Terrence Deak

Prior Stressor Exposure Sensitizes LPS-Induced Cytokine Production

Exposure to stressors often alters the subsequent responsiveness of many systems. The present study tested whether prior exposure to inescapable tailshock (IS) alters the interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, or IL-6 response to an injection of bacterial endotoxin (lipopolysaccharide; LPS). Rats were exposed to IS or remained as home cage controls (HCC); 24 h later animals were injected i.p. with either 10 microg/kg LPS or equilvolume sterile saline. IS significantly increased plasma TNF-alpha, IL-1beta, and pituitary, hypothalamus, hippocampus, cerebellum IL-1beta 1 h, but not 2 h, after LPS, compared to controls. Additional animals were injected with LPS or saline 4, 10, or 21 days after exposure to IS and tail vein blood was collected and assayed for IL-1beta. An enhanced plasma IL-1beta response occurred 4 days after IS, but was gone by 10 days. These results suggest that exposure to IS sensitizes the innate immune response to LPS by resulting in either a larger or a more rapid induction of proinflammatory cytokines.

Timecourse and corticosterone sensitivity of the brain, pituitary, and serum interleukin-1β protein response to acute stress

Activation of peripheral immune cells leads to increases of interleukin-1beta (IL-1beta) mRNA, immunoreactivity, and protein levels in brain and pituitary. Furthermore, IL-1beta in brain plays a role in mediating many of the behavioral, physiological, and endocrine adjustments induced by immune activation. A similarity between the consequences of immune activation and exposure to stressors has often been noted, but the potential relationship between stress and brain IL-1beta has received very little attention. A prior report indicated that exposure to inescapable tailshocks (IS) raised levels of brain IL-1beta protein 2 h after IS, but only in adrenalectomized (and basal corticosterone replaced) subjects. The studies reported here explore this issue in more detail. A more careful examination revealed that IL-1beta protein levels in hypothalamus were elevated by IS in intact subjects, although adrenalectomy, ADX (with basal corticosterone replacement) exaggerated this effect. IL-1beta protein increases were already present immediately after the stress session, both in the hypothalamus and in other brain regions in adrenalectomized subjects, and no longer present 24 h later. Furthermore, IS elevated levels of IL-1beta protein in the pituitary, and did so in both intact and adrenalectomized subjects. IS also produced increased blood levels of IL-1beta, but only in adrenalectomized subjects. Finally, the administration of corticosterone in an amount that led to blood levels in adrenalectomized subjects that match those produced by IS, inhibited the IS-induced rise in IL-1beta in hypothalamus and pituitary, but not in other brain regions or blood.

Stress-induced increases in hypothalamic IL-1: a systematic analysis of multiple stressor paradigms.

Exposure to stressors such as footshock, tailshock, and immobilization have been shown to induce hypothalamic IL-1 production, while other stressors such as restraint, maternal separation, social isolation, and predator exposure have no effect on hypothalamic IL-1 levels. This disparity of findings has led to considerable controversy regarding the ability of stressors to induce hypothalamic IL-1 expression. Thus, the goal of the following experiments was to examine hypothalamic IL-1 responses in adult male Sprague-Dawley rats following exposure to a diverse set of stressors. Our data indicate that exposure to 2h of restraint in a Plexiglas tube, glucoprivic challenge induced by administration of 2-deoxyglucose (2-DG), or insulin-induced hypoglycemia all fail to alter hypothalamic IL-1 levels despite robust activation of the pituitary-adrenal response. However, when restraint was administered on an orbital shaker or in combination with insulin-induced hypoglycemia, robust increases in hypothalamic IL-1 were observed. No effects of glucoprivic (2-DG) challenge were observed when combined with restraint, indicating some specificity in the hypothalamic IL-1 response to stress. We also provide a preliminary validation of the ELISA detection method for IL-1, showing that (a) Western blot analyses confirmed strong immunopositive banding at the apparent molecular weight of both mature IL-1beta and the IL-1beta prohormone, and (b) footshock led to a two-fold increase in mRNA for IL-1 in the hypothalamus as detected by RT-PCR. These data provide novel insight into the characteristics of a stressor that may be necessary for the observation of stress-induced increases in hypothalamic IL-1.

