Nanotherapeutic approach for opiate addiction using DARPP-32 gene silencing in an animal model of opiate addiction.

Opiates act on the dopaminergic system of the brain and perturb 32 kDa dopamine and adenosine 3', 5'-monophosphate-regulated phosphoprotein (DARPP-32) function. The DARPP-32 mediated inhibition of protein phosphatase-1 (PP-1) and modulation of transcriptional factor CREB is critical to the changes in neuronal plasticity that result in behavioral responses during drug abuse. To investigate the role of DARPP-32 mediated signaling on withdrawal behavior in a rat model of opiate addiction, we used intracerebral administration of gold nanorods (GNR) complexed to DARPP-32 siRNA to silence DARPP-32 gene expression and measure its effects on the opiate withdrawal syndrome. We hypothesized that DARPP-32 siRNA will suppress the neurochemical changes underlying the withdrawal syndrome and therefore prevent conditioned place aversion by suppressing or removing the constellation of negative effects associated with withdrawal, during the conditioning procedure. Our results showed that opiate addicted animals treated with GNR-DARPP-32 siRNA nanoplex showed lack of condition place aversive behavior consequent to the downregulation of secondary effectors such as PP-1 and CREB which modify transcriptional gene regulation and consequently neuronal plasticity. Thus, nanotechnology based delivery systems could allow sustained knockdown of DARPP-32 gene expression which could be developed into a therapeutic intervention for treating drug addiction by altering reward and motivational systems and interfere with conditioned responses.

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.

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.

Differential kappa-opioid receptor expression on mouse lymphocytes at varying stages of maturation and on mouse macrophages after selective elicitation.

The combination of indirect immunofluorescent labeling and flow cytometry has proven to be a sensitive method for labeling of the kappa-opioid receptor on mouse thymocytes. In the present study, this labeling procedure was applied, along with phenotypic analysis, to mature immune cell populations to determine whether kappa-opioid receptor expression is present after immune cell maturation. Unfixed primary splenocytes from 6- to 8-week-old C57BL/6ByJ male mice were incubated with the fluorescein-containing, kappa-selective ligand fluorescein-conjugated 2-(3, 4-dichlorophenyl)-N-methyl-N-[1-(3-aminophenyl)-2-(1-pyrrolidinyl)eth yl]acetamide (FITC-AA). Amplification of FITC-AA binding to the kappa-opioid receptor was attained by adding a biotin-conjugated antifluorescein antibody, followed by extravidin-R-phycoerythrin. It has been shown previously that greater than 60% of immature thymocytes (CD4(+)/CD8(+)) demonstrated specific kappa-opioid receptor labeling. However, the present report shows that less than 25% of either T-helper or T-cytotoxic splenic lymphocytes expressed the kappa-opioid receptor. Likewise, only 16% of all splenic B lymphocytes were labeled for the kappa-opioid receptor. These findings demonstrate a decrease in kappa-opioid receptor expression on maturation of mouse lymphocytes. Interestingly, resident peritoneal macrophages showed a greater magnitude of specific receptor labeling, compared with either thymocytes or splenocytes, and approximately 50% of the resting Mphi expressed the kappa-opioid receptor. However, elicitation of Mphi with thioglycollate resulted in the complete loss of the expression of this receptor. Taken together, these findings demonstrate the diversity in the expression of the kappa-opioid receptor on immune cells at varying stages of differentiation, with preferential expression demonstrated by resident, peritoneal macrophages.

Detection of kappa opioid receptors on mouse thymocyte phenotypic subpopulations as assessed by flow cytometry.

Recent studies have shown kappa opioid receptor labeling on the R1EGO thymoma cell line by indirect immunofluorescence and flow cytometric analysis. The present study used a fluorescein-labeled arylacetamide (FITC-AA), a kappa opioid ligand, in conjunction with biotin-conjugated anti-fluorescein IgG and extravidin-R-phycoerythrin (PE), along with double-labeling with antibodies against specific immune cell surface markers to determine which subpopulation(s) of thymocytes express the kappa opioid receptor. Thymocytes, isolated from 6- to 8-week-old C57BL/6ByJ mice, incubated with FITC-AA followed by the PE amplification procedure, demonstrated labeling of the kappa opioid receptor. This labeling was inhibited 55 +/- 4% above background by excess nor-binaltorphimine (nor-BNI), a kappa selective antagonist. This kappa opioid receptor positive population consisted of 58 +/- 2% of all gated thymocytes. Phenotypic characterization determined that not only were 64 +/- 3% of the gated thymocytes CD4+/kappa opioid receptor positive, but 60 +/- 1% of all thymocytes were CD8+/kappa opioid receptor positive. Two subpopulations of CD3+ thymocytes, consisting of both mature and immature cells, also displayed labeling for the kappa opioid receptor. Double-labeling of thymocytes with anti-CD4 and anti-CD8 antibodies demonstrated 82 +/- 0.5% of these cells were of the double-positive phenotype. Therefore, these findings demonstrate that the thymocytes, which express the kappa opioid receptor, are predominantly of the immature CD4+/CD8+ phenotype. Collectively, these findings not only establish the presence of the kappa opioid receptor on immune cells involved in opioid responsiveness, but further indicate that this technique allows for the identification of distinct lymphocyte subpopulations which express the receptor.

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.

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.