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Inhibition of peripheral NF-κB activation by central action of α-melanocyte-stimulating hormone
Journal of Neuroimmunology 99 Ž1999. 211–217 www.elsevier.comrlocaterjneuroim
Inhibition of peripheral NF-kB activation by central action of a-melanocyte-stimulating hormone Takashi Ichiyama a,b, Tetsuhiro Sakai c , Anna Catania d , Gregory S. Barsh e, Susumu Furukawa b, James M. Lipton a,) a
Department of Physiology, UniÕersity of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines BlÕd., Dallas, TX 75235-9040, USA b Department of Pediatrics, Yamaguchi UniÕersity School of Medicine, Ube 755-8505, Japan c Department of Anesthesiology, UniÕersity of Hirosaki School of Medicine, Hirosaki 036-8562, Japan d III DiÕision of Internal Medicine, IRCCS Ospedale Maggiore, 20122 Milan, Italy e Department of Pediatrics, Stanford UniÕersity School of Medicine, Howard Hughes Medical Institute, Stanford, CA 94305-5428, USA Received 26 January 1999; received in revised form 8 June 1999; accepted 9 June 1999
Abstract With the rise in the field of neuroimmunomodulation research, there is increased recognition of the influence of the nervous system and neuropeptides in peripheral disease. The neuropeptide a-melanocyte-stimulating hormone Ž a-MSH. is a neuroimmunomodulatory agent that modulates production of proinflammatory cytokines and inhibits peripheral inflammation via actions on CNS receptors. We examined whether central a-MSH operates by inhibiting activation of the nuclear factor kappa B ŽNF-kB. that is essential to the expression of proinflammatory cytokines and development of inflammation in the periphery. Electrophoretic mobility shift assays of nuclear extracts from the murine foot pad injected with TNF-a demonstrated that centrally administered a-MSH does inhibit NF-kB activation. Western blot analysis revealed that this inhibition was linked to central a-MSH-induced preservation of expression of Ik Ba protein in the peripheral tissue. The NF-kB and Ik Ba effects were inhibited in mice with spinal cord transection. Intraperitoneal Žip. injection of the nonspecific b-adrenergic receptor blocker propranolol, and of a specific b 2-adrenergic receptor antagonist, likewise prevented these effects of central a-MSH; blockade of cholinergic, a-adrenergic, or b 1-adrenergic receptors did not. Centrally administered a-MSH inhibited peripheral NF-kB activation and Ik Ba degradation even in mice with nonfunctional melanocortin 1 receptors ŽMC1R.. These findings indicate that a-MSH can act centrally to inhibit NF-kB activation in peripheral acute inflammation via a descending neural pathway. The pathway involves b 2-adrenergic receptors, but does not require activation of MC1R within the brain. q 1999 Elsevier Science B.V. All rights reserved. Keywords: a-Melanocyte-stimulating hormone; NF-kB; Inflammation; Melanocortin receptor; Spinal cord transection; b 2 -Adrenoreceptor
1. Introduction The neuropeptide a-melanocyte-stimulating hormone Ž a-MSH. exerts anti-inflammatory effects through inhibition of production and action of proinflammatory cytokines ŽLipton and Catania, 1997, 1998.. a-MSH is a pro-opiomelanocortin derivative which shares the 1–13 amino acid sequence with adrenocorticotropic hormone; this peptide occurs in the pituitary, brain, skin, circulation, and other sites ŽCatania and Lipton, 1993.. a-MSH is a melanocortin and five melanocortin receptor subtypes have been identified ŽMC1R through MC5R. ŽChhajlani and ) Corresponding author. Tel.: q1-214-648-2357; fax: q1-214-6484703; E-mail: [email protected] ŽJ. Lipton.
Wikberg, 1992; Mountjoy et al., 1992; Gantz et al., 1993a,b; Barrett et al., 1994.. It is thought that the local anti-inflammatory effects of a-MSH are mediated through MC1R on neutrophils, monocytermacrophages, and on brain cells ŽCatania et al., 1996; Rajora et al., 1996, 1997; Wong et al., 1997.. In addition, a-MSH exerts an anti-inflammatory effect on peripheral inflammation through descending neural pathways ŽLipton et al., 1991; Ceriani et al., 1994; Macaluso et al., 1994.. It has not been established which receptors mediate the central a-MSH-induced inhibition of peripheral inflammation. Nuclear factor kappa B ŽNF-kB. is a pivotal transcription factor for genes that encode proinflammatory cytokines such as IL-1, IL-6, IL-8, and TNF-a ŽCollart et al., 1990; Libermann and Baltimore, 1990; Hiscott et al., 1993;
0165-5728r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 9 9 . 0 0 1 2 2 - 8
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Matsusaka et al., 1993; Kunsch et al., 1994; Roulston et al., 1995.. The primary form of NF-kB consists of a heterodimer of p50 and p65 that is bound by members of the Ik B family, including Ik Ba , in cytoplasm ŽBaeuerle and Henkel, 1994; Baldwin, 1996.. NF-kB activation requires degradation of Ik Ba protein ŽGrimm and Baeuerle, 1993; Henkel et al., 1993.. Phosphorylation of Ik Ba by drugs, cytokines, bacterial products, and viruses leads to Ik Ba degradation, translocation of NF-kB to the nucleus, and transcription of proinflammatory cytokine genes ŽKumar et al., 1994; Brown et al., 1995.. a-MSH inhibits NF-kB activation and Ik Ba degradation in cultured cells ŽManna and Aggarwal, 1998.. We tested the hypothesis that central a-MSH modulates peripheral acute inflammation by inhibiting NF-kB activation through descending neural pathways stemming from MC1R within the brain.
