ABSTRACT
BACKGROUND AND PURPOSE: Nabiximols (Sativex® ) is a cannabinoid-based compound used for the treatment of moderate to severe spasticity in multiple sclerosis (MS). The aim of the study was to investigate the effect of the administration of Nabiximols on blood transcriptome profile of patients with MS and to interpret it in the context of pathways and networks. METHODS: Whole-genome expression profiling was performed in whole blood of 33 subjects with MS at baseline and after 4 weeks of drug treatment. Patients were classified as responders (n = 19) and non-responders (n = 14). Pathway and network analyses on genes modulated by the drug were performed, followed by in vitro stimulation of peripheral blood mononuclear cells with pro-inflammatory agents to support the immunomodulatory properties of the drug. RESULTS: Individual effect size was modest; however, we observed a downregulation of several immune-related pathways after 4 weeks of treatment, which was more pronounced when restricting analyses to responders. Interesting hub molecules functionally related to the immune system emerged from network analysis, including NFKB1, FYN, MAP14 and TP53. The immunomodulatory properties of the drug were confirmed through in vitro assays in peripheral blood mononuclear cells collected from patients with MS. CONCLUSIONS: Our findings support the immunomodulatory activity of cannabinoids in patients with MS. Further studies in more specific cell types are needed to refine these results.
Subject(s)
Cannabidiol/therapeutic use , Down-Regulation , Dronabinol/therapeutic use , Multiple Sclerosis/drug therapy , Adult , Cannabidiol/pharmacology , Dronabinol/pharmacology , Drug Combinations , Female , Humans , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/immunology , Male , Middle Aged , Multiple Sclerosis/immunologyABSTRACT
The endocannabinoid system (ECS), comprising the cannabinoid receptors (CBR), their ligands, and enzymes controlling the turnover of endocannabinoids, has been suggested to be involved in male reproductive function. As information is scarce on the expression of the ECS in human male reproductive tissues, this study aimed to investigate by means of molecular biology (RT-PCR) and immunohistochemistry/immunofluorescence the expression and distribution of CB1 and CB2, GPR55 (an orphan G protein-coupled receptor that recognises cannabinoid ligands) and FAAH (isoforms 1 and 2) in the human seminal vesicles (SV). The specimens expressed PCR products corresponding to CB1 (66 bp), CB2 (141 bp), GPR55 (112 bp), FAAH1 (260 bp) and FAAH2 (387 bp). Immumohistochemistry revealed dense expression of CB1, CB2 and GPR55 located to the pseudo-stratified columnar epithelium and varicose nerves (also characterised by the expression of vasoactive intestinal polypeptide and calcitonin gene-related peptide). Cytosolic staining for FAAH1 and FAAH2 was seen in cuboidal cells of all layers of the epithelium. No immunoreactivity was detected in the smooth musculature or nerve fibres. CB1, CB2, GPR55, FAAH1 and FAAH2 are highly expressed in the human SV. Considering their localisation, the ECS may be involved in epithelial homeostasis, secretory function or autonomic mechano-afferent signalling.
Subject(s)
Amidohydrolases/metabolism , Endocannabinoids/metabolism , Receptors, Cannabinoid/metabolism , Receptors, G-Protein-Coupled/metabolism , Seminal Vesicles/metabolism , Aged , Epithelial Cells/metabolism , Fluorescent Antibody Technique , Humans , Immunohistochemistry , Male , Middle Aged , Seminal Vesicles/pathologyABSTRACT
OBJECTIVE: This study sought to develop a novel animal model to study the impact of nerve-sparing radical hysterectomy (NSRH) on female genital blood flow. DESIGN: In vivo animal study. POPULATION: Thirty Sprague-Dawley female rats. MATERIALS AND METHODS: Female rats underwent either unilateral pelvic nerve (PN) crush (PNC; n = 9), or crush of both the PNs and all efferent nerves in the pelvic plexus ('clock-nerve crush', CNC; n = 9). Under anaesthesia, we electrically stimulated the crushed PN at 3 and 10 days after crush while monitoring blood pressure and recording clitoral and vaginal blood flows by laser Doppler. Uninjured PNs were stimulated as an internal control. Twelve additional rats were assigned either to bilateral PNC or sham surgery, and genital tissues were processed 10 days after injury for in vitro analysis. MAIN OUTCOME MEASURES: Genital blood flow, nNOS, eNOS, collagen I-III. RESULTS: Stimulation of the crushed PN in both groups subjected to PNC and CNC induced significantly lower peak genital blood flow at 3 and 10 days (P < 0.05) compared to stimulation of the non-crushed control PN. The immunofluorescence and Western blot analyses revealed that all injured rats exhibited more vaginal collagen III and collagen I than rats did that ad undergone sham surgeries (P < 0.05). PCN reduced nNOS expression in both clitoral and vaginal tissue. CONCLUSIONS: Based on our study it may be hypothesised that NSRH might cause reductions of genital blood flow and vaginal fibrosis due to neurapraxia of the pelvic nerve and reductions of nNOS nerve fibres in clitoral and distal vaginal tissue. TWEETABLE ABSTRACT: Pelvic nerve neurapraxia during nerve-sparing radical hysterectomy could lead to sexual arousal dysfunction.