Evidence that brief stress may induce the acute phase response in rats

Exposing rats to a single session of inescapable tail shock (IS) reduces corticosteroid binding globulin (CBG) 24 h later (Fleshner et al., Endocrinology 136: 5336-5342, 1995). The present experiments examined whether reductions in CBG are differentially affected by controllable vs. identical uncontrollable tail shock, are mediated by IS-induced glucocorticoid elevation, or reflect IS-induced activation of the acute phase response and whether IS produces fever. The results demonstrate that 1) equivalent reductions in CBG are observed in response to escapable tail shock or yoked IS, 2) IS-induced CBG reduction is not blocked by adrenalectomy in rats that receive basal corticosteroid replacement or by pretreatment with RU-38486, and 3) IS appears to activate the acute phase response, since IS reduces serum levels of an acute-phase negative reactant (CBG), increases serum levels of acute-phase positive reactants (haptoglobin and alpha 1-acid glycoprotein), and increases core body temperature 20-24 h later.Exposing rats to a single session of inescapable tail shock (IS) reduces corticosteroid binding globulin (CBG) 24 h later (Fleshner et al., Endocrinology 136: 5336-5342, 1995). The present experiments examined whether reductions in CBG are differentially affected by controllable vs. identical uncontrollable tail shock, are mediated by IS-induced glucocorticoid elevation, or reflect IS-induced activation of the acute phase response and whether IS produces fever. The results demonstrate that 1) equivalent reductions in CBG are observed in response to escapable tail shock or yoked IS, 2) IS-induced CBG reduction is not blocked by adrenalectomy in rats that receive basal corticosteroid replacement or by pretreatment with RU-38486, and 3) IS appears to activate the acute phase response, since IS reduces serum levels of an acute-phase negative reactant (CBG), increases serum levels of acute-phase positive reactants (haptoglobin and α1-acid glycoprotein), and increases core body temperature 20-24 h later.

Prior stressor exposure primes the HPA axis

Exposure to stressors often alters the subsequent responsiveness of many systems. The present study tested whether prior exposure to inescapable tailshock (IS) alters the corticosterone (CORT) or adrenocorticotropin hormone (ACTH) response to either an injection of bacterial endotoxin (lipopolysaccharide; LPS) or subsequent placement on a pedestal. Rats were exposed to IS or remained as home cage controls (HCC). 1, 4, 10, or 21 days later animals were injected i.p. with either 10 microg/kg LPS or equivolume sterile saline. Prior IS significantly increased plasma CORT 1 h, but not 2 or 5 h after LPS, compared to controls 1, 4, and 10 days, but not 21 days after IS. Exposure to IS 24 h earlier also significantly increased plasma ACTH 1 h after LPS. Additional animals were placed on a pedestal 24 h after IS, and plasma CORT was measured 15, 30, and 60 min later. IS significantly increased plasma CORT 15 min after pedestal exposure, but not after 30 or 60 min. These results suggest that exposure to IS sensitizes the CORT and ACTH response to subsequent HPA activation.

Gene expression changes in the hypothalamus provide evidence for regionally-selective changes in IL-1 and microglial markers after acute stress