2. Materials and methods 2.1. Animals These experiments were approved by the local Internal Review Board for Animal Research. Male C57BLr6 mice ŽSimonsen Laboratories, Gilroy, CA. were housed in a 24 " 18C environment in groups not exceeding five animals per cage Ž28 = 18 = 13 cm3 .. They were allowed to acclimatize to standard lighting and temperature conditions with food and water freely available before experiments began. Mice with nonfunctional MC1R ŽMC-1rerMC-1re. originated from stock bred by Dr. G. S. Barsh of Stanford University School of Medicine ŽStanford, CA.. Characterization of these mice was described previously ŽRobbins et al., 1993.. In brief, the mice have nonfunctional MC1R resulting from a frameshift mutation between the fourth and fifth transmembrane domains. 2.2. Experimental protocol Mice were anesthetized with 10% pentobarbital sodium solution Ž1 mgrmouse, 50 mgrml, Nembutal sodium solution; Abbott Laboratories, North Chicago, IL.. Animals received an intracerebroventricular Žicv. injection of a-MSH Ž40 mg dissolved in 20 ml saline., or 20 ml saline using techniques previously described ŽHaley and McCormick, 1957.. Recombinant human TNF-a ŽR & D systems, Minneapolis, MN. dissolved in saline Ž20 ng, 20 ml. was injected into one rear foot pad and saline Ž20 ml. was injected into the other using 28-gauge allergy test syringes ŽSherwood, St. Louis, MO.. Spinal cord transection was induced by a standardized crushing of lumbar vertebrae with a hemostat ŽMacaluso et al., 1994.. In the experiments proper, mice were tested after cord section when the anesthesia had worn off: none
showed behavioral or motor responses to pinching of the hind feet with a hemostat; all had complete paralysis of the hind limbs. Several agents were injected intraperitoneally Žip. to pharmacologically block certain receptors: atropine Ž300 mg; Sigma, St. Louis, MO., a muscarinic receptor blocker; phentolamine Ž300 mg; Sigma., an agent that competes for occupancy of a-adrenergic receptors; propranolol Ž300 mg; Sigma., a nonspecific competitive antagonist for badrenergic receptors; atenolol Ž300 mg; Sigma., a b 1adrenergic receptor antagonist; butoxamine Ž300 mg; Sigma., a b 2-adrenergic receptor blocker. At the indicated time points, foot pads were surgically removed and immediately frozen in liquid nitrogen. 2.3. Nuclear extracts Nuclear extracts were harvested from frozen foot pads using a previously published procedure ŽDeryckere and Gannon, 1994.. The protein concentration of the supernatant containing nuclear extracts was determined using Bio-Rad ŽHercules, CA. protein concentration reagent. Nuclear extracts were stored at y808C. 2.4. Electrophoretic Mobility Shift Assay (EMSA) Double-stranded oligonucleotides for the consensus binding sites of NF-kB Ž5X-AGTTGAGGGGACTTTCCCAGGC-3X ; Promega, Madison, WI. were labeled using Klenow fragment ŽLife Technologies, Gaithersburg, MD. in the presence of 100 mCi of w a-32 PxdCTP Ž3000 Cirmmol; NEN, Boston, MA. and unlabeled dATP, dGTP, and dTTP at 100 mM in a final volume of 50 ml. Unincorporated nucleotides were removed using a G-25 ŽPharmacia, Piscataway, NJ. spin column. Five micrograms of nuclear proteins were preincubated with binding buffer Ž50 mgrml polywdI–dCx, 5% glycerol, 10 mM Tris–HCl, pH 7.8, 1 mM EDTA, 40 mM KCl, and 1 mM DTT. on ice for 15 min before addition of the radiolabeled oligonucleotide probe and incubation at room temperature for 20 min. Specificity of the binding reaction was determined in competition assays by co-incubating with 100-fold molar excess of unlabeled NF-kB or AP-1 consensus oligonucleotide ŽPromega., and by supershift assay with polyclonal rabbit anti-NF-kB-p50 and anti-NF-kB-p65 antibody ŽSanta Cruz Biotechnology, Santa Cruz, CA.. Reaction products were separated in a 4% polyacrylamide gel. Dried gels were analyzed by autoradiography. Autoradiograms were analyzed by densitometry ŽBio-Rad.. 2.5. Western blot analysis We followed a modification of a previously described procedure ŽLentsch et al., 1997.. Frozen foot pads were homogenized in lysis buffer Ž10 mM HEPES, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, 0.5 mM PMSF,
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Fig. 1. Representative EMSA demonstrating effect of a-MSH on NF-kB activation 0.5, 1, and 3 h after intradermal injection of TNF-a Ž20 ng in 20 ml saline. into foot pad of the mouse. NF-kB activation was induced by TNF-a, but not saline Ž20 ml., in mice given icv saline. Centrally administered a-MSH Ž40 mg in 20 ml saline. inhibited NF-kB activation at each indicated time. Ss injection of saline into the foot pad with centrally administered saline; T s injection of TNF-a into the foot pad with centrally administered saline; TqMs injection of TNF-a into the foot pad with centrally administered a-MSH.
and 1.0 mgrml each of the following protease inhibitors: aprotinin, antipain, leupeptin, chymostatin, and pepstatin A. and were centrifuged at 4000 rpm for 2 min. Supernatants were transferred to 1.5-ml tubes. Protein concentrations were determined with BCA assay ŽPierce, Rockford, IL.. Samples Ž20 mg total protein. were separated in a denaturing 10% polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. After four additional washes in TBST Ž40 mM Tris–HCl, pH 7.6, 300 mM NaCl, and 0.5% Tween 20., membranes were incubated in a 1:1000 dilution of rabbit polyclonal anti-Ik Ba ŽSanta Cruz Biotechnology. in TBST containing 5% nonfat dry milk at room temperature for 2 h. After four washes in TBST, membranes were incubated in a 1:5000 dilution of horseradish peroxidase-conjugated donkey anti-rabbit IgG ŽAmersham, Arlington Heights, IL. for 1 h at room temperature. After four washes in TBST, immunoreactive
Fig. 2. Representative EMSA indicating specificity of NF-kB oligonucleotide in experiments on nuclear extract from murine foot pad 1 h after TNF-a injection. Control Žlane 1.; gel shift band disappeared in competition assays when 100-fold excess unlabeled NF-kB oligonucleotide was added Žlane 2.; unlabeled AP-1 oligonucleotide had no effect Žlane 3.; co-incubation of nuclear extracts with anti-NF-kB-p50 polyclonal antibody Žlane 4. or with anti-NF-kB-p65 polyclonal antibody Žlane 5. caused supershift of the NF-kBrDNA complex.
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Fig. 3. Representative EMSA showing effect of spinal cord transection on a-MSH-induced inhibition of NF-kB activation induced by TNF-a. In mice with spinal cord transection, centrally administered a-MSH had no effect on TNF-a-induced NF-kB activation of foot pad at each indicated time.
proteins were detected using enhanced chemiluminescence ŽAmersham. and analyzed by autoradiography.
3. Results 3.1. Inhibition by centrally administered a-MSH of NF-k B actiÕation in the periphery EMSA of nuclear extracts from foot pads injected with TNF-a demonstrated NF-kB activation ŽFig. 1.. NF-kB activation was observed throughout the 3-h test period in
Fig. 4. Effect of pharmacological blockade on inhibition of NF-kB activation by centrally administered a-MSH. All samples taken 3 h after TNF-a injected into the foot pad. ŽA. Intraperitoneal injection of a nonspecific b-blocker propranolol Ž300 mg. inhibited the suppressive effect of a-MSH on NF-kB activation by TNF-a. A muscarinic receptor blocker atropine Ž300 mg. or an agent that competes for occupancy of a-adrenergic receptors phentolamine Ž300 mg. did not alter the effects of a-MSH. ŽB. Blockade of b 2-adrenoreceptor with butoxamine Ž300 mg. inhibited the suppressive effect of a-MSH on NF-kB activation, but blockade of b1 -adrenergic receptor with atenolol Ž300 mg. did not.
