immature BMDCs from males expressed glucocorticoid receptor (GR) and androgen receptor (AR) proteins compared with females (males vs females, mean [SD]: GR = 68.75 [7.27] vs 43.61 [13.97], P = NS; AR = 75.99 [15.38] vs 8.25 [1.88], P = 0.002), whereas higher numbers of immature BMDCs from females expressed PR protein compared with males (females vs males: PR = 74.19 [12.11] vs 14.14 [4.55], P = 0.043). These differences were not found at the level of transcription (females vs males: GR = 0.088 vs 0.073, P = NS; AR = 0.076 vs 0.069, P = NS; PR = 0.075 vs 0.065, P = NS). Compared with those from females, mature BMDCs from males produced higher quantities of cytokines (tumor necrosis factor-alpha [TNF-alpha], interleukin [IL]-1beta, IL-10) (females vs males: TNF-alpha = 920.0 [79.25] vs 1100.61 [107.97], P = NS; IL-1beta = 146.60 [38.04] vs 191.10 [10.47], P = NS; IL-10 = 167.25 [4.50] vs 206.15 [23.48], P = NS). Conversely, BMDCs from females were more sensitive to progesterone, as indicated by a more dramatic reduction in proinflammatory cytokine secretion (females vs males, highest concentration of progesterone: TNF-alpha = 268.94 [28.59] vs 589.91 [100.98], P = 0.04; IL-1beta = 119.50 [10.32] vs 154.35 [6.22], P = NS). CONCLUSIONS These findings suggest that progesterone effects on DCs in rodents may be more pronounced in females than in males, and this is likely due to differences in PR protein expression. Our observations may help elucidate disparities in the incidence and severity of autoimmune disorders between females and males, and the role specific steroid hormones play in regulating immune responses.

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.

Tissue expression of steroid hormone receptors is associated with differential immune responsiveness

Glucocorticoids have been used as treatments against a number of diseases, especially autoimmune/inflammatory conditions in which the immune system is overactive. These treatments have varying degrees of responsiveness among individuals and in different tissues (including brain); therefore, it is important to determine what could account for these differences. In this study, we evaluated expression of stress hormone receptors in immune cells from lymphoid and non-lymphoid tissues (including brain) as a possible explanation. We analyzed leukocytes (CD45(+)) in kidney, liver, spleen, and thymus tissues from healthy mice for expression of the receptor for stress hormone (glucocorticoid-GR) as well as other steroid hormones (androgen-AR, progesterone-PR) and found that all tissues expressed these steroid hormone receptors but with varying patterns. To determine whether tissue-specific differences were related to immune cell composition, we examined steroid hormone receptor expression in T lymphocytes from each of these tissues and found similar patterns of expression in these cells regardless of tissue source. Because glucocorticoids can also impact brain function, we further examined expression of the stress hormone receptor in brain tissue and found GR expressed in immune cells at this site. In order to investigate the potential impact in an area of neuropathology, we utilized a mouse model of West Nile Virus (WNV). We observed pathological changes in brains of WNV-infected animals and T lymphocytes in the areas of inflammation; however, these cells did not express GR. These data indicate that tissue-specific differences in steroid hormone receptor expression by immune cells could determine responsiveness to steroid hormone treatment.

Flow Cytometry as a Tool for Measurement of Steroid Hormone Receptor Protein Expression in Leukocytes

Measurement of protein expression in live, intact cells using flow cytometry (FC) has been employed for several decades in the areas of immunology, cell biology, and molecular biology. More recently, this technique has found appreciation in applied scientific fields, including cancer biology and endocrinology, to serve as a tool for identifying cells more likely to respond to specific treatments. FC, also referred to as fluorescence-activated cell sorting (FACS), is an antibody-based method that provides the user with an ability to identify proteins expressed on surfaces of cells as well as in the cytoplasm, including steroid hormone receptors. This technique is most useful for examining specific cell types in a heterogeneous population and therefore can be used to identify cells more likely to respond to treatments based on expression of the appropriate receptor. Isolation of purified subpopulations for further manipulation and investigation of functional capacity is also possible using a cell sorter, which uses similar technology to isolate cells for use by the researcher. This is especially important for studying responses of less abundant cell populations in tissues that express high levels of a target protein or receptor of interest. Furthermore, FACS analysis is clinically useful to identify and isolate responsive cell populations, which may be less appreciable in whole tissues because of the diluting effects of surrounding, nonresponding cell types. Immune cells are commonly utilized as a source of cell populations in the FC technique and have previously been shown to express steroid hormone receptors and respond to steroid hormone treatment. Here, we demonstrate that FC is a useful tool for identifying immune cells expressing steroid hormone receptor protein. This method can also be easily expanded to include other, nonimmune cell populations to address specific research questions related to steroid hormone receptor biology.