Karen Bulloch

The role of adrenocorticoids as modulators of immune function in health and disease: neural, endocrine and immune interactions.

Bruce S. McEwen , Christine A. Biron , Kenneth W. Brunson , Karen Bulloch , William H. Chambers , Firdaus S. Dhabhar , Ronald H. Goldfarb , Richard P. Kitson , Andrew H. Miller , Robert L. Spencer , Jay M. Weiss d a Laboratory of Neuroendocrinology, Rockefeller UniÕersity, 1230 York AÕenue, Box 165, New York, NY 10021, USA b DiÕision of Biology and Medicine, Brown UniÕersity, ProÕidence, RI, USA c Pittsburgh Cancer Institute, UniÕersity of Pittsburgh, School of Medicine, Pittsburgh, PA, USA d Department of Psychiatry and BehaÕioral Sciences, Emory UniÕersity School of Medicine, Atlanta, GA, USA

Ovarian steroids and the brain Implications for cognition and aging

Article abstract-Ovarian steroids have many effects on the brain throughout the lifespan, beginning during gestation and continuing into senescence. These hormones affect areas of the brain that are not primarily involved in reproduction, such as the basal forebrain, hippocampus, caudate putamen, midbrain raphe, and brainstem locus coeruleus. Here we discuss three effects of estrogens and progestins that are especially relevant to memory processes and identify hormonal alterations associated with aging and neurodegenerative diseases. First, estrogens and progestins regulate synaptogenesis in the CA1 region of the hippocampus during the 4- to 5-day estrous cycle of the female rat. Formation of new excitatory synapses is induced by estradiol and involves N-methyl-D-aspartate (NMDA) receptors, whereas synaptic downregulation involves intracellular progestin receptors. Second, there are developmentally programmed sex differences in the hippocampal structure that may help to explain why male and female rats use different strategies to solve spatial navigation problems. During the period of development when testosterone is elevated in the male, aromatase and estrogen receptors are transiently expressed in the hippocampus. Recent data on behavior and synapse induction strongly suggest that this pathway is involved in the masculinization or defeminization of hippocampal structure and function. Third, ovarian steroids have effects throughout the brain, including effects on brainstem and midbrain catecholaminergic neurons, midbrain serotonergic pathways, and the basal forebrain cholinergic system. Regulation of the serotonergic system appears to be linked to the presence of estrogen- and progestin-sensitive neurons in the midbrain raphe, whereas the ovarian steroid influence on cholinergic function involves induction of choline acetyltransferase and acetylcholinesterase according to a sexually dimorphic pattern. Because of these widespread influences on these various neuronal systems, it is not surprising that ovarian steroids produce measurable cognitive effects after ovariectomy and during aging. NEUROLOGY 1997;48(Suppl 7): S8-S15

Microglia Derived from Aging Mice Exhibit an Altered Inflammatory Profile

Microglia play a critical role in neurodegenerative diseases and in the brain aging process. Yet, little is known about the functional dynamics of microglia during aging. Thus, using young and aging transgenic mice expressing enhanced‐green fluorescent protein (EGFP) under the promoter of the c‐fms gene for macrophage‐colony stimulating factor receptor, we evaluated invivo‐induced inflammatory responses of EGFP‐expressing microglia sorted by flow cytometry. Aging microglia were characterized by the presence of lipofuscin granules, decreased processes complexity, altered granularity, and increased mRNA expression of both pro‐inflammatory (TNFα, IL‐1β, IL‐6) and anti‐inflammatory (IL‐10, TGFβ1) cytokines. Following lipopolysaccharide (LPS) challenge (1 mg/kg, 3 h), aging microglia exhibit increased basal expression of TNFα, IL‐1β, IL‐6, and IL‐10. Yet, the fold‐over‐basal LPS response remained constant across age, implying that the inflammatory machinery in aging microglia is functional and adjusted to the basal state. Gender differences were not overall observed across the treatments (age, LPS). The low but sustained production of pro‐inflammatory cytokines by aging microglia may have a profound impact in the brain aging process.