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Fig. 5. Representative Western blot demonstrating effect of centrally administered a-MSH on TNF-a-induced Ik Ba degradation in the murine foot pad. Expression of Ik Ba protein decreased 1 h after injection of TNF-a Ž20 ng in 20 ml saline.. Centrally administered a-MSH Ž40 mg in 20 ml saline. preserved expression of Ik Ba protein.
the TNF-a-treated foot pads of mice given central injection of saline. The gel shift bands of nuclear extracts from the foot pads injected with TNF-a were reduced by centrally administered a-MSH ŽFig. 1.. Densitometry showed an inhibition of approximately 40%. Specificity of the NF-kB consensus oligonucleotide probe was confirmed in experiments on nuclear extracts from the foot pads stimulated with TNF-a for 1 h. The intensity of the gel shift band was markedly diminished in the presence of 100-fold molar excess of unlabeled NF-kB oligonucleotide, but not by a 100-fold molar excess of unlabeled AP-1 oligonucleotide ŽFig. 2.. Assays with anti-NF-kB-p50 and anti-NFk B-p65 antibody revealed supershift of the gel shift band, confirming the identity and specificity of the band ŽFig. 2.. 3.2. Influence of spinal cord transection and drugs on central a-MSH-induced inhibition of peripheral NF-k B actiÕation Spinal cord transection abolished the inhibitory effect of centrally administered a-MSH on NF-kB activation induced by TNF-a injection into the foot pads ŽFig. 3.. In experiments designed to test the contribution of certain neurotransmitter receptors to the effect of centrally administered a-MSH, propranolol, a nonspecific b-blocker propranolol suppressed the inhibitory effects of a-MSH on NF-kB activation. However, a muscarinic receptor blocker Žatropine., or an agent that competes for occupancy of a-adrenergic receptors Žphentolamine., did not ŽFig. 4A.. After positive effects were observed with propranolol, selective antagonists of b-adrenoreceptors were tested. Blockade of b 2-adrenergic receptor with butoxamine inhibited the effect of a-MSH on NF-kB activation; block-
Fig. 6. Effect of spinal cord transection on centrally administered aMSH-induced protection of Ik Ba degradation induced by TNF-a. In mice with spinal cord transection, centrally administered a-MSH had no effect on Ik Ba degradation in the foot pad 1 h after local injection of TNF-a.
Fig. 7. Effect of pharmacological blockade on centrally administered a-MSH-induced preservation of Ik Ba degradation 1 h after local injection of TNF-a. Intraperitoneal injection of a nonspecific b-blocker propranolol Ž300 mg. or b 2-adrenoreceptor antagonist butoxamine Ž300 mg. inhibited the effect of a-MSH on Ik Ba protein. Other agents; a muscarinic receptor blocker Žatropine, 300 mg., an agent that competes for occupancy of a-adrenergic receptors Žphentolamine, 300 mg. or a b1 -adrenoreceptor antagonist Žatenolol, 300 mg., did not.
ade of b 1-adrenergic receptor with atenolol was ineffective ŽFig. 4B.. 3.3. Restriction of Ik Ba degradation in the foot pads by centrally administered a-MSH Western blot analysis of extracts of foot pads from mice given icv saline revealed that expression of Ik Ba protein decreased 1 h after TNF-a injection, but returned 1.5 h after. Centrally administered a-MSH protected the expression of Ik Ba protein at 1 h after TNF-a injection ŽFig. 5.. 3.4. Influence of a-MSH effect on Ik Ba degradation by spinal cord transection and drugs In mice with spinal cord transection, centrally administered a-MSH did not prevent characteristic degradation of Ik Ba protein in the foot pads 1 h after TNF-a injection
Fig. 8. EMSA from nuclear extract of foot pads of MC-1rerMC-1re mice demonstrated that centrally administered a-MSH inhibited NF-kB activation induced by TNF-a much as in normal mice Žsee Fig. 1..
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Fig. 9. Western blot analysis of extract of foot pads of MC-1rerMC-1re mice indicated that centrally administered a-MSH protected expression of Ik Ba protein 1 h after TNF-a injection.
ŽFig. 6.. In experiments on the contribution of certain neurotransmitter receptors to the effect of centrally administered a-MSH, ip administration of propranolol and of butoxamine suppressed the inhibitory effects of a-MSH on Ik Ba degradation; injection of a muscarinic receptor blocker Žatropine., an a-adrenoreceptor antagonist Žphentolamine., or a b 1-adrenoreceptor antagonist Žatenolol. did not ŽFig. 7.. 3.5. Effects of a-MSH on NF-k B actiÕation and Ik Ba degradation in MC-1rer MC-1re mice In MC-1rerMC-1re mice, centrally administered aMSH inhibited NF-kB activation induced by TNF-a in the foot pad throughout the experimental period, much as in control mice ŽFig. 8.. Densitometry showed an inhibition of approximately 40%. In these mice, centrally administered a-MSH also preserved expression of Ik Ba protein in the foot pads 1 h after TNF-a injection, a pattern similar to that in normal mice ŽFig. 9..
4. Discussion Inflammation is characteristic in many diseases of the periphery including arthritis, inflammatory bowel disease, asthma, and vascular disease. Therapy for these disorders has not progressed rapidly and perhaps a new avenue for control via agents that act on the CNS will be beneficial. Our results suggest that targeting CNS a-MSH receptors could improve peripheral inflammation. Our data demonstrate that centrally administered aMSH inhibits NF-kB activation and Ik Ba degradation in peripheral acute inflammation. Intact descending neural pathways are necessary for this influence of centrally administered a-MSH, but the specific melanocortin receptor subtype MC1R within the brain is not. In addition, our experiments on pharmacological blockade of peripheral neural transmission revealed that b 2-adrenergic neurotransmission is essential to the effect of central a-MSH on peripheral acute inflammation. Our previous observations indicated that central a-MSH modulates peripheral inflammation ŽLipton et al., 1991; Ceriani et al., 1994; Macaluso et al., 1994.. a-MSH given icv inhibited ear edema of mice induced by topical picryl chloride ŽLipton et al., 1991.. Centrally administered aMSH likewise suppressed ear edema induced by intrader-