Recent work from our laboratory and others has shown that certain stressors increase expression of the pro-inflammatory cytokine interleukin-1beta (IL-1) in the hypothalamus. The first goal of the following studies was to assess the impact of acute stress on other key inflammatory factors, including both cytokines and cell surface markers for immune-derived cells resident to the CNS in adult male Sprague Dawley rats exposed to intermittent footshock (80 shocks, 90 s variable ITI, 5 s each). While scattered changes in IL-6 and GFAP were observed in the hippocampus and cortex, we found the hypothalamus to be exquisitely sensitive to the effects of footshock. At the level of the hypothalamus, mRNA for IL-1 and CD14 were significantly increased, while at the same time CD200R mRNA was significantly decreased. A subsequent experiment demonstrated that propranolol (20mg/kg i.p.) blocked the increase in IL-1 and CD14 mRNA observed in the hypothalamus, while the decrease in CD200R was unaffected by propranolol. Interestingly, inhibition of glucocorticoid synthesis via injection of metyrapone (50mg/kg s.c.) plus aminoglutethimide (100mg/kg s.c.) increased basal IL-1 mRNA and augmented IL-1 and CD14 expression provoked by footshock. Injection of minocycline, a putative microglial inhibitor, blocked the IL-1 response to footshock, while CD14 and CD200R were unaffected. Together, these gene expression changes (i) provide compelling evidence that stress may provoke neuroinflammatory changes that extend well beyond isolated changes in a single cytokine; (ii) suggest opposing roles for classic stress-responsive factors (norepinephrine and corticosterone) in the modulation of stress-related neuroinflammation; (iii) indicate microglia within the hypothalamus may be key players in stress-related neuroinflammation; and (iv) provide a potential mechanism (increased CD14) by which acute stress primes reactivity to later immune challenge.

Discrimination between changes in glucocorticoid receptor expression and activation in rat brain using western blot analysis.

These studies investigated autoregulation of glucocorticoid receptor (GR) protein expression and activation in rat brain using western blot methodology. By comparing GR immunoblot reactivity present in various tissue subcellular preparations we were able to discriminate between corticosterone-induced changes in GR activation or GR protein expression. Our cytosolic tissue preparation yielded a similar pattern of treatment effects on relative GR as measured by receptor binding assay or western blot. In both cases, short-term adrenalectomy (18 h) produced no change in cytosolic GR. On the other hand, long-term adrenalectomy (3-14 days) resulted in a large increase in cytosolic GR, whereas acute (1-2 h) treatment with high dose corticosterone produced a large decrease in cytosolic GR. Western blot measurement of GR levels in a nuclear extract or whole-cell extract from the same brains indicated that acute corticosterone treatment produced a large increase in nuclear GR and no change in whole-cell GR. Thus, all of the decrease in cytosolic GR observed after acute corticosterone treatment could be accounted for by receptor redistribution to the nuclear tissue fraction as opposed to rapid receptor protein downregulation. Long-term treatment of rats with adrenalectomy or high dose corticosterone produced a large increase and decrease, respectively, in whole-cell GR, indicating genuine changes in receptor protein expression. These studies indicate that in vivo regulation of GR protein expression in the rat brain can be studied using western blot analysis of a whole-cell tissue preparation. This procedure has an important advantage over receptor binding studies in that GR protein expression can be measured in adrenal-intact rats. These studies also support the validity of using cytosolic receptor binding assays to estimate relative changes in GR occupation/activation when appropriate comparison groups are included.

Exposure to forced swim stress does not alter central production of IL-1

In recent years, there has been increasing recognition that pro-inflammatory cytokines play a role in behavioral and physiological alterations produced by exposure to psychological stressors. Indeed, increases in central IL-1 production have been observed following stressors such as inescapable tailshock and social isolation, while no changes in IL-1 have been observed following other stressors (e.g., exposure to a predator). The goal of the following work was to establish whether exposure to the forced swim test (FST), a commonly used animal model of behavioral despair/depression, leads to an increase in central or peripheral production of IL-1. Briefly, adult male Sprague-Dawley rats (n=8 per group) were forced to swim for 15-30 min (25 degrees C) and killed at various intervals (ranging from immediately to 24 h) following stressor termination. Brains (hippocampus, hypothalamus, posterior cortex) and multiple peripheral tissues (pituitary, adrenals, spleen, plasma) were then dissected and frozen for subsequent measurement of IL-1 using a commercially available enzyme-linked immunosorbent assay. No observable increases in IL-1 were found in rats that were forced to swim acutely, or in rats that were re-exposed to the forced swim stressor 24 h later. These data suggest that exposure to forced swim does not lead to an increase in central production of IL-1, suggesting that the central IL-1 system is unlikely to play a role in mediating behavioral consequences of this stressor. However, these data do not exclude the possibility that other pro-inflammatory cytokines (such as IL-6 and TNF-alpha) might be produced in response to forced swim exposure.