Subject(s)
Hypogastric Plexus/injuries , Hysterectomy/adverse effects , Hysterectomy/methods , Peripheral Nerve Injuries/prevention & control , Vagina/blood supply , Vagina/pathology , Animals , Blotting, Western , Clitoris/metabolism , Collagen Type I/metabolism , Collagen Type III/metabolism , Electric Stimulation , Female , Fibrosis , Fluorescent Antibody Technique , Laser-Doppler Flowmetry , Models, Animal , Nitric Oxide Synthase/metabolism , Pelvis/innervation , Peripheral Nerve Injuries/complications , Peripheral Nerve Injuries/etiology , Rats, Sprague-Dawley , Regional Blood Flow , Vagina/metabolismABSTRACT
Improving treatment of advanced melanoma may require the development of effective strategies to overcome resistance to different anti-tumor agents and to counteract relevant pro-tumoral mechanisms in the microenvironment. Here we provide preclinical evidence that these goals can be achieved in most melanomas, by co-targeting of oncogenic and death receptor pathways, and independently of their BRAF, NRAS, p53 and PTEN status. In 49 melanoma cell lines, we found independent susceptibility profiles for response to the MEK1/2 inhibitor AZD6244, the PI3K/mTOR inhibitor BEZ235 and the death receptor ligand TRAIL, supporting the rationale for their association. Drug interaction analysis indicated that a strong synergistic anti-tumor activity could be achieved by the three agents and the AZD6244-TRAIL association on 20/21 melanomas, including cell lines resistant to the inhibitors or to TRAIL. Mechanistically, synergy was explained by enhanced induction of caspase-dependent apoptosis, mitochondrial depolarization and modulation of key regulators of extrinsic and intrinsic cell death pathways, including c-FLIP, BIM, BAX, clusterin, Mcl-1 and several IAP family members. Moreover, silencing experiments confirmed the central role of Apollon downmodulation in promoting the apoptotic response of melanoma cells to the combinatorial treatments. In SCID mice, the AZD6244-TRAIL association induced significant growth inhibition of a tumor resistant to TRAIL and poorly responsive to AZD6244, with no detectable adverse events on body weight and tissue histology. Reduction in tumor volume was associated not only with promotion of tumor apoptosis but also with suppression of the pro-angiogenic molecules HIF1α, VEGFα, IL-8 and TGFß1 and with inhibition of tumor angiogenesis. These results suggest that synergistic co-targeting of oncogenic and death receptor pathways can not only overcome melanoma resistance to different anti-tumor agents in vitro but can also promote pro-apoptotic effects and inhibition of tumor angiogenesis in vivo.
Subject(s)
Antineoplastic Agents/administration & dosage , Melanoma/drug therapy , Receptors, Death Domain/metabolism , Animals , Apoptosis/drug effects , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Benzimidazoles/administration & dosage , Cell Line, Tumor , Drug Resistance, Neoplasm , Drug Synergism , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Melanoma/genetics , Melanoma/metabolism , Melanoma/physiopathology , Mice , Mice, SCID , Neovascularization, Pathologic , Receptors, Death Domain/genetics , TNF-Related Apoptosis-Inducing Ligand/administration & dosageABSTRACT
BACKGROUND AND PURPOSE: α1 -adrenoceptor (-AR) antagonists may facilitate ureter stone passage in humans. We aimed to study effects by the α1 A -AR selective antagonist silodosin (compared to tamsulosin and prazosin) on ureter pressures in a rat model of ureter obstruction, and on contractions of human and rat isolated ureters. EXPERIMENTAL APPROACH: After ethical approval, ureters of male rats were cannulated beneath the kidney pelvis for in vivo ureteral intraluminal recording of autonomous peristaltic pressure waves. A partial ureter obstruction was applied to the distal ureter. Mean arterial blood pressure (MAP) was recorded. Approximate clinical and triple clinical doses of the α1 -AR antagonists were given intravenously. Effects by the α1 -AR antagonists on isolated human and rat ureters were studied in organ baths. KEY RESULTS: Intravenous silodosin (0.1-0.3 mg kg(-1) ) or prazosin (0.03-0.1 mg kg(-1) ) reduced obstruction-induced increases in intraluminal ureter pressures by 21-37% or 18-40% respectively. Corresponding effects by tamsulosin (0.01 or 0.03 mg kg(-1) ) were 9-20%. Silodosin, prazosin and tamsulosin reduced MAP by 10-12%, 25-26% (P < 0.05), or 18-25% (P < 0.05) respectively. When effects by the α1 A -AR antagonists on obstruction-induced ureter pressures were expressed as a function of MAP, silodosin had six- to eightfold and 2.5- to eightfold better efficacy than tamsulosin or prazosin respectively. Silodosin effectively reduced contractions of both human and rat isolated ureters. CONCLUSIONS AND IMPLICATIONS: Silodosin inhibits contractions of the rat and human isolated ureters and has excellent functional selectivity in vivo to relieve pressure-load of the rat obstructed ureter. Silodosin as pharmacological ureter stone expulsive therapy should be clinically further explored.