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mal injection of IL-1b, IL-8, leukotriene B4, and plateletactivating factor ŽCeriani et al., 1994; Macaluso et al., 1994.. Our present data suggest a molecular mechanism underlying these prior observations. b-Adrenergic agonists are known to have anti-inflammatory effects ŽGreen, 1972; Maling et al., 1974; Svensjo¨ et al., 1977; Inagaki et al., 1989.. Catecholamines acting predominantly on b-adrenergic receptors inhibited increased permeability of the mouse peritoneum induced by local injection of acetic acid ŽGreen, 1972.. b-Adrenoreceptor stimulants inhibited hind paw edema induced by histamine, serotonin, bradykinin or compound 48r80 ŽMaling et al., 1974. and also inhibited bradykinin-induced macromolecular leakage ŽSvensjo¨ et al., 1977.. Further, such stimulants inhibited increased vascular permeability induced by mediators of allergic reactions ŽInagaki et al., 1989.. Our previous data demonstrated that the inhibitory effects of centrally administered a-MSH on hind paw edema induced in the mouse by kappa carrageenan were inhibited by a nonspecific b-adrenergic receptor blocker and a specific b 2-adrenergic receptor antagonist ŽMacaluso et al., 1994.. Superoxide generation by neutrophils was down-regulated by b-adrenergic stimulation ŽBarnett et al., 1997.. Catecholamines inhibited TNF production during concomitant LPS stimulation through b-adrenoreceptor activation ŽGuirao et al., 1997.. Further, TNF and IL-6 promoter activities induced by LPS were inhibited by b 2-adrenoreceptor activation ŽNakamura et al., 1998.. These findings and our observations with butoxamine strongly indicate that b 2-adrenergic receptors are important in the modulation of inflammation. a-MSH modulates inflammation by inhibiting production of proinflammatory cytokines, nitric oxide, prostaglandin E2, and neopterin ŽWeiss et al., 1991; Star et al., 1995; Weidenfeld et al., 1995; Rajora et al., 1996, 1997; Wong et al., 1997.. Recent research indicated that NF-kB activation and Ik Ba degradation were inhibited by a-MSH in various cells ŽManna and Aggarwal, 1998.. Elevations in cyclic AMP inhibit NF-kB-mediated transcription in human monocytic cells and endothelial cells ŽOllivier et al., 1996. and cyclic AMP accumulation is known to be stimulated by b 2-adrenoreceptor agonists ŽRoberts and Summers, 1998.. We therefore suggest that centrally administered a-MSH acts to inhibit transcriptional activation of NF-kB in peripheral inflammation through b 2-adrenoreceptor-mediated increases in intracellular cyclic AMP level. Melanocortin receptors MC1R, MC3R, MC4R, and MC5R have been identified in brain tissue ŽLipton and Catania, 1997.. It was believed that a-MSH exerts its local anti-inflammatory action through MC1R on peripheral cells, but we demonstrated that MC1R in the brain is not required for centrally administered a-MSH to exert its anti-inflammatory action through descending neural pathways. Centrally and systemically administered a-MSH suppressed LPS-induced fever, but not plasma levels of
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IL-6 or corticosterone, through action on MC3R and MC4R within the brain ŽHuang et al., 1997, 1998.. It is therefore possible that several receptor subtypes mediate the influence of centrally administered a-MSH on peripheral inflammation. 5. Summary Our data indicate that a-MSH injected centrally can restrict Ik Ba degradation and subsequent NF-kB activation in peripheral acute inflammation. Spinal cord transections revealed that an intact descending neural pathway is necessary for centrally administered a-MSH to exert these effects. Pharmacological studies indicate that b 2-adrenoreceptors in the periphery are likewise important in the anti-inflammatory pathway activated by centrally administered a-MSH. Experiments on MC-1rerMC-1re mice demonstrated that the anti-inflammatory effect of centrally administered a-MSH on peripheral inflammation does not depend solely on mediation by MC1R within the brain. References Baeuerle, P.A., Henkel, T., 1994. Function and activation of NF-kB in the immune system. Annu. Rev. Immunol. 12, 141–179. Baldwin, A.S. Jr., 1996. The NF-kB and Ik B proteins: new discoveries and insights. Annu. Rev. Immunol. 14, 649–683. Barnett, C.C. Jr., Moore, E.E., Partrick, D.A., Silliman, C.C., 1997. b-Adrenergic stimulation down-regulates neutrophil priming for superoxide generation, but not elastase release. J. Surg. Res. 70, 166– 170. Barrett, P., MacDonald, A., Helliwell, R., Davidson, G., Morgan, P., 1994. Cloning and expression of a new member of the melanocytestimulating hormone receptor family. J. Mol. Endocrinol. 12, 203–213. Brown, K., Gerstberger, S., Carlson, L., Franzoso, G., Siebenlist, U., 1995. Control of Ik B-a proteolysis by site-specific, signal-induced phosphorylation. Science 267, 1485–1488. Catania, A., Lipton, J.M., 1993. a-Melanocyte stimulating hormone in the modulation of host reactions. Endocr. Rev. 14, 564–576. Catania, A., Rajora, N., Capsoni, F., Minonzio, F., Star, R.A., Lipton, J.M., 1996. The neuropeptide a-MSH has specific receptors on neutrophils and reduces chemotaxis in vitro. Peptides 17, 675–679. Ceriani, G., Macaluso, A., Catania, A., Lipton, J.M., 1994. Central neurogenic antiinflammatory action of a-MSH: modulation of peripheral inflammation induced by cytokines and other mediators of inflammation. Neuroendocrinology 59, 138–143. Chhajlani, V., Wikberg, J.E., 1992. Molecular cloning and expression of the human melanocyte stimulating hormone receptor cDNA. FEBS Lett. 309, 417–420. Collart, M.A., Baeuerle, P., Vassalli, P., 1990. Regulation of tumor necrosis factor alpha transcription in macrophages: involvement of four k B-like motifs and of constitutive and inducible forms of NF-kB. Mol. Cell. Biol. 10, 1498–1506. Deryckere, F., Gannon, F., 1994. A one-hour minipreparation technique for extraction of DNA-binding proteins from animal tissues. Biotechniques 16, 405. Gantz, I., Konda, Y., Tashiro, T., Shimoto, Y., Miwa, H., Munzert, G., Watson, S.J., DelValle, J., Yamada, T., 1993a. Molecular cloning of a novel melanocortin receptor. J. Biol. Chem. 268, 8246–8250. Gantz, I., Miwa, H., Konda, Y., Shimoto, Y., Tashiro, T., Watson, S.J., DelValle, J., Yamada, T., 1993b. Molecular cloning, expression, and
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T. Ichiyama et al.r Journal of Neuroimmunology 99 (1999) 211–217 Matsusaka, T., Fujikawa, K., Nishio, Y., Mukaida, N., Matsushima, K., Kishimoto, T., Akira, S., 1993. Transcription factors NF-IL6 and NF-kB synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8. Proc. Natl. Acad. Sci. U. S. A. 90, 10193–10197. Mountjoy, K.G., Robbins, L.S., Mortrud, M.T., Cone, R.D., 1992. The cloning of a family of genes that encode the melanocortin receptors. Science 257, 1248–1251. Nakamura, A., Johns, E.J., Imaizumi, A., Abe, T., Kohsaka, T., 1998. Regulation of tumour necrosis factor and interleukin-6 gene transcription by b 2 -adrenoceptor in the rat astrocytes. J. Neuroimmunol. 88, 144–153. Ollivier, V., Parry, G.C.N., Cobb, R.R., de Prost, D., Mackman, N., 1996. Elevated cyclic AMP inhibits NF-kB-mediated transcription in human monocytic cells and endothelial cells. J. Biol. Chem. 271, 20828–20835. Rajora, N., Ceriani, G., Catania, A., Star, R.A., Murphy, M.T., Lipton, J.M., 1996. a-MSH production, receptors, and influence on neopterin in a human monocytermacrophage cell line. J. Leukocyte Biol. 59, 248–253. Rajora, N., Boccoli, G., Burns, D., Sharma, S., Catania, A.P., Lipton, J.M., 1997. a-MSH modulates local and circulating tumor necrosis factor-a in experimental brain inflammation. J. Neurosci. 17, 2181– 2186. Robbins, L.S., Nadeau, J.H., Johnson, K.R., Kelly, M.A., Roselli-Rehfuss, L., Baack, E., Mountjoy, K.G., Cone, R.D., 1993. Pigmentation phenotypes of variant extension locus alleles result from point mutations that alter MSH receptor function. Cell 72, 827–834.
