Sex-, Stress-, and Sympathetic Post-Ganglionic Neuron-Dependent Changes in the Expression of Pro- and Anti-Inflammatory Mediators in Rat Dural Immune Cells.

BACKGROUND: Migraine attacks are associated with sterile inflammation of the dura. Immune cells are a primary source of inflammatory mediators, and we therefore sought to further explore the link between dural immune cells and migraine. OBJECTIVE: Based on the observations that migraine is more common in women than in men, stress is the most common trigger for a migraine attack, and sympathetic post-ganglionic innervation of the dura enables local control of dural immune cells, we hypothesized that stress shifts the balance of inflammatory mediator expression in dural immune cells toward those that trigger a migraine attack, where these changes are larger in females and dependent, at least in part, on sympathetic post-ganglionic innervation of the dura. Our objective was to test this hypothesis. METHODS: Dura were obtained from naïve or stressed, intact or surgically sympathectomized, adult male and female rats. Dura were assessed immediately or 24 hours after termination of 4 continuous days of unpredictable, mild stressors. Following enzymatic digestion of each dura, myeloid and lymphoid-derived dural immune cells were isolated by fluorescence-activated cell sorting for semi-quantitative polymerase chain reaction analysis. RESULTS: In myeloid-derived dural immune cells, there was an increase in pro-inflammatory mediator mRNA following stress, particularly in females, which remained elevated with a 24-hour delay after stress. There was a stress-induced decrease in anti-inflammatory mediator mRNA immediately after stress in females, but not males. The stress-induced changes were attenuated in sympathectomized females. In lymphoid-derived dural immune cells, there was a persistent increase in pro-inflammatory mediator mRNA following stress, particularly in females. A stress-induced increase in anti-inflammatory mediator mRNA was also observed in both males and females, and was further attenuated in sympathectomized females. CONCLUSIONS: Consistent with our hypothesis, there is a stress-induced shift in the balance of pro- and anti-inflammatory mediator expression in dural immune cells that is more pronounced in females, and is dependent, at least in part, on sympathetic post-ganglionic innervation in females. This shift in the balance of inflammatory mediator expression may not only play an important role in triggering migraine attacks, but also suggests it may be possible, if not necessary, to employ different strategies to most effectively treat migraine in men and women.

Influence of pain treatment by epidural fentanyl and bupivacaine on homing of opioid-containing leukocytes to surgical wounds.

Endogenous opioids released from leukocytes extravasating into injured tissue can interact with peripheral opioid receptors to inhibit nociception. Animal studies have shown that the homing of opioid-producing leukocytes to the injured site is modulated by spinal blockade of noxious input. This study investigated whether epidural analgesia (EDA) influences the migration of beta-endorphin (END) and/or met-enkephalin (ENK)-containing leukocytes into the subcutaneous wound tissue of patients undergoing abdominal surgery. In part I patients received general anesthesia combined either with intra- and postoperative EDA (with bupivacaine and fentanyl) or with postoperative patient controlled intravenous analgesia (PCIA; with the opioid piritramide). In part II patients received general anesthesia combined with either epidural fentanyl or bupivacaine which was continued postoperatively. Samples of cutanous and subcutanous tissue were taken from the wound site at the beginning, at the end and at various times after surgery, and were examined by immunohistochemistry for the presence of END and ENK. We found that (i) epidural bupivacaine, fentanyl and PCIA provided similar and clinically acceptable postoperative pain relief; (ii) compared to PCIA, epidural bupivacaine or fentanyl did not change the gross inflammatory reaction within the surgical wound; (iii) opioid-containing leukocytes were almost absent in normal subcutaneous tissue but migrated to the inflamed wound tissue in ascending numbers within a few hours, reaching a peak at about 24 h after surgery; (iv) compared to PCIA, EDA resulted in significantly decreased homing of END-containing leukocytes to the injured site at 24 h after surgery; and (v) the magnitude of this decrease was similar regardless of the epidural medication. These findings suggest that nociceptive but not sympathetic neurons are primarily involved in the attraction of opioid-containing leukocytes during early stages of inflammation.

A fundamental bimodal role for neuropeptide Y1 receptor in the immune system.