Subject(s)
Indoles/pharmacology , Prazosin/pharmacology , Ureter/drug effects , Ureteral Obstruction/drug therapy , Adrenergic alpha-1 Receptor Antagonists/administration & dosage , Adrenergic alpha-1 Receptor Antagonists/pharmacology , Animals , Arterial Pressure/drug effects , Dose-Response Relationship, Drug , Humans , Indoles/administration & dosage , Male , Muscle Contraction/drug effects , Prazosin/administration & dosage , Rats , Rats, Sprague-Dawley , Species Specificity , Sulfonamides/administration & dosage , Sulfonamides/pharmacology , Tamsulosin , Ureter/metabolism , Ureter/pathology , Ureteral Obstruction/pathologyABSTRACT
To study the role of cytokines that are relevant in cancer cachexia syndrome due to intracerebral tumours, mice were injected with human A431 epidermoid carcinoma, OVCAR3 ovarian carcinoma and GBLF glioma cells comparing intracerebral (i.c.) and systemic (i.p. or s.c.) routes of implantation. Anorexia and weight loss developed within 7-10 days in mice injected i.c. with A431 or OVCAR3 cells well before a large tumour developed, while i.c.-injected GBLF cells did not induce cachexia until day 20, when the tumour was large. By contrast, mice injected i.p. or s.c. developed tumours without evidence of anorexia. Thus, intracerebrally-growing A431 and OVCAR3 resulted in cancer cachexia independent of tumour mass, and we investigated their cytokine pattern. Serum levels of murine and human cytokines are not predictive of cancer cachexia development. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis revealed in the brain of i.c.-injected A431 tumour-bearing mice expression of human interleukin-(IL-)1alpha, IL-1beta and LIF in all samples and IL-6 in two of four samples while in i.c.-injected OVCAR3 tumour-bearing animals IL-6, and LIF were detected in all samples and tumour necrosis factor-alpha (TNFalpha) in two of four samples. Only LIF was expressed in brains of mice injected with GBLF cells. Murine IL-6 was increased only in the brains of A431-bearing mice. Only mice injected i.c. simultaneously with a monoclonal antibody (mAb) directed against the murine IL-6 receptor and OVCAR3 cells, but not those with mAb and A431 cells, showed a significant increase in survival time with a partial and temporary attenuation of cachexia symptoms. These results suggest that IL-6 in OVCAR3 model may be important cachectogenic factor when centrally released by even a limited number of tumour cells.
Subject(s)
Brain Neoplasms/metabolism , Cachexia/metabolism , Cytokines/physiology , Neoplasms/metabolism , Animals , Anorexia/metabolism , Body Weight , Brain/metabolism , Central Nervous System/metabolism , Cytokines/blood , Enzyme-Linked Immunosorbent Assay , Feeding Behavior , Female , Growth Inhibitors/blood , Growth Inhibitors/metabolism , Humans , Interleukin-1/blood , Interleukin-1/metabolism , Interleukin-6/blood , Interleukin-6/metabolism , Leukemia Inhibitory Factor , Lymphokines/blood , Lymphokines/metabolism , Mice , Mice, Nude , Neoplasm Transplantation , RNA, Messenger/metabolism , Rats , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Tumor Cells, CulturedABSTRACT
Severe infection or tissue invasion can provoke a catabolic response, leading to severe metabolic derangement, cachexia, and even death. Macrophage migration inhibitory factor (MIF) is an important regulator of the host response to infection. Released by various immune cells and by the anterior pituitary gland, MIF plays a critical role in the systemic inflammatory response by counterregulating the inhibitory effect of glucocorticoids on immune-cell activation and proinflammatory cytokine production. We describe herein an unexpected role for MIF in the regulation of glycolysis. The addition of MIF to differentiated L6 rat myotubes increased synthesis of fructose 2,6-bisphosphate (F2,6BP), a positive allosteric regulator of glycolysis. Increased expression of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) enhanced F2,6BP production and, consequently, cellular lactate production. The catabolic effect of TNF-alpha on myotubes was mediated by MIF, which served as an autocrine stimulus for F2, 6BP production. TNF-alpha administered to mice decreased serum glucose levels and increased muscle F2,6BP levels; pretreatment with a neutralizing anti-MIF mAb completely inhibited these effects. Anti-MIF also prevented hypoglycemia and increased muscle F2,6BP levels in TNF-alpha-knockout mice that were administered LPS, supporting the intrinsic contribution of MIF to these inflammation-induced metabolic changes. Taken together with the recent finding that MIF is a positive, autocrine stimulator of insulin release, these data suggest an important role for MIF in the control of host glucose disposal and carbohydrate metabolism.
