Biomolecular characterization of 3500-year-old ancient Egyptian mummification balms from the Valley of the Kings.

Ancient Egyptian mummification was practiced for nearly 4000 years as a key feature of some of the most complex mortuary practices documented in the archaeological record. Embalming, the preservation of the body and organs of the deceased for the afterlife, was a central component of the Egyptian mummification process. Here, we combine GC-MS, HT-GC-MS, and LC-MS/MS analyses to examine mummification balms excavated more than a century ago by Howard Carter from Tomb KV42 in the Valley of the Kings. Balm residues were scraped from now empty canopic jars that once contained the mummified organs of the noble lady Senetnay, dating to the 18th dynasty, ca. 1450 BCE. Our analysis revealed balms consisting of beeswax, plant oil, fats, bitumen, Pinaceae resins, a balsamic substance, and dammar or Pistacia tree resin. These are the richest, most complex balms yet identified for this early time period and they shed light on balm ingredients for which there is limited information in Egyptian textual sources. They highlight both the exceptional status of Senetnay and the myriad trade connections of the Egyptians in the 2nd millennium BCE. They further illustrate the excellent preservation possible even for organic remains long removed from their original archaeological context.

The role of alveolar macrophages in the pathogenesis of aspiration pneumonitis.

RATIONALE: A robust TNFalpha response is seen following aspiration of food particles, while there is only a modest response to acid. OBJECTIVES: To examine the direct effects of acid and particulate components of gastric content on local and systemic macrophages. METHODS: Pathogen-free Long-Evans rats were injured with intratracheal instillation of normal saline (SHAM), low pH saline (ACID), small non-acidic particles (SNAP) or acidified particles (CASP). The alveolar (local) and the peritoneal (systemic) macrophages were harvested following the injury. MEASUREMENTS: We examined the phagocytic activity and TNFalpha release by the alveolar and peritoneal macrophages following in vivo and in vitro exposure to acid and/or food particles. TNFalpha release by macrophages was examined in response to E. coli lipopolysaccharide (LPS) stimulation. MAIN RESULTS: In rats injured with gastric particles, the number of the mononuclear cells was higher than those obtained from acid-injured animals. Both in vivo and in vitro exposure of the alveolar macrophages to SNAP resulted in increased production of TNFalpha within 8 hours. Transient exposure of the alveolar macrophages to a low pH environment suppressed LPS-induced production of this cytokine. Additionally, the phagocytic activity of the alveolar macrophages was inhibited by in vitro exposure of the macrophages to acid. CONCLUSIONS: We conclude that the two components of gastric aspiration have diverse effects on local and systemic macrophages. Although there is a synergy between acid and gastric particulate in producing an acute lung injury, the modulatory effects of these injuries on the alveolar macrophages are averse.

An antidepressant mechanism of desipramine is to decrease tumor necrosis factor-alpha production culminating in increases in noradrenergic neurotransmission.

The monoamine theory of depression proposes decreased bioavailability of monoamines, such as norepinephrine (NE), as the underlying cause of depression. Thus, the antidepressant efficacy of NE-reuptake inhibitors such as desipramine is attributed to increases in synaptic concentrations of NE. The time difference between inhibition of reuptake and therapeutic efficacy, however, argues against this being the primary mechanism. If desipramine elicits its therapeutic efficacy by increasing NE release, in turn, increasing activation of the alpha(2)-adrenergic autoinhibitory receptor, then mimicking this increase with an exogenous agonist (clonidine) should support or even enhance the efficacy of the antidepressant. Intriguingly, simultaneous administration of clonidine with desipramine prevented the cellular and behavioral effects elicited by desipramine alone, in both acute and chronic administration paradigms. These results suggest the involvement of additional factor(s) in the mechanism of antidepressant action of this drug. Desipramine administration results in a virtual ablation of neuron-derived tumor necrosis factor-alpha (TNF), thus implicating an essential role of TNF in the therapeutic efficacy of this antidepressant. Additionally, following chronic administration of desipramine, TNF-regulation of NE release is transformed, from inhibition to facilitation. Here, we demonstrate that a transformation in TNF-regulation of NE release in the brain is a key element in the efficacy of this antidepressant. Interestingly, an increase in neurotransmission prior to the antidepressant's effect on TNF production prevents the efficacy of the antidepressant drug. Thus, the efficacy of desipramine is due to decreased levels of TNF in the brain induced by this drug, ultimately modifying noradrenergic neurotransmission.