Psychological conditions, including stress, compromise immune defenses. Although this concept is not novel, the molecular mechanism behind it remains unclear. Neuropeptide Y (NPY) in the central nervous system is a major regulator of numerous physiological functions, including stress. Postganglionic sympathetic nerves innervating lymphoid organs release NPY, which together with other peptides activate five Y receptors (Y1, Y2, Y4, Y5, and y(6)). Using Y1-deficient (Y1(-/-)) mice, we showed that Y1(-/-) T cells are hyperresponsive to activation and trigger severe colitis after transfer into lymphopenic mice. Thus, signaling through Y1 receptor on T cells inhibits T cell activation and controls the magnitude of T cell responses. Paradoxically, Y1(-/-) mice were resistant to T helper type 1 (Th1) cell-mediated inflammatory responses and showed reduced levels of the Th1 cell-promoting cytokine interleukin 12 and reduced interferon gamma production. This defect was due to functionally impaired antigen-presenting cells (APCs), and consequently, Y1(-/-) mice had reduced numbers of effector T cells. These results demonstrate a fundamental bimodal role for the Y1 receptor in the immune system, serving as a strong negative regulator on T cells as well as a key activator of APC function. Our findings uncover a sophisticated molecular mechanism regulating immune cell functions that can lead to stress-induced immunosuppression.

Short exposure of maturing, bone marrow-derived dendritic cells to norepinephrine: impact on kinetics of cytokine production and Th development.

The information gathered by dendritic cells (DC) during the innate immune response to a pathogen is determinant for the type of adaptive response. Here we show that short-term (3 h) exposure of bone marrow-derived DC to norepinephrine (NE), at the beginning of lipopolysaccharide (LPS) or keyhole limpet hemocyanin (KLH) stimulation hampers IL-12 production and increases IL-10 release. The NE effect was mediated by both beta- and alpha2-adrenergic receptors. The capacity of NE-exposed DC to produce IL-12 upon CD40 cross-linking as well as to stimulate allogeneic T-helper (Th) lymphocytes was reduced. Adoptive transfer of NE-exposed DC induced a Th2 slanted response in vivo. Thus, a brief NE exposure of antigen-stimulated DC seems to limit their Th1 polarizing properties. Noteworthy, the ganglionic blocker pentolinium administered in mice before skin sensitization with fluoroscein isothiocyanate (FITC) could increase the Th1-type response in the draining lymph nodes. Our results suggest that the extent of Th differentiation in the response to an antigen might be influenced by the local sympathetic nervous activity in the early phase of dendritic cell stimulation.

Noradrenergic inhibition of TNF upregulation in hemorrhagic shock.

UNLABELLED: The early stress responses to hemorrhagic shock, trauma and endotoxicosis are associated with an early proinflammatory response characterized by increased gene expression of proinflammatory cytokines, PMN influx and accumulation in the lung and apoptosis. The central role of the neuroendocrine system in modulating these proinflammatory responses has been strongly suggested by recent studies. OBJECTIVES: To examine the role of noradrenergic innervation in modulating the early increase in lung and spleen content of TNF-alpha in response to fixed-pressure (40 mm Hg) hemorrhage in vivo. METHODS: Conscious unrestrained nonheparinized male Sprague-Dawley rats (n = 42) were randomized to receive intraperitoneally either 6-hydroxy-dopamine (6-OHDA; chemical sympathectomy, SNSx) or saline (0.3 ml) prior to undergoing hemorrhage followed by fluid resuscitation with lactated Ringer's solution. Animals were sacrificed at completion of the resuscitation period and tissue samples (lung and spleen) excised for determination of TNF-alpha content, myeloperoxidase activity and apoptosis. RESULTS: Hemorrhage resulted in an immediate marked elevation in circulating epinephrine and norepinephrine levels (10- and 2-fold, respectively), increasing their plasma ratio to 6:1. SNSx depleted tissue stores of norepinephrine (80%), did not alter basal plasma levels of epi- or norepinephrine or the hemorrhage-induced rise in epinephrine, but completely prevented the rise in circulating norepinephrine. Hemorrhage increased lung and spleen contents of TNF-alpha (55 and 72%, respectively). SNSx significantly enhanced the hemorrhage-induced rise in lung TNF in response to hemorrhage. CONCLUSIONS: These results show a suppressive role for noradrenergic innervation on the hemorrhage-induced increase in tissue TNF-alpha content in vivo. We speculate that the effects of norepinephrine are protective from tissue injury but are likely to contribute to the generalized immunosuppression following trauma.