Subject(s)
Fructosediphosphates/biosynthesis , Glucose/metabolism , Lactic Acid/biosynthesis , Macrophage Migration-Inhibitory Factors/metabolism , Animals , Cell Line , Cell Movement/physiology , Glycolysis/drug effects , Humans , Liver/metabolism , Macrophage Migration-Inhibitory Factors/pharmacology , Macrophages/metabolism , Mice , Muscles/metabolism , Phosphofructokinase-2 , Phosphoric Monoester Hydrolases/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Rats , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
Administration of interleukin IL-1 induces acute-phase response and inhibition of gastric secretion more efficiently when administered intracerebroventricularly (i.c.v.) than when the same dose of IL-1 is administered systemically. In this study we describe the pharmacokinetics of IL-1beta, administered centrally or systemically, in the serum or in peripheral tissues. IL-1beta administered i.c.v. resulted in higher peak IL-1beta concentrations, and lasted longer, than intravenous (i.v.) or intraperitoneal (i.p.) administration. Higher IL-1beta levels in the liver and heart were observed after i. c.v. administration (compared to the i.p. or i.v. route). Our data suggest that centrally injected IL-1 induces higher circulating and hepatic IL-1 levels and contributes to the fact that the i.c.v. route of administration is particularly effective in inducing a liver acute-phase response.
Subject(s)
Brain/drug effects , Interleukin-1/pharmacology , Acute-Phase Proteins/metabolism , Animals , Brain/metabolism , Injections, Intraperitoneal , Injections, Intravenous , Injections, Intraventricular , Interleukin-1/pharmacokinetics , Interleukin-6/blood , Male , Metabolic Clearance Rate , Mice , Mice, Inbred Strains , Serum Amyloid A Protein/metabolism , Tissue DistributionABSTRACT
Cancer cells maintain a high glycolytic rate even in the presence of oxygen, a phenomenon first described over 70 years ago and known historically as the Warburg effect. Fructose 2,6-bisphosphate is a powerful allosteric regulator of glycolysis that acts to stimulate the activity of 6-phosphofructo-1-kinase (PFK-1), the most important control point in mammalian glycolysis. The steady state concentration of fructose 2,6-bisphosphate in turn depends on the activity of the enzyme 6-phosphofructo-2-kinase (PFK-2)/fructose-2, 6-bisphosphatase, which is expressed in several tissue-specific isoforms. We report herein the identification of a gene product for this enzyme that is induced by proinflammatory stimuli and which is distinguished by the presence of multiple copies of the AUUUA mRNA instability motif in its 3'-untranslated end. This inducible gene for PFK-2 is expressed constitutively in several human cancer cell lines and was found to be required for tumor cell growth in vitro and in vivo. Inhibition of inducible PFK-2 protein expression decreased the intracellular level of 5-phosphoribosyl-1-pyrophosphate, a product of the pentose phosphate pathway and an important precursor for nucleic acid biosynthesis. These studies identify a regulatory isoenzyme that may be essential for tumor growth and provide an explanation for long-standing observations concerning the apparent coupling of enhanced glycolysis and cell proliferation.
Subject(s)
Gene Expression Regulation, Neoplastic , Neoplasms/genetics , Neoplasms/metabolism , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , 3' Untranslated Regions/genetics , Allosteric Regulation , Amino Acid Sequence , Base Sequence , Cell Division , Cell Transformation, Neoplastic , Cloning, Molecular , Glycolysis , Humans , Molecular Sequence Data , Neoplasms/pathology , Phosphofructokinase-2 , RNA, Messenger/analysis , RNA, Messenger/genetics , Sequence Alignment , Tumor Cells, CulturedABSTRACT
Fas is a receptor of the tumor necrosis factor (TNF)/ nerve growth factor (NGF) receptor superfamily that mediates apoptosis and some inflammatory changes. As the central administration of TNF is known to activate the hypothalamus-pituitary-adrenal axis (HPAA) and to induce peripheral responses including induction of serum interleukin (IL)-6 and serum amyloid A (SAA), we investigated the effects of intracerebroventricular (i.c.v.) administration of agonist anti-Fas monoclonal antibody Jo2. Centrally administered anti-Fas (1 microg/mouse, i.c.v.) induced elevated levels of corticosterone, IL-6, and SAA comparable to those observed after i.c.v. administration of recombinant murine TNF. On the other hand, administration of murine NGF did not elevate serum corticosterone or IL-6, but induced SAA. Thus, Fas can trigger a centrally mediated anti-inflammatory response (HPAA activation) and induce a peripheral acute-phase response comparable to that induced with TNF, whereas NGF induces only acute-phase proteins.