Tumor necrosis factor expressed by primary hippocampal neurons and SH-SY5Y cells is regulated by alpha(2)-adrenergic receptor activation.

Neuron expression of the cytokine tumor necrosis factor-alpha (TNF), and the regulation of the levels of TNF by alpha(2)-adrenergic receptor activation were investigated. Adult rat hippocampal neurons and phorbol ester (PMA)-differentiated SH-SY5Y cells were examined. Intracellular levels of TNF mRNA accumulation, as well as TNF protein and that released into the supernatant were quantified by in situ hybridization, immunocytochemistry and bioanalysis, respectively. Both neuron cultures demonstrated constitutive production of TNF. Activation of the alpha(2)-adrenergic receptor increased intracellular levels of TNF mRNA and protein in SH-SY5Y cells after addition of graded concentrations of the selective agonist, Brimonidine (UK-14304) to parallel cultures. Intracellular levels of mRNA were increased in a concentration-dependent fashion within 15 min of UK-14304 addition and were sustained during 24 hr of receptor activation. In addition, the levels of TNF in the supernatant were increased in both types of neuron cultures within 15 min of alpha(2)-adrenergic receptor activation. Furthermore, levels of TNF significantly increased in the supernatants of both neuron cultures after potassium-induced depolarization. A reduction in this depolarization-induced release occurred in hippocampal neuron cultures after exposure to the sympathomimetic tyramine with media replacement to deplete endogenous catecholamines. This finding reveals a role for endogenous catecholamines in the regulation of TNF production. Potassium-induced depolarization resulted in the release of TNF in hippocampal neuron cultures within 15 min but not until 24 hr in SH-SY5Y cultures demonstrating a temporally mediated event dependent upon cell type. Neuron expression of TNF, regulated by alpha(2)-adrenergic receptor activation demonstrates not only how a neuron controls its own production of this pleiotropic cytokine, but also displays a normal role for neurons in directing the many functions of TNF.

Clonidine suppresses plasma and cerebrospinal fluid concentrations of TNF-alpha during the perioperative period.

UNLABELLED: The analgesic properties of alpha(2)-agonists are well known. In experimental models, tumor necrosis factor (TNF)-alpha regulates adrenergic responses in the brain. Constitutive TNF-alpha, in brain regions involved in pain perception, is decreased after the administration of clonidine. We investigated patients undergoing lower-extremity revascularization. Seven patients were treated with clonidine 0.2 mg per os (low), and three patients received 0.4 mg per os clonidine (high) before surgery. Eight patients received placebo and served as controls. Continuous spinal anesthesia was provided by insertion of a pliable catheter into the subarachnoid space. Baseline plasma and cerebrospinal fluid (CSF) samples were obtained before injection of local anesthetic. Samples were analyzed for TNF-alpha using a biologic assay. Systemic and central release of catecholamines were assessed by high-pressure liquid chromatography measurement of norepinephrine in plasma and CSF, vanillylmandelic acid and methoxy hydroxyl phenyl glycol in 24-h urinary excretion, respectively. Clonidine 0.2 mg pretreatment decreased TNF-alpha concentrations both in plasma and CSF. Patients receiving clonidine had lower pain visual analog scale scores and required less morphine compared with the Placebo group (P < 0.01). Preoperative administration of clonidine decreased catecholamine release in the periphery, as well as in the central nervous system. A smaller norepinephrine concentration in plasma and CSF, and less secretion of vanillylmandelic acid (P < 0.01) and methoxy hydroxyl phenyl glycol in the urine, were observed. Larger dose clonidine (0.4 mg) resulted in no detectable TNF-alpha in CSF. These results suggest that an interaction between TNF-alpha and the function of adrenergic neurons in the central nervous system may contribute to the sedative and analgesic effects of adrenergic agonists. IMPLICATIONS: Preoperative administration of clonidine decreases both plasma and cerebrospinal fluid concentrations of inflammatory cytokines, resulting in perioperative analgesia and decreased sympathetic tone.

Antidepressant drug-induced alterations in neuron-localized tumor necrosis factor-alpha mRNA and alpha(2)-adrenergic receptor sensitivity.