Tracking the estrogen receptor in neurons: Implications for estrogen-induced synapse formation

Estrogens (E) and progestins regulate synaptogenesis in the CA1 region of the dorsal hippocampus during the estrous cycle of the female rat, and the functional consequences include changes in neurotransmission and memory. Synapse formation has been demonstrated by using the Golgi technique, dye filling of cells, electron microscopy, and radioimmunocytochemistry. N-methyl-d-aspartate (NMDA) receptor activation is required, and inhibitory interneurons play a pivotal role as they express nuclear estrogen receptor alpha (ERα) and show E-induced decreases of GABAergic activity. Although global decreases in inhibitory tone may be important, a more local role for E in CA1 neurons seems likely. The rat hippocampus expresses both ERα and ERβ mRNA. At the light microscopic level, autoradiography shows cell nuclear [3H]estrogen and [125I]estrogen uptake according to a distribution that primarily reflects the localization of ERα-immunoreactive interneurons in the hippocampus. However, recent ultrastructural studies have revealed extranuclear ERα immunoreactivity (IR) within select dendritic spines on hippocampal principal cells, axon terminals, and glial processes, localizations that would not be detectable by using standard light microscopic methods. Based on recent studies showing that both types of ER are expressed in a form that activates second messenger systems, these findings support a testable model in which local, non-genomic regulation by estrogen participates along with genomic actions of estrogens in the regulation of synapse formation.

Steroid hormone receptor expression and function in microglia

Steroid hormones such as glucocorticoids and estrogens are well‐known regulators of peripheral immune responses and also show anti‐inflammatory properties in the brain. However, the expression of steroid hormone receptors in microglia, the pivotal immune cell that coordinates the brain inflammatory response, is still controversial. Here we use real time RT‐PCR to show that microglia, isolated from adult fms‐EGFP mice by FACS, express glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and estrogen receptor alpha (ERα). GR was the most abundant steroid hormone receptor transcript in microglia. The presence of GR and ERα immunoreactivity was further confirmed in vivo at the ultrastructural level. To understand the role of steroid hormone receptors during the inflammation process, we evaluated the expression of steroid hormone receptors after inflammatory challenge and found a significant down‐regulation of GR, MR, and ERα in microglia. Finally, we tested the immunomodulatory properties of estrogens and glucocorticoids. Estradiol benzoate did not have any significant impact on the inflammatory profile of ex vivo sorted microglia, either in resting conditions or after challenge. Furthermore, corticosterone was a more consistent anti‐inflammatory agent than 17β‐estradiol in vitro. Our results support the hypothesis that adult microglia are a direct target of steroid hormones and that glucocorticoids, through the predominant expression of GR and MR, are the primary steroid hormone regulators of microglial inflammatory activity. The down‐regulation of steroid hormone receptors after LPS challenge may serve as a prerequisite to suppressing the anti‐inflammatory actions of endogenous steroid hormones on the immune system, and contribute to a sustained activation of microglia.

CD11c/EYFP transgene illuminates a discrete network of dendritic cells within the embryonic, neonatal, adult, and injured mouse brain.

The CD11c enhanced yellow fluorescent protein (EYFP) transgenic mouse was constructed to identify dendritic cells in the periphery (Lindquist et al. [ 2004 ] Nat. Immunol. 5:1243–1250). In this study, we used this mouse to characterize dendritic cells within the CNS. Our anatomic results showed discrete populations of EYFP+ brain dendritic cells (EYFP+ bDC) that colocalized with a small fraction of microglia immunoreactive for Mac‐1, Iba‐1, CD45, and F4/80 but not for NeuN, Dcx, NG2 proteoglycan, or GFAP. EYFP+ bDC, isolated by fluorescent activated cell sorting (FACS), expressed mRNA for the Itgax (CD11c) gene, whereas FACS anlaysis of EYFP+ bDC cultures revealed the presence of CD11c protein. Light microscopy studies revealed that EYFP+ bDC were present in the embryonic CNS when the blood–brain barrier is formed and postnatally when brain cells are amenable to culturing. In adult male mice, EYFP+ bDC distribution was prominent within regions of the CNS that 1) are subject to structural plasticity and neurogenesis, 2) receive sensory and humoral input from the external environment, and 3) lack a blood–brain barrier. Ultrastructural analysis of EYFP+ bDC in adult neurogenic niches showed their proximity to developing neurons and a morphology characteristic of immune/microglia cells. Kainic acid‐induced seizures revealed that EYFP+ bDC responded to damage of the hippocampus and displayed morphologies similar to those described for seizure‐activated EGFP+ microglia in the hippocampus of cfms (CSF‐1R) EGFP mice. Collectively, these findings suggest a new member of the dendritic cell family residing among the heterogeneous microglia population. J. Comp. Neurol. 508:687–710, 2008.