Subject(s)
Antibodies, Monoclonal/pharmacology , Apolipoproteins/biosynthesis , Hypothalamo-Hypophyseal System/drug effects , Interleukin-6/biosynthesis , Pituitary-Adrenal System/drug effects , Serum Amyloid A Protein/biosynthesis , fas Receptor/immunology , Acute-Phase Proteins/biosynthesis , Animals , Antibodies, Monoclonal/administration & dosage , Corticosterone/blood , Injections, Intraventricular , Ligands , Male , Mice , Receptors, Nerve Growth Factor/metabolism , Receptors, Tumor Necrosis Factor/metabolismABSTRACT
Ciliary neurotrophic factor (CNTF) inhibits the production of tumor necrosis factor (TNF) in lipopolysaccharide (LPS)-treated mice and protects against LPS lethality when coadministered with its soluble receptor (sCNTFR alpha). Both of these activities are abolished in adrenalectomized (ADX) mice. LPS-induced pulmonary polymorphonuclear neutrophil (PMN) infiltration and nitric oxide (NO) production were also inhibited by CNTF + sCNTFR alpha but not by CNTF alone. sCNTFR alpha did not alter the clearance or tissue distribution of CNTF. Furthermore, CNTF variants coadministered with sCNTFR alpha protected against LPS toxicity in a manner related to their affinity for the beta components of CNTFR. Thus, inhibition of TNF production and protection against LPS lethality by CNTF/sCNTFR alpha require an intact hypothalamus-pituitary-adrenal axis (HPAA) and may be mediated by endogenous glucocorticoids. This protective effect is, at least in part, due to the inhibition of PMN infiltration and NO production, and appears to be mediated by cells displaying only beta-receptor subtypes.
Subject(s)
Disease Models, Animal , Nerve Tissue Proteins/therapeutic use , Neuroprotective Agents , Receptor Protein-Tyrosine Kinases/physiology , Receptors, Nerve Growth Factor/physiology , Shock, Septic/prevention & control , Adrenalectomy , Animals , Ciliary Neurotrophic Factor , Humans , Lipopolysaccharides/toxicity , Lung/enzymology , Male , Metabolic Clearance Rate , Mice , Mice, Inbred BALB C , Nerve Tissue Proteins/pharmacokinetics , Nitrates/blood , Nitrites/blood , Peroxidase/metabolism , Receptor, Ciliary Neurotrophic Factor , Recombinant Proteins , Tissue Distribution , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
Autoimmunity and oxidative/excitotoxic damage are considered as possible pathogenetic mechanisms in amyotrophic lateral sclerosis (ALS). As tumor necrosis factor (TNF) is implicated in autoimmune diseases, including experimental autoimmune encephalomyelitis, and can be neurotoxic, we studied TNF production in a proposed animal model of ALS, the mnd mouse. These mice develop symptoms (progressive weakness of the limbs) as late as at 7 months of age. We measured TNF in serum, brain and spinal cord of mnd mice at 3 and 7 months of age. TNF was detectable in the brain and spinal cord (but not in the serum) at 7 months, while no TNF was detected in mnd mice at 3 months (asymptomatic) or in control mice of the same genetic background and the same age. Immunohistochemistry confirmed localization of TNF-alpha in motor neurons situated in the ventral horn of the spinal cord of 7-month old mnd mice. These results suggest the possibility of testing inhibitors of TNF production in this disease.
Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Motor Neurons/metabolism , Spinal Cord/metabolism , Tumor Necrosis Factor-alpha/metabolism , Animals , Disease Models, Animal , Immunohistochemistry , Male , Mice , Mice, Neurologic Mutants , Motor Neurons/pathologyABSTRACT
In previous work, we reported that chlorpromazine inhibits tumor necrosis factor (TNF) production in endotoxin lipopolysaccharide-treated mice, and protects against lipopolysaccharide toxicity. Chlorpromazine is used as an antipsychotic and has several effects on the central nervous system. It acts on different neurotransmitter receptors and has other biochemical activities some of which, like inhibition of phospholipase A2, might be responsible for the inhibitory effect on TNF production. To investigate the role of these actions in the inhibition of TNF production by chlorpromazine, we have synthesized some chlorpromazine derivatives that do not have central activities. Some of these analogs have lost their affinity for various receptors and their phospholipase A2 inhibitory activity, but still inhibit TNF production. No correlation was found between TNF inhibition and the ability to inhibit nitric oxide (NO) synthase, whereas a good correlation was evident between TNF inhibition and antioxidant activity.