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Roberts, S.J., Summers, R.J., 1998. Cyclic AMP accumulation in rat soleus muscle: stimulation by b 2 - but not b 3-adrenoceptors. Eur. J. Pharmacol. 348, 53–60. Roulston, A., Lin, R., Beauparlant, P., Wainberg, M.A., Hiscott, J., 1995. Regulation of human immunodeficiency virus type 1 and cytokine gene expression in myeloid cells by NF-kBrRel transcription factors. Microbiol. Rev. 59, 481–505. Star, R.A., Rajora, N., Huang, J., Stock, R.C., Catania, A., Lipton, J.M., 1995. Evidence of autocrine modulation of macrophage nitric oxide synthase by a-melanocyte-stimulating hormone. Proc. Natl. Acad. Sci. U. S. A. 92, 8016–8020. Svensjo, ¨ E., Persson, C.G.A., Rutili, G., 1977. Inhibition of bradykinin induced macromolecular leakage from post-capillary venules by a b 2-adrenoreceptor stimulant, terbutaline. Acta Physiol. Scand. 101, 504–506. Weidenfeld, J., Crumeyrolle-Arias, M., Haour, F., 1995. Effect of bacterial endotoxin and interleukin-1 on prostaglandin biosynthesis by the hippocampus of mouse brain: role of interleukin-1 receptors and glucocorticoids. Neuroendocrinology 62, 39–46. Weiss, J.M., Sundar, S.K., Cierpial, M.A., Ritchie, J.C., 1991. Effects of interleukin-1 infused into brain are antagonized by a-MSH in a dose-dependent manner. Eur. J. Pharmacol. 192, 177–179. Wong, K.Y., Rajora, N., Boccoli, G., Catania, A., Lipton, J.M., 1997. A potential mechanism of local anti-inflammatory action of alphamelanocyte-stimulating hormone within the brain: modulation of tumor necrosis factor-alpha production by human astrocytic cells. Neuroimmunomodulation 4, 37–41.
Inhibition of peripheral NF-kB activation by central action of a-melanocyte-stimulating hormone Takashi Ichiyama a,b, Tetsuhiro Sakai c , Anna Catania d , Gregory S. Barsh e, Susumu Furukawa b, James M. Lipton a,) a
Department of Physiology, UniÕersity of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines BlÕd., Dallas, TX 75235-9040, USA b Department of Pediatrics, Yamaguchi UniÕersity School of Medicine, Ube 755-8505, Japan c Department of Anesthesiology, UniÕersity of Hirosaki School of Medicine, Hirosaki 036-8562, Japan d III DiÕision of Internal Medicine, IRCCS Ospedale Maggiore, 20122 Milan, Italy e Department of Pediatrics, Stanford UniÕersity School of Medicine, Howard Hughes Medical Institute, Stanford, CA 94305-5428, USA Received 26 January 1999; received in revised form 8 June 1999; accepted 9 June 1999
Abstract With the rise in the field of neuroimmunomodulation research, there is increased recognition of the influence of the nervous system and neuropeptides in peripheral disease. The neuropeptide a-melanocyte-stimulating hormone Ž a-MSH. is a neuroimmunomodulatory agent that modulates production of proinflammatory cytokines and inhibits peripheral inflammation via actions on CNS receptors. We examined whether central a-MSH operates by inhibiting activation of the nuclear factor kappa B ŽNF-kB. that is essential to the expression of proinflammatory cytokines and development of inflammation in the periphery. Electrophoretic mobility shift assays of nuclear extracts from the murine foot pad injected with TNF-a demonstrated that centrally administered a-MSH does inhibit NF-kB activation. Western blot analysis revealed that this inhibition was linked to central a-MSH-induced preservation of expression of Ik Ba protein in the peripheral tissue. The NF-kB and Ik Ba effects were inhibited in mice with spinal cord transection. Intraperitoneal Žip. injection of the nonspecific b-adrenergic receptor blocker propranolol, and of a specific b 2-adrenergic receptor antagonist, likewise prevented these effects of central a-MSH; blockade of cholinergic, a-adrenergic, or b 1-adrenergic receptors did not. Centrally administered a-MSH inhibited peripheral NF-kB activation and Ik Ba degradation even in mice with nonfunctional melanocortin 1 receptors ŽMC1R.. These findings indicate that a-MSH can act centrally to inhibit NF-kB activation in peripheral acute inflammation via a descending neural pathway. The pathway involves b 2-adrenergic receptors, but does not require activation of MC1R within the brain. q 1999 Elsevier Science B.V. All rights reserved. Keywords: a-Melanocyte-stimulating hormone; NF-kB; Inflammation; Melanocortin receptor; Spinal cord transection; b 2 -Adrenoreceptor
1. Introduction The neuropeptide a-melanocyte-stimulating hormone Ž a-MSH. exerts anti-inflammatory effects through inhibition of production and action of proinflammatory cytokines ŽLipton and Catania, 1997, 1998.. a-MSH is a pro-opiomelanocortin derivative which shares the 1–13 amino acid sequence with adrenocorticotropic hormone; this peptide occurs in the pituitary, brain, skin, circulation, and other sites ŽCatania and Lipton, 1993.. a-MSH is a melanocortin and five melanocortin receptor subtypes have been identified ŽMC1R through MC5R. ŽChhajlani and ) Corresponding author. Tel.: q1-214-648-2357; fax: q1-214-6484703; E-mail: [email protected] ŽJ. Lipton.
Wikberg, 1992; Mountjoy et al., 1992; Gantz et al., 1993a,b; Barrett et al., 1994.. It is thought that the local anti-inflammatory effects of a-MSH are mediated through MC1R on neutrophils, monocytermacrophages, and on brain cells ŽCatania et al., 1996; Rajora et al., 1996, 1997; Wong et al., 1997.. In addition, a-MSH exerts an anti-inflammatory effect on peripheral inflammation through descending neural pathways ŽLipton et al., 1991; Ceriani et al., 1994; Macaluso et al., 1994.. It has not been established which receptors mediate the central a-MSH-induced inhibition of peripheral inflammation. Nuclear factor kappa B ŽNF-kB. is a pivotal transcription factor for genes that encode proinflammatory cytokines such as IL-1, IL-6, IL-8, and TNF-a ŽCollart et al., 1990; Libermann and Baltimore, 1990; Hiscott et al., 1993;
0165-5728r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 9 9 . 0 0 1 2 2 - 8
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Matsusaka et al., 1993; Kunsch et al., 1994; Roulston et al., 1995.. The primary form of NF-kB consists of a heterodimer of p50 and p65 that is bound by members of the Ik B family, including Ik Ba , in cytoplasm ŽBaeuerle and Henkel, 1994; Baldwin, 1996.. NF-kB activation requires degradation of Ik Ba protein ŽGrimm and Baeuerle, 1993; Henkel et al., 1993.. Phosphorylation of Ik Ba by drugs, cytokines, bacterial products, and viruses leads to Ik Ba degradation, translocation of NF-kB to the nucleus, and transcription of proinflammatory cytokine genes ŽKumar et al., 1994; Brown et al., 1995.. a-MSH inhibits NF-kB activation and Ik Ba degradation in cultured cells ŽManna and Aggarwal, 1998.. We tested the hypothesis that central a-MSH modulates peripheral acute inflammation by inhibiting NF-kB activation through descending neural pathways stemming from MC1R within the brain.
