γ2-melanocyte-stimulating hormone suppression of systemic inflammatory responses to endotoxin is...

Ž .Journal of Neuroimmunology 120 2001 67–77www.elsevier.comrlocaterjneuroim

g -Melanocyte-stimulating hormone suppression of systemic2

inflammatory responses to endotoxin is associated with modulationof central autonomic and neuroendocrine activities

Yun Xia a, Jarl E.S. Wikberg b, Teresa L. Krukoff a,)

a Department of Cell Biology, Faculty of Medicine and Dentistry, and Center for Neuroscience, UniÕersity of Alberta, Edmonton, AB, Canada T6G 2H7b Department of Pharmaceutical Pharmacology, Box 591, Biomedical Centre, S-751-24 Uppsala, Sweden

Received 1 June 2001; received in revised form 23 July 2001; accepted 1 August 2001

Abstract

Central autonomic and neuroendocrine activities are important components of the host response to bacterial inflammation. WeŽ .demonstrate that intravenous infusion of g -melanocyte-stimulating hormone g -MSH , a potent autonomic regulating peptide, prevents2 2

Ž .lipopolysaccharide LPS -induced hypotension and tachycardia, and modulates the ACTH response to endotoxin. In the hypothalamicparaventricular nucleus, a major neuroendocrine and autonomic center, g -MSH inhibits LPS-induced increases in CRF mRNA levels, but2

does not suppress LPS-augmented arginine vasopressin heteronuclear RNA expression. In the locus coeruleus, a brainstem noradrenergiccenter, g -MSH inhibits LPS-induced increases in tyrosine hydroxylase mRNA levels. g -MSH inhibits LPS-induced IL-1b gene2 2

expression in the brain, pituitary and thymus, and prevents increases in plasma NO levels. These findings reveal that g -MSH attenuates2

systemic inflammatory responses to endotoxin and suggest that modulation of central autonomic and neuroendocrine activities byg -MSH contributes to its anti-inflammatory effects. q 2001 Elsevier Science B.V. All rights reserved.2

Keywords: Melanocortins; Host responses; Sympathetic activity; Cardiovascular; Hypothalamic–pituitary–adrenal axis

1. Introduction

Gram-negative bacteria-induced inflammation leads toŽ .the release of lipopolysaccharide LPS , a fragment of the

bacterial cell wall, into the local tissue and circulation.Ž .LPS exposure endotoxemia triggers systemic inflamma-

tory responses that result in hypotension, leukocyte infiltra-tion, fever and changes in metabolic activities. In responseto inflammatory challenge, activation of the autonomic

Ž .nervous system ANS plays a vital role in developing andmodulating the host response, including the cardiovascularresponse, body temperature, homeostasis, and behavior.ANS activity also participates in release of stress hor-mones, such as increases in circulating levels of adrenocor-

Ž . Žticotropic hormone ACTH and corticosterone Plotsky et.al., 1989; Besedovsky and Del Rey, 1996 . Moreover,

activation of the ANS modulates immune responses, in-cluding immune cell traffic, antigen processing and cy-

) Corresponding author. Tel.: q1-780-492-4996; fax: q1-780-492-0450.

Ž .E-mail address: [email protected] T.L. Krukoff .

Žtokine production Madden and Felten, 1995; Elenkov et.al., 2000 .

Systemic inflammatory responses cause tissue injuryand organ failure, which are associated with high mortal-ity. Amplification of inflammatory responses is due tohemodynamic disturbance and excessive immune re-

Ž .sponses Dellinger, 1999; Glauser, 2000 , both of whichare influenced by activation of autonomic activity. Inhibi-tion of inappropriate inflammatory responses by regulatingautonomic activity may have beneficial effects, as experi-mental sympathectomy or pharmacological manipulationof the ANS activity alter immune responses to pathogensŽ .Elenkov et al., 2000 , and stimulation of the vagus nerveattenuates LPS-induced systemic inflammatory responsesŽ .Borovikova et al., 2000 .

Ž . Žg -Melanocyte stimulating hormone g -MSH Naka-2 2.nishi et al., 1979 , derived from the N-terminal of pro-

opiomelanocortin, is a potent central ANS-regulating pep-Ž .tide Gruber and Callahan, 1989 . Previous studies have

shown that g -MSH causes a dose-dependent increase of2

arterial pressure, heart rate, and cerebral blood flow afterŽ . Žintravenous i.v. administration in conscious rats Klein et

.al., 1985; Sun et al., 1992; De Wildt et al., 1995 . The

0165-5728r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.Ž .PII: S0165-5728 01 00408-8

( )Y. Xia et al.rJournal of Neuroimmunology 120 2001 67–7768

pressor and cardioaccelerator effects of g -MSH depend2

on the maintenance of sympathetic drive to peripheralcardiovascular tissues and activation of the arginine vaso-

Ž . Žpressin AVP system Gruber and Callahan, 1989; Ver-.steeg et al., 1998 . At low doses, g -MSH has a nutriuretic2