Subject(s)
Antioxidants/pharmacology , Chlorpromazine/analogs & derivatives , Chlorpromazine/pharmacology , Tumor Necrosis Factor-alpha/biosynthesis , Animals , Blood Pressure/drug effects , Depression, Chemical , Enzyme Inhibitors/pharmacology , In Vitro Techniques , Lipopolysaccharides/pharmacology , Male , Mice , Mice, Inbred BALB C , Motor Activity/drug effects , Nitric Oxide Synthase/antagonists & inhibitors , Phospholipases A/antagonists & inhibitors , Phospholipases A2 , Rats , Rats, Wistar , Receptors, Neurotransmitter/drug effects , Receptors, Neurotransmitter/metabolism , SwineABSTRACT
The aim of this work was to study the relative role of the two TNF receptors (p55 and p75) in the central actions of TNF, studying the elevation of serum corticosterone (CS) and IL-6 levels after injection of recombinant murine (rm)TNF (intracerebroventricularly (i.c.v.)) in normal or p55-deficient (p55 -/-) mice. rmTNF induced high serum IL-6 levels and doubled serum CS in normal mice, whereas no elevation of serum IL-6 or CS was induced in p55 -/- mice. However, a normal CS response was observed in p55 -/- mice after LPS (2.5 microg, i.c.v.). p55 -/- mice also responded, although to a lesser extent than p55 +/+, in terms of LPS-induced IL-6 production. We also injected two agonist Abs specific for the two receptors, alpha p55 and alpha p75. While alpha p55 injected i.c.v. induced a marked elevation in CS and IL-6, alpha p75 induced CS (although less than alpha p55) but no IL-6. rmTNF, which binds both receptors, was more potent in inducing IL-6 and CS than injection of rhTNF, which in mice binds only p55. Finally, we investigated the role of p55 and p75 in IL-6 induction by TNF in a murine brain endothelioma. The results resembled closely those obtained in vivo: rmTNF was more potent than rhTNF and only alpha p55, and not alpha p75, induced IL-6 production. These data indicate that p55 plays a major role in TNF activation of the hypothalamus-pituitary-adrenal axis and in the centrally mediated induction of peripheral IL-6 by TNF, but p75, despite having little IL-6 inductive properties by itself, seems to potentiate p55 induction of IL-6.
Subject(s)
Corticosterone/blood , Interleukin-6/blood , Receptors, Tumor Necrosis Factor/physiology , Tumor Necrosis Factor-alpha/administration & dosage , Animals , Blood-Brain Barrier , Cells, Cultured , Cerebral Ventricles , Endothelium, Vascular/metabolism , Humans , Male , Mice , Mice, Inbred Strains , Recombinant ProteinsABSTRACT
Cardiotrophin-1 (CT-1) is a member of the gp130 family of cytokines that includes interleukin-6, interleukin-11, ciliary neurotrophic factor, leukemia inhibitory factor, and oncostatin M. As interleukin-6, leukemia inhibitory factor, and ciliary neurotrophic factor were previously reported to inhibit the production of tumor necrosis factor (TNF), we studied the effect of CT-1 on serum and heart TNF levels in mice treated with lipopolysaccharide (100 ng/mouse, iv). Co-treatment with CT-1 (5 micrograms/mouse intravenously) markedly inhibit TNF production both in serum and in the heart. The effect of CT-1 seems to be direct as it also inhibited TNF production when added to whole mouse blood cultured with lipopolysaccharide. Thus, CT-1 might play a protective role in some TNF-mediated diseases.