The pleiotropic cytokine tumor necrosis factor-alpha (TNF) and alpha(2)-adrenergic receptor activation regulate norepinephrine (NE) release from neurons in the central nervous system. The present study substantiates the role of TNF as a neuromodulator and demonstrates a reciprocally permissive relationship between the biological effects of TNF and alpha(2)-adrenergic receptor activation as a mechanism of action of antidepressant drugs. Immunohistochemical analysis and in situ hybridization reveal that administration of the antidepressant drug desipramine decreases the accumulation of constitutively expressed TNF mRNA in neurons of the rat brain. Superfusion and electrical field stimulation were applied to a series of rat hippocampal brain slices to study the regulation of [(3)H]NE release. Superfusion of hippocampal slices obtained from rats chronically administered the antidepressant drug zimelidine demonstrates that TNF-mediated inhibition of [(3)H]NE release is transformed, such that [(3)H]NE release is potentiated in the presence of TNF, an effect that occurs in association with alpha(2)-adrenergic receptor activation. However, chronic zimelidine administration does not alter stimulation-evoked [(3)H]NE release, whereas chronic desipramine administration increases stimulation-evoked [(3)H]NE release and concomitantly decreases alpha(2)-adrenergic autoreceptor sensitivity. Collectively, these data support the hypothesis that chronic antidepressant drug administration alters alpha(2)-adrenergic receptor-dependent regulation of NE release. Additionally, these data demonstrate that administration of dissimilar antidepressant drugs similarly transform alpha(2)-adrenergic autoreceptors that are functionally associated with the neuromodulatory effects of TNF, suggesting a possible mechanism of action of antidepressant drugs.

Interactions between the alpha(2)-adrenergic and the prostaglandin response in the regulation of macrophage-derived tumor necrosis factor.

Mediators such as prostaglandin E(2) (PGE(2)) and norepinephrine (NE) regulate macrophage (Mφ) responsiveness. Activation of alpha(2)-adrenergic receptors on Mφ potentiates lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNFalpha) production. PGE(2) inhibits LPS-stimulated TNFalpha production and gene expression, a response that can be desensitized by pretreatment of Mφ with PGE(2). We have determined that concomitant pretreatment of Mφ with PGE(2) and the alpha(2)-adrenergic agonist UK-14304 (UK) can prevent the PGE(2)-induced desensitization. PGE(2) concentration-effect curves have been determined for the inhibition of LPS-stimulated TNFalpha production by murine peritoneal Mφ. The addition of 10 nM UK to Mφ in culture significantly shifts the PGE(2) concentration-effect curve to the right; pretreatment of Mφ with UK significantly shifts the PGE(2) concentration-effect curve to the left; and pretreatment with the cyclooxygenase inhibitor, indomethacin, increases the maximum response of PGE(2). Preincubation of Mφ with PGE(2) (0.5 h) followed by washing significantly shifts the subsequent PGE(2) concentration-effect curve to the right. Concomitant preincubation of Mφ with PGE(2) and UK prevents this rightward shift, an effect that is blocked by the alpha(2)-adrenergic receptor antagonist yohimbine. Northern blot analysis demonstrates that UK increases LPS-induced TNFalpha mRNA accumulation, and this is blocked by yohimbine, while PGE(2) decreases TNFalpha mRNA accumulation. Preincubation of Mφ with PGE(2) prevents PGE(2) regulation of TNFalpha mRNA, and concomitant preincubation of Mφ with PGE(2) and UK reverses this effect. These investigations support the role of NE as a regulator of Mφ TNFalpha production, a response that has functional interactions with Mφ sensitivity to PGE(2).

Antidepressant drug administration modifies the interactive relationship between alpha(2)-adrenergic sensitivity and levels of TNF in the rat brain.

A reciprocally permissive interaction occurs between cellular responses elicited by the pleiotropic cytokine tumor necrosis factor-alpha (TNF) and alpha(2)-adrenergic receptor activation, such that each may adapt in response to modifications in the other's effects. Changes in presynaptic adrenergic sensitivity as well as neuronal sensitivity to TNF have been implicated in the mechanism of action of antidepressant drugs. The present study examines the influence of alpha(2)-adrenergic receptor activation on levels of TNF in regions of the brain associated with adrenergic function and the expression of mood. Additionally, the role of TNF as a neuromodulator is demonstrated by in vivo microinfusion of rrTNF proximal to the hippocampus. Administration to rats of an alpha(2)-adrenergic receptor agonist (clonidine) decreases levels of TNF in homogenates of rat locus coeruleus and hippocampus within 7.5 min. Chronic (14 days) administration of the antidepressant drugs desipramine or zimelidine transforms alpha(2)-adrenergic receptor-dependent decreases in TNF levels to increases in levels of TNF in the locus coeruleus. This transformation to an increase in total levels of TNF also occurs, although transiently, in the hippocampus following acute (1 day) antidepressant drug administration. The effect of TNF on presynaptic alpha(2)-adrenergic sensitivity was also investigated. Field stimulation of hippocampal slices from rats microinfused with rrTNF proximal to the hippocampus for 14 days demonstrates a decrease in fractional release of [3H]NE and an increase in alpha(2)-adrenergic autoreceptor sensitivity. These data demonstrate a mutual dependence between alpha(2)-adrenergic receptor activation and levels of TNF in the central nervous system that would culminate in an increase in neurotransmitter release following antidepressant drug administration.