2. Materials and methods 2.1. Animals These experiments were approved by the local Internal Review Board for Animal Research. Male C57BLr6 mice ŽSimonsen Laboratories, Gilroy, CA. were housed in a 24 " 18C environment in groups not exceeding five animals per cage Ž28 = 18 = 13 cm3 .. They were allowed to acclimatize to standard lighting and temperature conditions with food and water freely available before experiments began. Mice with nonfunctional MC1R ŽMC-1rerMC-1re. originated from stock bred by Dr. G. S. Barsh of Stanford University School of Medicine ŽStanford, CA.. Characterization of these mice was described previously ŽRobbins et al., 1993.. In brief, the mice have nonfunctional MC1R resulting from a frameshift mutation between the fourth and fifth transmembrane domains. 2.2. Experimental protocol Mice were anesthetized with 10% pentobarbital sodium solution Ž1 mgrmouse, 50 mgrml, Nembutal sodium solution; Abbott Laboratories, North Chicago, IL.. Animals received an intracerebroventricular Žicv. injection of a-MSH Ž40 mg dissolved in 20 ml saline., or 20 ml saline using techniques previously described ŽHaley and McCormick, 1957.. Recombinant human TNF-a ŽR & D systems, Minneapolis, MN. dissolved in saline Ž20 ng, 20 ml. was injected into one rear foot pad and saline Ž20 ml. was injected into the other using 28-gauge allergy test syringes ŽSherwood, St. Louis, MO.. Spinal cord transection was induced by a standardized crushing of lumbar vertebrae with a hemostat ŽMacaluso et al., 1994.. In the experiments proper, mice were tested after cord section when the anesthesia had worn off: none
showed behavioral or motor responses to pinching of the hind feet with a hemostat; all had complete paralysis of the hind limbs. Several agents were injected intraperitoneally Žip. to pharmacologically block certain receptors: atropine Ž300 mg; Sigma, St. Louis, MO., a muscarinic receptor blocker; phentolamine Ž300 mg; Sigma., an agent that competes for occupancy of a-adrenergic receptors; propranolol Ž300 mg; Sigma., a nonspecific competitive antagonist for badrenergic receptors; atenolol Ž300 mg; Sigma., a b 1adrenergic receptor antagonist; butoxamine Ž300 mg; Sigma., a b 2-adrenergic receptor blocker. At the indicated time points, foot pads were surgically removed and immediately frozen in liquid nitrogen. 2.3. Nuclear extracts Nuclear extracts were harvested from frozen foot pads using a previously published procedure ŽDeryckere and Gannon, 1994.. The protein concentration of the supernatant containing nuclear extracts was determined using Bio-Rad ŽHercules, CA. protein concentration reagent. Nuclear extracts were stored at y808C. 2.4. Electrophoretic Mobility Shift Assay (EMSA) Double-stranded oligonucleotides for the consensus binding sites of NF-kB Ž5X-AGTTGAGGGGACTTTCCCAGGC-3X ; Promega, Madison, WI. were labeled using Klenow fragment ŽLife Technologies, Gaithersburg, MD. in the presence of 100 mCi of w a-32 PxdCTP Ž3000 Cirmmol; NEN, Boston, MA. and unlabeled dATP, dGTP, and dTTP at 100 mM in a final volume of 50 ml. Unincorporated nucleotides were removed using a G-25 ŽPharmacia, Piscataway, NJ. spin column. Five micrograms of nuclear proteins were preincubated with binding buffer Ž50 mgrml polywdI–dCx, 5% glycerol, 10 mM Tris–HCl, pH 7.8, 1 mM EDTA, 40 mM KCl, and 1 mM DTT. on ice for 15 min before addition of the radiolabeled oligonucleotide probe and incubation at room temperature for 20 min. Specificity of the binding reaction was determined in competition assays by co-incubating with 100-fold molar excess of unlabeled NF-kB or AP-1 consensus oligonucleotide ŽPromega., and by supershift assay with polyclonal rabbit anti-NF-kB-p50 and anti-NF-kB-p65 antibody ŽSanta Cruz Biotechnology, Santa Cruz, CA.. Reaction products were separated in a 4% polyacrylamide gel. Dried gels were analyzed by autoradiography. Autoradiograms were analyzed by densitometry ŽBio-Rad.. 2.5. Western blot analysis We followed a modification of a previously described procedure ŽLentsch et al., 1997.. Frozen foot pads were homogenized in lysis buffer Ž10 mM HEPES, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, 0.5 mM PMSF,
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Fig. 1. Representative EMSA demonstrating effect of a-MSH on NF-kB activation 0.5, 1, and 3 h after intradermal injection of TNF-a Ž20 ng in 20 ml saline. into foot pad of the mouse. NF-kB activation was induced by TNF-a, but not saline Ž20 ml., in mice given icv saline. Centrally administered a-MSH Ž40 mg in 20 ml saline. inhibited NF-kB activation at each indicated time. Ss injection of saline into the foot pad with centrally administered saline; T s injection of TNF-a into the foot pad with centrally administered saline; TqMs injection of TNF-a into the foot pad with centrally administered a-MSH.
and 1.0 mgrml each of the following protease inhibitors: aprotinin, antipain, leupeptin, chymostatin, and pepstatin A. and were centrifuged at 4000 rpm for 2 min. Supernatants were transferred to 1.5-ml tubes. Protein concentrations were determined with BCA assay ŽPierce, Rockford, IL.. Samples Ž20 mg total protein. were separated in a denaturing 10% polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. After four additional washes in TBST Ž40 mM Tris–HCl, pH 7.6, 300 mM NaCl, and 0.5% Tween 20., membranes were incubated in a 1:1000 dilution of rabbit polyclonal anti-Ik Ba ŽSanta Cruz Biotechnology. in TBST containing 5% nonfat dry milk at room temperature for 2 h. After four washes in TBST, membranes were incubated in a 1:5000 dilution of horseradish peroxidase-conjugated donkey anti-rabbit IgG ŽAmersham, Arlington Heights, IL. for 1 h at room temperature. After four washes in TBST, immunoreactive
Fig. 2. Representative EMSA indicating specificity of NF-kB oligonucleotide in experiments on nuclear extract from murine foot pad 1 h after TNF-a injection. Control Žlane 1.; gel shift band disappeared in competition assays when 100-fold excess unlabeled NF-kB oligonucleotide was added Žlane 2.; unlabeled AP-1 oligonucleotide had no effect Žlane 3.; co-incubation of nuclear extracts with anti-NF-kB-p50 polyclonal antibody Žlane 4. or with anti-NF-kB-p65 polyclonal antibody Žlane 5. caused supershift of the NF-kBrDNA complex.