Subject(s)
Cytokines/blood , Cytokines/pharmacology , Heart/drug effects , Lipopolysaccharides/pharmacology , Myocardium/metabolism , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/biosynthesis , Animals , Cells, Cultured , Male , Mice , Mice, Inbred StrainsABSTRACT
Nitric oxide (NO) plays a key role in the pathophysiology of inflammation and sepsis. The regulation of the peripheral inducible NO synthase (iNOS-responsible for the massive NO synthesis in inflammation) has been extensively studied in sepsis, but little is known about the actual NO production and its dependence on the location of the primary stimulus (endotoxin, LPS). We measured the activation of the NO pathway after a central (intracerebroventricular) or systemic (intravenous) low dose of LPS (2.5 micrograms/mouse) in three ways: the accumulation of its stable end products (nitrites/nitrates) in the circulation, the induction of iNOS mRNA and the decrease in sodium nitroprusside-dependent ADP ribosylation of proteins in the liver and brain. Plasma nitrites/nitrates increased after LPS by either route. iNOS mRNA was induced in the liver after intravenous and, to a lower extent, in the brain after intracerebroventricular LPS. Ex vivo ADP ribosylation was decreased in both organs after both administration routes, although to different degrees (higher in the liver after intravenous and in the brain after intracerebroventricular administration), suggesting that NO had been produced in the periphery and in the brain after both routes of LPS administration, despite the fact that no LPS is expected to reach the brain after peripheral low-dose injection. Our data thus demonstrate a cross-talk between periphery and brain in the regulation of NO by LPS. Additionally, the possibility of iNOS-independent NO synthesis stimulated by LPS is implied by the discrepancy between the amount of local NO production suggested by ADP ribosylation and the iNOS mRNA levels.
Subject(s)
Lipopolysaccharides/administration & dosage , Lipopolysaccharides/pharmacology , Nitric Oxide/biosynthesis , Actins/drug effects , Actins/metabolism , Adenosine Diphosphate Ribose/metabolism , Animals , Blotting, Northern , Brain/drug effects , Brain/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/drug effects , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Injections, Intravenous , Injections, Intraventricular , Liver/drug effects , Liver/metabolism , Male , Mice , Mice, Inbred Strains , Nitrates/blood , Nitric Oxide/blood , Nitrites/blood , Nitroprusside/pharmacology , RNA, Messenger/biosynthesis , RNA, Messenger/drug effects , RNA, Messenger/genetics , Sulfhydryl Reagents/pharmacology , Time FactorsABSTRACT
The present study was designed to assess the effect of N,N-dimethyl-3-[(1-benzyl-1H-indazol-3-yl)ossi]-1-propana mine (benzydamine) on in vivo and in vitro production of inflammatory cytokines. Benzydamine inhibited tumour necrosis factor-alpha (TNF-alpha) production in vitro by human lipopolysaccharide-stimulated monocytes with an ED50 of approximately 25 microM (12 donors). Under the same conditions, benzydamine had modest or no effect on production of interleukin (IL-1), IL-6 and IL-8. Inhibition of TNF-alpha production was not restricted to LPS in that similar results were obtained using inactivated streptococci. Inhibition of TNF production was associated with a modest (about 30% at 50 microM, 7 donors) reduction of mRNA. A similar inhibition of TNF-alpha production was also detected with mouse peritoneal macrophages. With mouse cells benzydamine also substantially inhibited IL-1 production in vitro. In vivo treatment with benzydamine (40 mg/kg s.c.) protected mice against LPS lethality. Protection against septic shock was observed when benzydamine was administered before or concomitantly with LPS. Protection against LPS toxicity was associated with a marked reduction of serum levels of TNF-alpha and IL-1 beta, whereas IL-6 was unaffected. Inhibition of inflammatory cytokine production may play a role in the anti-inflammatory activity of benzydamine and provide suggestions for novel therapeutic applications.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Benzydamine/pharmacology , Cytokines/biosynthesis , Inflammation Mediators/antagonists & inhibitors , Animals , Blotting, Northern , Cells, Cultured , Cytokines/antagonists & inhibitors , Female , Humans , Lipopolysaccharides/antagonists & inhibitors , Lipopolysaccharides/toxicity , Macrophages, Peritoneal/metabolism , Mice , Mice, Inbred C3H , RNA, Messenger/biosynthesis , RNA, Messenger/metabolism , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
IL-1 plays an important role in the pathophysiologic responses to infection and inflammation, in part by mediating its own production and that of other proinflammatory cytokines. However, the relative contribution of IL-1 alpha and IL-1 beta to the inflammatory response has not been well clarified. Using IL-1 beta-deficient (IL-1 beta -/-) mice, we investigated the specific role of IL-1 beta in the in vivo and in vitro response to LPS. No differences between IL-1 beta +/+ and IL-1 beta -/- mice were observed in circulating levels for IL-1 alpha, IL-6, or TNF-alpha after the systemic administration of either a low (5 micrograms/kg) or high (5 mg/kg) dose of LPS. IL-1 beta -/- mice also had a normal response to LPS in terms of activation of the hypothalamus-pituitary-adrenal axis, hypoglycemia, serum amyloid A production, and anorexia. IL-1 beta -/- mice were normally sensitive to the lethal effect of LPS and were protected against LPS toxicity when pretreated with low-dose LPS. However, in vitro, peritoneal macrophages from IL-1 beta -/- mice stimulated with LPS produced significantly less IL-1 alpha than macrophages from IL-1 beta +/+ mice (p < 0.05). No differences in IL-6 or TNF-alpha synthesis were observed between macrophages from IL-1 beta +/+ and IL-1 beta -/- mice. In summary, our results suggest that either IL-1 beta is not essential for the in vivo systemic response to LPS or that its role can be fulfilled by other cytokines with overlapping activities.