Brain-derived TNFalpha: involvement in neuroplastic changes implicated in the conscious perception of persistent pain.

The pleiotropic cytokine tumor necrosis factor-alpha (TNFalpha) is implicated in the development of persistent pain through its actions in the periphery and in the central nervous system (CNS). Activation of the alpha(2)-adrenergic receptor is associated with modulation of pain, possibly through its autoregulatory effect on norepinephrine (NE) release in the CNS. The present study employs a chronic constriction nerve injury (CCI) pain model to demonstrate the interactive role of presynaptic sensitivity to TNFalpha and the alpha(2)-adrenergic autoreceptor in the pathogenesis of neuropathic pain. Accumulation of TNFalpha is increased initially in a region of the brain containing the locus coeruleus (LC) at day 4 post-ligature placement, followed by an increase in TNFalpha in the hippocampus at day 8 post-ligature placement, coincident with hyperalgesia. Levels of TNFalpha in the thoraco-lumbar spinal cord are also increased at day 8 post-ligature placement. Concurrently, alpha(2)-adrenergic receptor and TNFalpha-induced inhibition of NE release are increased, and stimulated NE release is decreased in superfused hippocampal slices isolated at day 8 post-ligature placement. Stimulated NE release is also decreased in spinal cord slices (lumbar region) from animals undergoing CCI, although in contrast to that which occurs in the hippocampus, alpha(2)-adrenergic receptor inhibition of NE release is not changed. These results indicate an important role that TNFalpha plays in adrenergic neuroplastic changes in a region of the brain that, among its many functions, appears to be a crucial link in the conscious perception of pain. We predict that neuroplastic changes, involving increased functional responses of alpha(2)-adrenergic autoreceptors and increased presynaptic sensitivity to TNFalpha, culminate in decreased NE release in the CNS. These neuroplastic changes provide a mechanism for the role of CNS-derived TNFalpha in the pathogenesis of persistent pain.

Brain-derived TNFalpha mediates neuropathic pain.

Neuropathic pain is a chronic pain state that develops a central component following acute nerve injury. However, the pathogenic mechanisms involved in the expression of this central component are not completely understood. We have investigated the role of brain-associated TNF in the evolution of hyperalgesia in the chronic constriction injury (CCI) model of neuropathic pain. Thermal nociceptive threshold has been assessed in rats (male, Sprague-Dawley) that have undergone loose, chromic gut ligature placement around the sciatic nerve. Total levels of TNF in regions of the brain, spinal cord and plasma have been assayed (WEHI-13VAR bioassay). Bioactive TNF levels are elevated in the hippocampus. During the period of injury, hippocampal noradrenergic neurotransmission demonstrates a decrease in stimulated norepinephrine (NE) release, concomitant with elevated hippocampal TNF levels. Continuous intracerebroventricular (i.c.v.) microinfusion of TNF-antibodies (Abs) starting at four days, but not six days, following ligature placement completely abolishes the hyperalgesic response characteristic of this model, as assessed by the 58 degrees C hot-plate test. Antibody infusion does not decrease spinal cord or plasma levels of TNF. Continuous i.c.v. microinfusion of rrTNF alpha exacerbates the hyperalgesic response by ligatured animals, and induces a hyperalgesic response in animals not receiving ligatures. Likewise, field-stimulated hippocampal adrenergic neurotransmission is decreased upon continuous i.c.v. microinfusion of TNF. These results indicate an important role of brain-derived TNF, both in the pathology of neuropathic pain, as well as in fundamental pain perception.

Streptococcal histone induces murine macrophages To produce interleukin-1 and tumor necrosis factor alpha.