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Fig. 3. Representative EMSA showing effect of spinal cord transection on a-MSH-induced inhibition of NF-kB activation induced by TNF-a. In mice with spinal cord transection, centrally administered a-MSH had no effect on TNF-a-induced NF-kB activation of foot pad at each indicated time.
proteins were detected using enhanced chemiluminescence ŽAmersham. and analyzed by autoradiography.
3. Results 3.1. Inhibition by centrally administered a-MSH of NF-k B actiÕation in the periphery EMSA of nuclear extracts from foot pads injected with TNF-a demonstrated NF-kB activation ŽFig. 1.. NF-kB activation was observed throughout the 3-h test period in
Fig. 4. Effect of pharmacological blockade on inhibition of NF-kB activation by centrally administered a-MSH. All samples taken 3 h after TNF-a injected into the foot pad. ŽA. Intraperitoneal injection of a nonspecific b-blocker propranolol Ž300 mg. inhibited the suppressive effect of a-MSH on NF-kB activation by TNF-a. A muscarinic receptor blocker atropine Ž300 mg. or an agent that competes for occupancy of a-adrenergic receptors phentolamine Ž300 mg. did not alter the effects of a-MSH. ŽB. Blockade of b 2-adrenoreceptor with butoxamine Ž300 mg. inhibited the suppressive effect of a-MSH on NF-kB activation, but blockade of b1 -adrenergic receptor with atenolol Ž300 mg. did not.
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Fig. 5. Representative Western blot demonstrating effect of centrally administered a-MSH on TNF-a-induced Ik Ba degradation in the murine foot pad. Expression of Ik Ba protein decreased 1 h after injection of TNF-a Ž20 ng in 20 ml saline.. Centrally administered a-MSH Ž40 mg in 20 ml saline. preserved expression of Ik Ba protein.
the TNF-a-treated foot pads of mice given central injection of saline. The gel shift bands of nuclear extracts from the foot pads injected with TNF-a were reduced by centrally administered a-MSH ŽFig. 1.. Densitometry showed an inhibition of approximately 40%. Specificity of the NF-kB consensus oligonucleotide probe was confirmed in experiments on nuclear extracts from the foot pads stimulated with TNF-a for 1 h. The intensity of the gel shift band was markedly diminished in the presence of 100-fold molar excess of unlabeled NF-kB oligonucleotide, but not by a 100-fold molar excess of unlabeled AP-1 oligonucleotide ŽFig. 2.. Assays with anti-NF-kB-p50 and anti-NFk B-p65 antibody revealed supershift of the gel shift band, confirming the identity and specificity of the band ŽFig. 2.. 3.2. Influence of spinal cord transection and drugs on central a-MSH-induced inhibition of peripheral NF-k B actiÕation Spinal cord transection abolished the inhibitory effect of centrally administered a-MSH on NF-kB activation induced by TNF-a injection into the foot pads ŽFig. 3.. In experiments designed to test the contribution of certain neurotransmitter receptors to the effect of centrally administered a-MSH, propranolol, a nonspecific b-blocker propranolol suppressed the inhibitory effects of a-MSH on NF-kB activation. However, a muscarinic receptor blocker Žatropine., or an agent that competes for occupancy of a-adrenergic receptors Žphentolamine., did not ŽFig. 4A.. After positive effects were observed with propranolol, selective antagonists of b-adrenoreceptors were tested. Blockade of b 2-adrenergic receptor with butoxamine inhibited the effect of a-MSH on NF-kB activation; block-
Fig. 6. Effect of spinal cord transection on centrally administered aMSH-induced protection of Ik Ba degradation induced by TNF-a. In mice with spinal cord transection, centrally administered a-MSH had no effect on Ik Ba degradation in the foot pad 1 h after local injection of TNF-a.
Fig. 7. Effect of pharmacological blockade on centrally administered a-MSH-induced preservation of Ik Ba degradation 1 h after local injection of TNF-a. Intraperitoneal injection of a nonspecific b-blocker propranolol Ž300 mg. or b 2-adrenoreceptor antagonist butoxamine Ž300 mg. inhibited the effect of a-MSH on Ik Ba protein. Other agents; a muscarinic receptor blocker Žatropine, 300 mg., an agent that competes for occupancy of a-adrenergic receptors Žphentolamine, 300 mg. or a b1 -adrenoreceptor antagonist Žatenolol, 300 mg., did not.
ade of b 1-adrenergic receptor with atenolol was ineffective ŽFig. 4B.. 3.3. Restriction of Ik Ba degradation in the foot pads by centrally administered a-MSH Western blot analysis of extracts of foot pads from mice given icv saline revealed that expression of Ik Ba protein decreased 1 h after TNF-a injection, but returned 1.5 h after. Centrally administered a-MSH protected the expression of Ik Ba protein at 1 h after TNF-a injection ŽFig. 5.. 3.4. Influence of a-MSH effect on Ik Ba degradation by spinal cord transection and drugs In mice with spinal cord transection, centrally administered a-MSH did not prevent characteristic degradation of Ik Ba protein in the foot pads 1 h after TNF-a injection
Fig. 8. EMSA from nuclear extract of foot pads of MC-1rerMC-1re mice demonstrated that centrally administered a-MSH inhibited NF-kB activation induced by TNF-a much as in normal mice Žsee Fig. 1..
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Fig. 9. Western blot analysis of extract of foot pads of MC-1rerMC-1re mice indicated that centrally administered a-MSH protected expression of Ik Ba protein 1 h after TNF-a injection.
ŽFig. 6.. In experiments on the contribution of certain neurotransmitter receptors to the effect of centrally administered a-MSH, ip administration of propranolol and of butoxamine suppressed the inhibitory effects of a-MSH on Ik Ba degradation; injection of a muscarinic receptor blocker Žatropine., an a-adrenoreceptor antagonist Žphentolamine., or a b 1-adrenoreceptor antagonist Žatenolol. did not ŽFig. 7.. 3.5. Effects of a-MSH on NF-k B actiÕation and Ik Ba degradation in MC-1rer MC-1re mice In MC-1rerMC-1re mice, centrally administered aMSH inhibited NF-kB activation induced by TNF-a in the foot pad throughout the experimental period, much as in control mice ŽFig. 8.. Densitometry showed an inhibition of approximately 40%. In these mice, centrally administered a-MSH also preserved expression of Ik Ba protein in the foot pads 1 h after TNF-a injection, a pattern similar to that in normal mice ŽFig. 9..