Cyclosporin A: effects on the secretory process and noradrenergic activity in the submandibular gland of the rat.

OBJECTIVES: The aim of the present work was to study the effect of long-term cyclosporine (CSA) administration on norepinephrine (NE) metabolism and adrenergic-evoked secretion in the rat submandibular gland (SMG). METHODS: Dose-response curves to adrenergic agonists (methoxamine, isoproterenol, NE) were performed in control and CSA (10 and 30 mg/kg every 2 days for 1 month)-treated rats after SMG duct cannulation. In SMG tissue neuronal NE uptake, release, synthesis and endogenous content were determined. In addition phosphoinositide intracellular signaling was also investigated. RESULTS: CSA administration caused an increase in salivary secretion evoked by methoxamine (alpha-adrenergic agonist) and NE but failed to modify salivation evoked by beta-adrenergic stimulation (isoproterenol). Long-term CSA administration decreased NE release and synthesis whereas it enhanced the amine uptake and phosphoinositide hydrolysis in the SMG. CONCLUSIONS: The administration of CSA for 30 days induced salivary gland sensitization likely mediated by diminished adrenergic input. Present results suggest that the decreased sympathetic activity evoked by long-term CSA administration in the rat SMG may lead to sensitization of the gland supported by increased phosphoinositide hydrolysis and enhanced adrenergic-evoked salivation.

Prolonged alpha-adrenergic stimulation causes changes in leukocyte distribution and lymphocyte apoptosis in the rat.

We have previously shown in the rat model that acutely or chronically increased peripheral catecholamines lead to suppression of lymphocyte responsiveness via alpha(2)-adrenoceptor activation. Here we investigated the effects of alpha-adrenergic treatment on total leukocyte numbers and proportions of leukocyte subsets in peripheral blood and lymphoid tissues. It was found that a 12-h treatment with subcutaneously implanted tablets, one containing norepinephrine (NE) and one propranolol, leads to an increase in total blood leukocyte counts, due to a pronounced increase in granulocytes. In contrast, the numbers of all classes of lymphocytes other than NK cells were decreased. This decrease in blood lymphocytes is apparently not due to redistribution, since in the thymus, spleen, mesenteric and peripheral lymph nodes, the total numbers of lymphocytes were decreased as well, without any changes in subpopulations. Analogous results were obtained with rats adrenalectomized before the catecholamine treatment. Animals that received the alpha-adrenergic treatment displayed significantly more apoptotic cells in the lymphoid organs, as determined by the TUNEL technique. In the spleen, the enhanced rate of apoptosis was confined to the white pulp; red pulp areas exhibited significantly fewer apoptotic cells. Thus, an increased alpha-adrenergic tone in rats led to a general loss of lymphocytes due to lymphocyte directed apoptosis that was independent of glucocorticoids.

Calcitonin gene-related peptide-like immunoreactivity in spinal cord and superior cervical ganglion of the Djungarian hamster (Phodopus sungorus).

The indirect immunofluorescent method was employed to investigate the distribution of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) in the spinal cord and superior cervical ganglion of the Djungarian hamster Phodopus sungorus. In cross-sections of the spinal cord, immunoreactive fibres and terminals were found in laminae 1 and 2 in high density, in the dorsolateral (Lissauer's) tract, in ventral and lateral horns, and in the area surrounding the central canal. A few CGRP-LI perikarya were seen in the ventral but not the dorsal horn. CGRP-LI was further observed in preganglionic sympathetic neurons which were labelled by retrograde axonal transport of fluoro-gold (FG) following injection of the substance unilaterally into the superior cervical ganglion. Preganglionic sympathetic neurons (PSN) were localized ipsilateral to the injection site mainly in the intermediolateral nucleus and the lateral funiculus of the upper thoracic segments. Most PSN exhibited CGRP-LI. Immunoreactive PSN were not seen contralaterally to the site of FG application nor in animals that did not receive injections. When the preganglionic fibres were ligated 4 days before perfusion, CGRP-LI cell bodies were found in preganglionic sympathetic neurons similar to the situation seen upon FG treatment. In the superior cervical ganglia of untreated hamsters, immunoreactive fibres were seen to enter the ganglion in which they terminated at non-immunoreactive principal ganglion cells. The present study, the first in a hamster species, describes the widespread distribution of CGRP in the spinal cord of P. sungorus and supports the view that considerable interspecies differences exist in occurrence and location of this neuropeptide.