The histone-like protein (HlpA) is highly conserved among streptococci. After lysis of streptococci in infected tissues, HlpA can enter the bloodstream and bind to proteoglycans in the glomerular capillaries of kidneys, where it can react with antibodies or stimulate host cell receptors. Deposits of streptococcal antigens in tissues have been associated with localized acute inflammation. In this study, we measured the ability of purified HlpA (5 to 100 microg/ml), from Streptococcus mitis, to induce the production of proinflammatory cytokines by cultured, murine peritoneal macrophages. The release of tumor necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1) was time and concentration dependent and was not diminished by the presence of polymyxin B. Exposure of macrophages to a mixture of HlpA and lipoteichoic acid resulted in a synergistic response in the production of both TNF-alpha and IL-1. Stimulation with a mixture of HlpA and heparin resulted in reduced cytokine production (50% less IL-1 and 76% less TNF-alpha) compared to that by cells incubated with HlpA alone. The inclusion of antibodies specific to HlpA in macrophage cultures during stimulation with HlpA did not affect the quantity of TNF-alpha or IL-1 produced. These observations suggest that streptococcal histone may contribute to tissue injury at infection sites by promoting monocytes/macrophages to synthesize and release cytokines that initiate and exacerbate inflammation. Streptococcus pyogenes, which can infect tissues in enormous numbers, may release sufficient amounts of HlpA to reach the kidneys and cause acute poststreptococcal glomerulonephritis.

Resident cardiac mast cells degranulate and release preformed TNF-alpha, initiating the cytokine cascade in experimental canine myocardial ischemia/reperfusion.

BACKGROUND: Neutrophil-induced cardiomyocyte injury requires the expression of myocyte intercellular adhesion molecule (ICAM)-1 and ICAM-1-CD11b/CD18 adhesion. We have previously demonstrated interleukin (IL)-6 activity in postischemic cardiac lymph; IL-6 is the primary stimulus for myocyte ICAM- 1 induction. Furthermore, we found that induction of IL-6 mRNA occurred very early on reperfusion of the infarcted myocardium. We hypothesized that the release of a preformed upstream cytokine induced IL-6 in leukocytes infiltrating on reperfusion. METHODS AND RESULTS: Constitutive expression of TNF-alpha and not IL-1beta was demonstrated in the normal canine myocardium and was localized predominantly in cardiac mast cells. Mast cell degranulation in the ischemic myocardium was documented by demonstration of a rapid release of histamine and TNF-alpha in the cardiac lymph after myocardial ischemia. Histochemical studies with FITC-labeled avidin demonstrated degranulating mast cells only in ischemic samples of canine myocardium. Immunohistochemistry suggested that degranulating mast cells were the primary source of TNF-alpha in the ischemic myocardium. In situ hybridization studies of reperfused myocardium localized IL-6 mRNA in infiltrating mononuclear cells and in mononuclear cells appearing in the postischemic cardiac lymph within the first 15 minutes of reperfusion. Furthermore, isolated canine mononuclear cells incubated with postischemic cardiac lymph demonstrated significant induction of IL-6 mRNA, which was partially blocked with a neutralizing antibody to TNF-alpha. CONCLUSIONS: Cardiac mast cells degranulate after myocardial ischemia, releasing preformed mediators, such as histamine and TNF-alpha. We suggest that mast cell-derived TNF-alpha may be a crucial factor in upregulating IL-6 in infiltrating leukocytes and initiating the cytokine cascade responsible for myocyte ICAM-1 induction and subsequent neutrophil-induced injury.

Adrenergic regulation of macrophage-derived tumor necrosis factor-alpha generation during a chronic polyarthritis pain model.

Increases in the levels of proinflammatory cytokines, such as TNF alpha, have been intricately linked with arthritis and the pathogenesis of several models of neuropathic pain. In addition, arthritis (as well as other types of persistent pain) is associated with increased sympathetic activity and alterations of other responses in autonomic nervous activity. Adrenergic regulation of LPS-stimulated TNF production by M phi isolated from rats with streptococcal-cell-wall (SCW)-induced arthritis has been examined. Serum TNF levels and the cellular composition of peritoneal exudates have also been assessed. M phi were obtained from: (1) normal control rats, (2) animals injected with complete Freund's adjuvant (CFA), 3 rats injected with SCW and arthritic, and (4) those injected with SCW, which failed to develop arthritis. Serum levels of TNF alpha in rats that develop arthritis are significantly greater (2.4 fold) than levels from the other groups. The proportion of OX19-positive T cell subpopulations are the same in peritoneal exudates from all groups. Immunocytochemical staining also reveals differences between M phi subgroups in the degree of activation. Peritoneal exudates from rats that develop arthritis contain a greater proportion of the high TNF producing subclass of M phi, as identified by positive ED3 staining (p < 0.001). In contrast, Ia antigen presenting M phi (OX6-positive) in the peritoneal exudate cells are only elevated in rats administered CFA. The selective blockade of adrenergic receptors by idazoxan or propranolol demonstrates that the constitutive involvement of either alpha 2 or beta-adrenergic regulation of M phi-derived TNF production is pronounced in rats with arthritis (p < 0.001). These investigations demonstrate a distinctive pattern of peripheral M phi populations in rats that develop chronic polyarthritic pain. We believe that identification of interactions between the adrenergic responses and proinflammatory cytokines will lead to the development of improved strategies to treat patients with chronic pain.