4. Discussion Inflammation is characteristic in many diseases of the periphery including arthritis, inflammatory bowel disease, asthma, and vascular disease. Therapy for these disorders has not progressed rapidly and perhaps a new avenue for control via agents that act on the CNS will be beneficial. Our results suggest that targeting CNS a-MSH receptors could improve peripheral inflammation. Our data demonstrate that centrally administered aMSH inhibits NF-kB activation and Ik Ba degradation in peripheral acute inflammation. Intact descending neural pathways are necessary for this influence of centrally administered a-MSH, but the specific melanocortin receptor subtype MC1R within the brain is not. In addition, our experiments on pharmacological blockade of peripheral neural transmission revealed that b 2-adrenergic neurotransmission is essential to the effect of central a-MSH on peripheral acute inflammation. Our previous observations indicated that central a-MSH modulates peripheral inflammation ŽLipton et al., 1991; Ceriani et al., 1994; Macaluso et al., 1994.. a-MSH given icv inhibited ear edema of mice induced by topical picryl chloride ŽLipton et al., 1991.. Centrally administered aMSH likewise suppressed ear edema induced by intrader-
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mal injection of IL-1b, IL-8, leukotriene B4, and plateletactivating factor ŽCeriani et al., 1994; Macaluso et al., 1994.. Our present data suggest a molecular mechanism underlying these prior observations. b-Adrenergic agonists are known to have anti-inflammatory effects ŽGreen, 1972; Maling et al., 1974; Svensjo¨ et al., 1977; Inagaki et al., 1989.. Catecholamines acting predominantly on b-adrenergic receptors inhibited increased permeability of the mouse peritoneum induced by local injection of acetic acid ŽGreen, 1972.. b-Adrenoreceptor stimulants inhibited hind paw edema induced by histamine, serotonin, bradykinin or compound 48r80 ŽMaling et al., 1974. and also inhibited bradykinin-induced macromolecular leakage ŽSvensjo¨ et al., 1977.. Further, such stimulants inhibited increased vascular permeability induced by mediators of allergic reactions ŽInagaki et al., 1989.. Our previous data demonstrated that the inhibitory effects of centrally administered a-MSH on hind paw edema induced in the mouse by kappa carrageenan were inhibited by a nonspecific b-adrenergic receptor blocker and a specific b 2-adrenergic receptor antagonist ŽMacaluso et al., 1994.. Superoxide generation by neutrophils was down-regulated by b-adrenergic stimulation ŽBarnett et al., 1997.. Catecholamines inhibited TNF production during concomitant LPS stimulation through b-adrenoreceptor activation ŽGuirao et al., 1997.. Further, TNF and IL-6 promoter activities induced by LPS were inhibited by b 2-adrenoreceptor activation ŽNakamura et al., 1998.. These findings and our observations with butoxamine strongly indicate that b 2-adrenergic receptors are important in the modulation of inflammation. a-MSH modulates inflammation by inhibiting production of proinflammatory cytokines, nitric oxide, prostaglandin E2, and neopterin ŽWeiss et al., 1991; Star et al., 1995; Weidenfeld et al., 1995; Rajora et al., 1996, 1997; Wong et al., 1997.. Recent research indicated that NF-kB activation and Ik Ba degradation were inhibited by a-MSH in various cells ŽManna and Aggarwal, 1998.. Elevations in cyclic AMP inhibit NF-kB-mediated transcription in human monocytic cells and endothelial cells ŽOllivier et al., 1996. and cyclic AMP accumulation is known to be stimulated by b 2-adrenoreceptor agonists ŽRoberts and Summers, 1998.. We therefore suggest that centrally administered a-MSH acts to inhibit transcriptional activation of NF-kB in peripheral inflammation through b 2-adrenoreceptor-mediated increases in intracellular cyclic AMP level. Melanocortin receptors MC1R, MC3R, MC4R, and MC5R have been identified in brain tissue ŽLipton and Catania, 1997.. It was believed that a-MSH exerts its local anti-inflammatory action through MC1R on peripheral cells, but we demonstrated that MC1R in the brain is not required for centrally administered a-MSH to exert its anti-inflammatory action through descending neural pathways. Centrally and systemically administered a-MSH suppressed LPS-induced fever, but not plasma levels of
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IL-6 or corticosterone, through action on MC3R and MC4R within the brain ŽHuang et al., 1997, 1998.. It is therefore possible that several receptor subtypes mediate the influence of centrally administered a-MSH on peripheral inflammation. 5. Summary Our data indicate that a-MSH injected centrally can restrict Ik Ba degradation and subsequent NF-kB activation in peripheral acute inflammation. Spinal cord transections revealed that an intact descending neural pathway is necessary for centrally administered a-MSH to exert these effects. Pharmacological studies indicate that b 2-adrenoreceptors in the periphery are likewise important in the anti-inflammatory pathway activated by centrally administered a-MSH. Experiments on MC-1rerMC-1re mice demonstrated that the anti-inflammatory effect of centrally administered a-MSH on peripheral inflammation does not depend solely on mediation by MC1R within the brain. References Baeuerle, P.A., Henkel, T., 1994. Function and activation of NF-kB in the immune system. Annu. Rev. Immunol. 12, 141–179. Baldwin, A.S. Jr., 1996. The NF-kB and Ik B proteins: new discoveries and insights. Annu. Rev. Immunol. 14, 649–683. Barnett, C.C. Jr., Moore, E.E., Partrick, D.A., Silliman, C.C., 1997. b-Adrenergic stimulation down-regulates neutrophil priming for superoxide generation, but not elastase release. J. Surg. Res. 70, 166– 170. Barrett, P., MacDonald, A., Helliwell, R., Davidson, G., Morgan, P., 1994. Cloning and expression of a new member of the melanocytestimulating hormone receptor family. J. Mol. Endocrinol. 12, 203–213. Brown, K., Gerstberger, S., Carlson, L., Franzoso, G., Siebenlist, U., 1995. Control of Ik B-a proteolysis by site-specific, signal-induced phosphorylation. Science 267, 1485–1488. Catania, A., Lipton, J.M., 1993. a-Melanocyte stimulating hormone in the modulation of host reactions. Endocr. Rev. 14, 564–576. Catania, A., Rajora, N., Capsoni, F., Minonzio, F., Star, R.A., Lipton, J.M., 1996. The neuropeptide a-MSH has specific receptors on neutrophils and reduces chemotaxis in vitro. Peptides 17, 675–679. Ceriani, G., Macaluso, A., Catania, A., Lipton, J.M., 1994. Central neurogenic antiinflammatory action of a-MSH: modulation of peripheral inflammation induced by cytokines and other mediators of inflammation. Neuroendocrinology 59, 138–143. Chhajlani, V., Wikberg, J.E., 1992. Molecular cloning and expression of the human melanocyte stimulating hormone receptor cDNA. FEBS Lett. 309, 417–420. Collart, M.A., Baeuerle, P., Vassalli, P., 1990. Regulation of tumor necrosis factor alpha transcription in macrophages: involvement of four k B-like motifs and of constitutive and inducible forms of NF-kB. Mol. Cell. Biol. 10, 1498–1506. Deryckere, F., Gannon, F., 1994. A one-hour minipreparation technique for extraction of DNA-binding proteins from animal tissues. Biotechniques 16, 405. Gantz, I., Konda, Y., Tashiro, T., Shimoto, Y., Miwa, H., Munzert, G., Watson, S.J., DelValle, J., Yamada, T., 1993a. Molecular cloning of a novel melanocortin receptor. J. Biol. Chem. 268, 8246–8250. Gantz, I., Miwa, H., Konda, Y., Shimoto, Y., Tashiro, T., Watson, S.J., DelValle, J., Yamada, T., 1993b. Molecular cloning, expression, and
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