Novel aspects of glucocorticoid actions.

Normal hypothalamic‐pituitary‐adrenal (HPA) axis activity leading to the rhythmic and episodic release of adrenal glucocorticoids (GCs) is essential for body homeostasis and survival during stress. Acting through specific intracellular receptors in the brain and periphery, GCs regulate behaviour, as well as metabolic, cardiovascular, immune and neuroendocrine activities. By contrast to chronic elevated levels, circadian and acute stress‐induced increases in GCs are necessary for hippocampal neuronal survival and memory acquisition and consolidation, as a result of the inhibition of apoptosis, the facilitation of glutamatergic neurotransmission and the formation of excitatory synapses, and the induction of immediate early genes and dendritic spine formation. In addition to metabolic actions leading to increased energy availability, GCs have profound effects on feeding behaviour, mainly via the modulation of orexigenic and anorixegenic neuropeptides. Evidence is also emerging that, in addition to the recognised immune suppressive actions of GCs by counteracting adrenergic pro‐inflammatory actions, circadian elevations have priming effects in the immune system, potentiating acute defensive responses. In addition, negative‐feedback by GCs involves multiple mechanisms leading to limited HPA axis activation and prevention of the deleterious effects of excessive GC production. Adequate GC secretion to meet body demands is tightly regulated by a complex neural circuitry controlling hypothalamic corticotrophin‐releasing hormone (CRH) and vasopressin secretion, which are the main regulators of pituitary adrenocorticotrophic hormone (ACTH). Rapid feedback mechanisms, likely involving nongenomic actions of GCs, mediate the immediate inhibition of hypothalamic CRH and ACTH secretion, whereas intermediate and delayed mechanisms mediated by genomic actions involve the modulation of limbic circuitry and peripheral metabolic messengers. Consistent with their key adaptive roles, HPA axis components are evolutionarily conserved, being present in the earliest vertebrates. An understanding of these basic mechanisms may lead to novel approaches for the development of diagnostic and therapeutic tools for disorders related to stress and alterations of GC secretion.

Long-term changes in mineralocorticoid and glucocorticoid receptor occupancy following exposure to an acute stressor

Stressors produce rapid activation of the hypothalamic-pituitary-adrenal axis, which typically resolves within 60-90 min following termination of the stressor. In addition, some stressors such as inescapable tailshock (IS) also produce elevated basal levels of corticosterone (CORT), and reduced serum levels of corticosteroid binding globulin (CBG). The elevated basal levels of CORT produced by IS are only observed at the trough of the circadian rhythm of CORT secretion, and are sustained for 2-3 days following stressor termination. The goal of the following experiments was to determine the extent to which the elevated basal levels of CORT observed following IS exposure produced greater corticosteroid receptor occupancy in the brain and pituitary. To do so, rats (n=8-10 per group) received either sham or bilateral adrenalectomy (with CORT replacement in their drinking water; 25 microg/ml) and were given 3 days to recover. Rats were then exposed to 100 ISs (1.6 mA, 5 s each) administered on a 60 s variable intertrial interval, or remained in their home cages. As seen previously, IS produced an increase in basal CORT (5 microg/dl) and a decrease in CBG (30% decrease). Rats were sacrificed 24 h following IS for trunk blood samples and brain dissections. IS exposure had very little effect on corticosteroid receptor protein expression as determined by mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) binding levels in ADX rats. In addition, no changes in whole cell GR levels (as detected by Western blot) were observed in sham rats exposed to IS. On the other hand, IS exposure led to greater occupancy of MR (ranging from 25%-50%) in hippocampus, hypothalamus, pituitary, and posterior cortex. IS also produced greater occupancy of GR (approximately 20%) in hypothalamus and posterior cortex. These long-term changes in corticosteroid receptor activation, evident 24 h after IS exposure, may be responsible for some of the long-term neural, behavioral and immune changes observed following this acute stress procedure.