Neuronal-associated tumor necrosis factor (TNF alpha): its role in noradrenergic functioning and modification of its expression following antidepressant drug administration.

Tumor necrosis factor-alpha (TNF alpha) and the alpha 2-adrenergic agonist clonidine regulate norepinephrine (NE) release from noradrenergic nerve terminals in the central nervous system (CNS). In the present study, superfusion and electrical field stimulation were applied to a series of rat hippocampal brain slices in order to investigate the regulation of [3H]-NE release. NE release had been previously determined to be decreased by TNF alpha in a concentration-dependent manner, an effect which was potentiated by the alpha 2-adrenergic antagonist idazoxan. Presently, we demonstrate that similar to alpha 2-adrenergic activation, TNF alpha regulation of NE release in a region of the brain rich in noradrenergic nerve terminals, is dependent upon the frequency of electrical stimulation applied to the hippocampal slice. Furthermore, immunoperoxidase staining has verified our previous findings of constitutive TNF alpha protein in the rat brain. Staining for TNF alpha appears to be largely localized to neurons and neuronal processes, further substantiating the proposal that TNF alpha is either synthesized de novo or is accumulated in and released by neurons. After administration of the tricyclic antidepressant desipramine, tissue sections obtained from the rat hippocampus and locus coeruleus are devoid of neuronal-associated TNF alpha immunoreactivity. TNF alpha localization in neurons and its modification of NE release comparable to alpha 2-adrenergic receptor activation, explains a functional role for the cytokine as a neuromodulator in the CNS.

Changes in noradrenergic sensitivity to tumor necrosis factor-alpha in brains of rats administered clonidine.

Tumor necrosis factor-alpha (TNF alpha) and the imidazoline clonidine modulate norepinephrine (NE) release from noradrenergic nerve terminals in the central nervous system. The present study demonstrates an intrinsic association between presynaptic alpha 2-adrenergic receptor sensitivity and TNF alpha responsiveness in governing this NE release. Superfusion and electrical field stimulation were applied to a series of rat hippocampal brain slices in order to study the regulation of [3H]-NE release. The alpha 2-adrenergic agonist clonidine and the cytokine TNF alpha concentration-dependently inhibit [3H]-NE release; whereas, the alpha 2-adrenergic antagonist idazoxan potentiates [3H]-NE release. The fractional release of [3H]-NE during field stimulation of control hippocampal slices was decreased by the addition of TNF alpha in a concentration-dependent manner, an effect which was potentiated by the alpha 2-adrenergic antagonist idazoxan; whereas, TNF alpha attenuated the concentration-dependent potentiating effect of idazoxan. Furthermore, constitutive TNF alpha, demonstrated to be present in several brain areas, was significantly decreased following administration of the alpha 2-adrenergic agonist clonidine (0.6 mg/kg, i.p., twice daily) to rats for either 1 or 14 days, without a change in TNF alpha mRNA accumulation. We next investigated whether the presynaptic sensitivity to TNF alpha was changed after clonidine administration to rats. TNF alpha enhanced, rather than inhibited, [3H]-NE release after 1 day of clonidine administration, while a suppressed sensitivity to TNF alpha was observed in the hippocampus after 14 days of clonidine administration. In addition, in the presence of idazoxan, TNF alpha potentiation of [3H]-NE release after 1 day clonidine administration was reversed to a decreased inhibition as compared to control slices exposed to idazoxan. Therefore, the temporary reversal in the presynaptic TNF alpha response after 1 day of clonidine administration illustrates a mechanism of action for its persistent antihypertensive effect, its transient sedative and antihyperpathic effects, and its acute ability to promote antidepressants. These results demonstrate a novel role for an immune mediator in the central nervous system, and demonstrates that presynaptic TNF alpha responsiveness is intimately associated with adrenergic receptor sensitivity.

Beta-adrenergic receptor regulation of macrophage-derived tumor necrosis factor-alpha production from rats with experimental arthritis.