Subject(s)
Interleukin-1/deficiency , Interleukin-1/genetics , Lipopolysaccharides/toxicity , Animals , Blood Glucose/drug effects , Blood Glucose/metabolism , Cells, Cultured , Corticosterone/blood , Cytokines/biosynthesis , Cytokines/drug effects , Female , Injections, Intraperitoneal , Interleukin-1/biosynthesis , Lethal Dose 50 , Lipopolysaccharides/antagonists & inhibitors , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Serum Amyloid A Protein/metabolismABSTRACT
Ciliary neurotrophic factor (CNTF) and interleukin-6 (IL-6) potentiate the elevation of serum corticosterone induced by suboptimal doses of interleukin-1 (IL-1). CNTF also potentiates IL-1-induced serum IL-6. Here, we report that four other cytokines (leukemia inhibitory factor [LIF], oncostatin M [OSM], interleukin-11 and cardiotrophin-1) also potentiated the elevation of serum corticosterone and IL-6 levels induced by IL-1. Furthermore, all the six cytokines studied induced the acute-phase protein serum amyloid A when administered alone. Because these cytokines differ both in structure and in function, but share gp130 as a subunit of their receptors, these results indicate that signaling through gp130 mediates potentiation of IL-1 activities. The potentiation of IL-1-induced serum corticosterone levels is not a consequence of the increased serum IL-6 observed after IL-1 administration. In fact, in IL-6 deficient mice, IL-1 increased serum corticosterone to a level comparable to that observed in wild-type mice. Thus, either endogenous IL-6 does not mediate IL-1-induced corticosterone increase, or its role may be fulfilled by other cytokines. To the extent that gp130-dependent cytokines may serve this role, they may be important feedback regulators of inflammation through the activation of the hypothalamus-pituitary-adrenal axis and the potentiation of acute-phase protein synthesis.
Subject(s)
Antigens, CD/physiology , Corticosterone/metabolism , Cytokines/pharmacology , Membrane Glycoproteins/physiology , Receptors, Cytokine/chemistry , Signal Transduction/drug effects , Acute-Phase Reaction , Animals , Antigens, CD/chemistry , Ciliary Neurotrophic Factor , Cytokine Receptor gp130 , Cytokines/metabolism , Drug Synergism , Growth Inhibitors/metabolism , Growth Inhibitors/pharmacology , Hypothalamo-Hypophyseal System/drug effects , Hypothalamo-Hypophyseal System/metabolism , Interleukin-1/metabolism , Interleukin-1/pharmacology , Interleukin-11/metabolism , Interleukin-11/pharmacology , Interleukin-6/metabolism , Interleukin-6/pharmacology , Leukemia Inhibitory Factor , Liver/drug effects , Liver/metabolism , Lymphokines/metabolism , Lymphokines/pharmacology , Male , Membrane Glycoproteins/chemistry , Mice , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/pharmacology , Oncostatin M , Peptides/metabolism , Peptides/pharmacology , Pituitary-Adrenal System/drug effects , Pituitary-Adrenal System/metabolism , Receptors, Cytokine/drug effects , Receptors, Cytokine/genetics , Recombinant Proteins/pharmacology , Serum Amyloid A Protein/metabolismABSTRACT
Glucocorticoids (GC) are known inhibitors of TNF production and are increased by endotoxin (LPS) through a stimulation of the hypothalamus-pituitary-adrenal axis (HPAA), suggesting a feedback mechanism. We tried different approaches to study the role of the HPAA and endogenous GC in the regulation of TNF production. Cyanoketone, a GC synthesis inhibitor, inhibited corticosterone (CS) induction by LPS and increased LPS-induced serum TNF levels. Similar results were obtained by pretreating mice with anticorticotropin-releasing hormone Abs. Administration of adrenocorticotropic hormone increased blood CS and inhibited LPS-induced serum TNF. TNF production by mouse blood stimulated in vitro with LPS was inhibited by addition of CS. Blood from stressed or adrenocorticotropic-releasing hormone-treated mice (in which CS levels are elevated) stimulated ex vivo with LPS produced significantly less TNF than blood from control mice. Normal TNF production was restored by the addition of the GC receptor antagonist RU 38486, indicating a role for the elevated endogenous CS levels in the inhibition of TNF production. These data indicate that the HPAA is a major regulator of TNF production.