Brain dendritic cells in ischemic stroke: time course, activation state, and origin

The immune response to stroke is comprised of inflammatory and regulatory processes. One cell type involved in both innate and adaptive immunity is the dendritic cell (DC). A DC population residing in the healthy brain (bDC) was identified using a transgenic mouse expressing enhanced yellow fluorescent protein (EYFP) under the promoter for the DC marker, CD11c (CD11c/EYFP Tg). To determine if bDC are involved in the immune response to cerebral ischemia, transient (40 min) middle cerebral artery occlusion (MCAO) followed by 6, 24, or 72 h reperfusion was conducted in CD11c/EYFP Tg mice. Our results demonstrated that DC accumulated in the ischemic hemisphere at 24 h post-MCAO-reperfusion, particularly in the border region of the infarct where T lymphocytes accrued. To distinguish resident bDC from the infiltrating peripheral DC, radiation chimeras [1. wild type (WT) hosts restored with CD11c/EYFP Tg bone marrow (BM) or 2. CD11c/EYFP Tg hosts restored with WT BM] were generated and examined by immunocytochemistry. These data confirmed that DC populating the core of the infarct at 72 h were of peripheral origin, whereas those in the border region were comprised primarily of resident bDC. The brain resident (CD45 intermediate) cells of CD11c/EYFP Tg mice were analyzed by flow cytometry. Compared to microglia, bDC displayed increased major histocompatibility class II (MHC II) and co-stimulatory molecules following MCAO-reperfusion. High levels of MHC II and the co-stimulatory molecule CD80 on bDC at 72 h corresponded to peak lymphocyte infiltration, and suggested a functional interaction between these two immune cell populations.

Brain dendritic cells: biology and pathology

Dendritic cells (DC) are the professional antigen-presenting cells of the immune system. In their quiescent and mature form, the presentation of self‐antigens by DC leads to tolerance; whereas, antigen presentation by mature DC, after stimulation by pathogen‐associated molecular patterns, leads to the onset of antigen-specific immunity. DC have been found in many of the major organs in mammals (e.g. skin, heart, lungs, intestines and spleen); while the brain has long been considered devoid of DC in the absence of neuroinflammation. Consequently, microglia, the resident immune cell of the brain, have been charged with many functional attributes commonly ascribed to DC. Recent evidence has challenged the notion that DC are either absent or minimal players in brain immune surveillance. This review will discuss the recent literature examining DC involvement within both the young and aged steady-state brain. We will also examine DC contributions during various forms of neuroinflammation resulting from neurodegenerative autoimmune disease, injury, and CNS infections. This review also touches upon DC trafficking between the central nervous system and peripheral immune compartments during viral infections, the new molecular technologies that could be employed to enhance our current understanding of brain DC ontogeny, and some potential therapeutic uses of DC within the CNS.

Acute in vivo exposure to interferon-γ enables resident brain dendritic cells to become effective antigen presenting cells

Dendritic cells (DC) are the professional antigen presenting cells (APC) that bridge the innate and adaptive immune system. Previously, in a CD11c/EYFP transgenic mouse developed to study DC functions, we anatomically mapped and phenotypically characterized a discrete population of EYFP+ cells within the microglia that we termed brain dendritic cells (bDC). In this study, we advanced our knowledge of the function of these cells in the CD11c/EYFP transgenic mouse and its chimeras, using acute stimuli of stereotaxically inoculated IFNγ or IL-4 into the CNS. The administration of IFNγ increased the number of EYFP+bDC but did not recruit peripheral DC into the CNS. IFNγ, but not IL-4, upregulated the expression levels of major histocompatibility class II (MHC-II). In addition, IFNγ-activated EYFP+bDC induced antigen-specific naïve CD4 T cells to proliferate and secrete Th1/Th17 cytokines. Activated bDC were also able to stimulate naïve CD8 T cells. Collectively, these data reveal the Th1 cytokine IFNγ, but not the Th2 cytokine IL4, induces bDC to up-regulate MHC-II and become competent APC.

Volumetric measurement of the hippocampus, the anterior cingulate cortex, and the retrosplenial granular cortex of the rat using structural MRI

MRI imaging of the rodent brain is a rapidly growing field in the neurosciences. Relatively limited information is available for regional volume determination. The present paper describes a reliable method for the assessment of the hippocampus, the anterior cingulate cortex, the retrosplenial granular cortex and the ventricles in rats. MRI scans were acquired using a 7 T magnet. The anatomical sampling method was found to be highly reliable with an intra-rater reliability of greater than 0.93. The current protocol should facilitate future in vivo neuroimaging research using animal models of neurodegenerative diseases.