Prostaglandin E2 (PGE2) and beta-adrenergic agonists can suppress lipopolysaccharide-induced tumor necrosis factor-alpha (TNF) production from elicited macrophages. We assessed the responsiveness of rat peritoneal macrophages to PGE2 and the beta-adrenergic agonist isoproterenol during immunologically-mediated arthritis. We assessed macrophage sensitivity to these mediators from resident macrophages and macrophages elicited with either streptococcal cell wall or complete Freund's adjuvant. Peritoneal macrophages were obtained from female Lewis rats that were (1) injected with complete Freund's adjuvant and non-arthritic (CFA); (2) injected with streptococcal cell wall and arthritic (ART); (3) injected with streptococcal cell wall and non-reactive (NON) and (4) non-elicited resident macrophages (RES). When challenged with graded concentrations of lipopolysaccharide (0.1 to 10,000 ng/ml), macrophages obtained from each group of rats released TNF in a concentration-dependent manner, with macrophages from arthritic rats (ART) producing the greatest amount of TNF (p < 0.001). While PGE2 suppressed lipopolysaccharide (100 ng/ml) stimulated TNF production in a concentration-dependent manner in all groups, the greatest sensitivity to PGE2 was observed with macrophages obtained from rats which received streptococcal cell wall when compared to both complete Freund's adjuvant-elicited and resident macrophages (p < 0.05). The beta-adrenergic agonist isoproterenol also inhibited lipopolysaccharide-stimulated TNF production from macrophages in all groups. In addition, the specific beta 2-adrenergic antagonist, ICI 118.551, shifted isoproterenol concentration-effect curves to the right (p < 0.01). Minimal responsiveness to isoproterenol was observed with resident peritoneal macrophages. Maximum isoproterenol-induced inhibition of TNF production was observed with complete Freund's adjuvant-elicited macrophages, and significantly less in macrophages of streptococcal cell wall-injected rats. Of particular interest, macrophages obtained from streptococcal cell wall-injected rats, which became arthritic, were significantly less sensitive to isoproterenol than those which did not develop arthritis (p < 0.02). In addition, these changes in sensitivity were not reflected by changes in the sensitivity of both CFA and ART groups to dibutyryl cAMP. The present study demonstrates a shift in the balance between inhibitory mediator responses in rats inoculated with one of two different adjuvants. These investigations support the role of PGE2 and a neurotransmitter as immunomodulating compounds which may effectively maintain an inflammatory lesion such as arthritis.

Temporal regulation by adrenergic receptor stimulation of macrophage (M phi)-derived tumor necrosis factor (TNF) production post-LPS challenge.

Macrophage (M phi) responsiveness can be regulated by various mediators, including those which emanate from, and mimic, the sympathetic nervous system. Whereas beta-adrenergic agonists suppress, alpha 2-adrenergic agonists augment lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNF) production and gene expressed. The susceptibility of M phi s to regulation of LPS-induced TNF production and mRNA accumulation was examined following beta-adrenergic and alpha 2-adrenergic receptor activation at specific time points post-LPS challenge. Complete Freund's adjuvant-elicited murine M phi s were incubated with LPS (30 ng/ml) in the presence or absence of adrenergic agonists or antagonists. We assessed the susceptibility of immunologically-activated M phi s to adrenergic receptor regulation: a) during the 1 h delay in the production of TNF after LPS-stimulation, and b) during the rapid increase in TNF production which follows. Disparate responsiveness of M phi s to adrenergic drugs was observed during this time course of TNF production and TNF mRNA accumulation. In particular, while the concomitant addition of an alpha 2-adrenergic antagonist and LPS resulted in 45% suppression of TNF production, this selective blockade of alpha 2-adrenergic receptors on M phi s was equally effective throughout the first 45 min post-LPS challenge. After this initial period, the alpha 2-adrenergic receptor became progressively less responsive as demonstrated by the delayed addition of yohimbine (10(-5) M) post-LPS challenge. The addition of the selective alpha 2-adrenergic agonist UK-14304 (10(-7) M) to LPS-activated M phi s augmented TNF mRNA accumulation. However, this augmentation was even greater when the addition of the alpha 2-adrenergic agonist was delayed post-LPS challenge. It was also shown that the beta-adrenergic agonist isoproterenol (10(-6) M) produced maximum suppression of TNF production within the first 1.5 h post-LPS challenge. Suppression by isoproterenol (10(-6) M) of TNF mRNA accumulation occurred throughout the 2-h period assessed post-LPS stimulation of M phi s. The decline in isoproterenol-induced regulation was accompanied by an elevation in beta 2-adrenergic receptor mRNA accumulation. Furthermore, suppression of TNF production induced by a maximum concentration of isoproterenol was observed at various LPS concentrations (0.001-1000 ng/ml), although this was not as pronounced a suppression as demonstrated for dibutyrl cAMP. These results demonstrate that the susceptibility of M phi s to adrenergic receptor regulation changes throughout the time period necessary for gene activation and ultimate release of TNF. Thus, the production of TNF during LPS-dependent disease states may be regulated by adrenergic mediators throughout different temporal windows, better explaining the role played by the nervous system.

Regulation of macrophage-derived tumor necrosis factor production by modification of adrenergic receptor sensitivity.

Catecholamines and prostaglandins are among the many diverse mediators which participate in an interactive communication between the nervous and immune systems. We have examined the response of murine peritoneal macrophages (M luminal diameter) to prostaglandin-E2 (PGE2) and the beta-adrenergic agonist isoproterenol. In the present study we found a relationship between the response elicited by PGE2 and a beta-adrenergic agonist, which in a fashion similar to the response of PGE2 on M luminal diameters suppresses lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) production. It has been established that exposure of M luminal diameters to PGE2 desensitizes the suppressive function of PGE2. In this study, prior exposure of M luminal diameters to a beta-adrenergic agonist and the effects on subsequent beta-adrenergic responses, as well as the relationship to PGE2 sensitivity was determined. Complete Freund's adjuvant-elicited M luminal diameters were incubated with or without either a beta-adrenergic agonist or antagonist. All groups of cells were then extensively washed, followed by incubation with LPS (100 ng/ml) with or without graded concentrations of PGE2 or the beta-adrenergic agonist isoproterenol. Supernatants were collected to determine TNF concentrations by a fibroblast cytolytic assay, and Northern blot analysis was used to determine changes in the regulation of TNF mRNA accumulation. Both isoproterenol and PGE2 inhibited LPS-stimulated TNF release and TNF mRNA accumulation. We have established M luminal diameters regulation of sensitivity to isoproterenol-induced suppression of TNF production. The isoproterenol concentration-effect curve was shifted to the right after pre-exposure of M luminal diameter to the beta-agonist, suggesting a desensitized beta-adrenergic receptor population. Further studies demonstrated that M luminal diameters pre-exposed to the beta-adrenergic antagonist, ICI 118.551, washed, and then challenged with LPS show an increased sensitivity for isoproterenol-induced suppression of TNF production. In addition, a decreased sensitivity of M luminal diameters to exogenous PGE2 was observed during the desensitization to the beta-adrenergic agonist. Although concomitant addition of isoproterenol increased PGE2-induced suppression of LPS-stimulated TNF production, M luminal diameter pre-exposed to isoproterenol (10(-6) M) demonstrated a decreased sensitivity for PGE2-induced suppression of LPS-stimulated TNF production and TNF mRNA accumulation. Our results show that the effects observed after acute administration of a mediator may be different when M luminal diameters have been previously exposed to that or other mediators. These investigations support a role for mediators released from the nervous system to regulate the release of a cytokine needed to maintain inflammatory responses.

Tumor necrosis factor-alpha: presynaptic sensitivity is modified after antidepressant drug administration.

Presynaptic adrenergic functioning was coupled to cytokine sensitivity in order to further establish the mechanism of action of a tricyclic antidepressant drug. Antidepressant administration of desipramine to rats twice-daily for 2 weeks increased hippocampal TNF levels and transformed the presynaptic TNF response. One day of desipramine administration resulted in increased locus coeruleus TNF mRNA accumulation and, simultaneously, hippocampal TNF levels escalated. The fractional release of [3H]norepinephrine during field stimulation of control hippocampal slices was decreased by the addition of TNF in a concentration-dependent manner, an effect which was potentiated by the alpha 2-adrenergic antagonist idazoxan. While no change in sensitivity to TNF was observed in the hippocampus after one day of desipramine administration, TNF enhanced, rather than inhibited [3H]norepinephrine release after 14 days. In addition, TNF potentiation of [3H]norepinephrine release after chronic desipramine administration was reversed in the presence of idazoxan to a greater inhibition than in control slices exposed to idazoxan. Therefore, TNF-induced regulation of [3H]norepinephrine release appears to be associated with an alteration of alpha 2-adrenergic receptor responsiveness. The reversal in presynaptic TNF responsiveness after 14 days of tricyclic antidepressant drug administration describes a mechanism of action for their delayed clinical effect.