Žaction mediated primarily through the renal nerves Chen.et al., 1997 . In addition to cardiovascular and renal ef-

fects, intraseptal or i.v. infusion of g -MSH inhibits LPS-2Ž .induced fever in the guinea pig Bock et al., 1994 ,

suggesting that g -MSH has central antipyretic actions2

during the inflammatory response. In this context, it isinteresting to note that a-MSH, an analogue of g -MSH,2

has potent anti-inflammatory actions in vivo and in vitroŽ .Catania et al., 1999; Lipton et al., 1999 . Taken together,we hypothesized that g -MSH has potential beneficial2

actions in treatment of systemic inflammatory responses.To study the actions of g -MSH in vivo, we induced2

systemic inflammation in rats with i.v. administration ofLPS. Changes in early inflammatory responses, includingcardiovascular responses, the hypothalamic–pituitary–

Ž .adrenal HPA response, IL-1b gene expression and plasmaŽ .nitric oxide NO production, were examined after g -MSH2

treatment. The potential central mechanisms mediating theeffects of g -MSH on inflammatory responses were inves-2

tigated.

2. Materials and methods

2.1. Animals

Ž .Male Sprague–Dawley rats 250–350 g were pur-chased from the Biological Animal Center, University ofAlberta. Food and water were given ad lib. The experimen-tal protocol was approved by the Animal Welfare Commit-tee at the University of Alberta.

2.2. Arterial and Õenous catheterization

Ž .As described previously Xia and Krukoff, 2001 , ratsŽwere anesthetized sodium pentobarbital; 60 mgrkg, i.p.;

.MTC Pharmaceuticals, Hamilton, Canada and instru-Ž .mented with indwelling arterial abdominal aorta and

Ž .venous inferior vena cava catheters. Capped free ends ofcatheters were guided beneath the skin, externalized be-tween the scapulae, and connected to stainless steel con-nectors with pedestals fixed on the skull. Rats were re-turned to their home cages and were allowed to recoverfrom the surgery for 4–5 days.

2.3. Experimental design

Ž .To record arterial pressure and heart rate HR , theŽarterial line was connected to a pressure transducer Trans-

.pac IV with a computer-assisted data recording module

ŽD1-150 RS using WINDAQ software DATAQ Instru-.ments, Akron, OH, USA . The venous line was connected

Žto a syringe filled with pyrogen-free isotonic saline 0.9%.NaCl . Rats were left undisturbed in their home cages for 1

Žh, and then subjected to one of the treatments ns five to.seven rats per group described below.

Ž . ŽGroups: a Vehicle group: i.v. infusion of saline 0.5mlrh; Harvard Apparatus infusion pump, Holliston, MA,

.USA throughout the experiment to control for g -MSH2Ž .infusion. A saline injection 0.3 ml , as a control for LPS,

was given 15 min after initiation of infusion.Ž .b g -MSHqÕehicle group: i.v. infusion of g -MSH2 2

ŽTyr–Val–Met–Gly–His–Phe–Arg–Trp–Asp–Arg–Phe–Ž .Gly Nakanishi et al., 1979 ; Peninsula Laboratories, Bel-

.mont, CA, USA at the rate of 50 nmolrkgrh in vehiclethroughout the experiment. A saline injection, as a controlfor LPS, was given 15 min after initiation of infusion. Thisdose of g -MSH was previously shown to increase the2

Žplasma concentration of immunoreactive g -MSH Chen et2.al., 1997 .

Ž .c VehicleqLPS group: i.v. infusion of saline fol-Žlowed by injection of LPS 50 mgrkg in 0.3 ml of saline,

.Escherichia coli, 055:B5; Sigma, St. Louis, MI, USA 15min after initiation of infusion. This dose of LPS waspreviously found to induce a transient depressor responseand tachycardia after i.v. administration to conscious ratsŽ .Xia and Krukoff, 2001 .

Ž .d g -MSHqLPS group: i.v. infusion of g -MSH2 2

followed by LPS injection as above. g -MSH has been2Ž .proposed to act through the melanocortin-3 MC receptor3

Ž .Roselli-Rehfuss et al., 1993 . Therefore, HS024, an antag-onist of melanocortin receptors, including MC1, MC3,

Ž .MC4 and MC5 receptors Wikberg, 1999 was employedin some experiments to determine if the actions of g -MSH2

are mediated via these melanocortin receptors. HS024ŽAc–Cys–Nle–Arg–His–DNal–Arg–Trp–Gly–Cys–

.NH ; Melacure Therapeutics, Uppsala, Sweden was in-2

fused at a rate of 50 nmolrkgrh in vehicle for 15 min and25 nmolrkgrh for the remainder of the experiment, fol-lowed by g -MSHqLPS as above. The nmol dose of2

HS024 was previously shown to block the increase inŽ .cAMP induced by a-MSH in vitro Kask et al., 1998 . To

control for any effects of HS024 itself on MAP or HR, agroup of rats received HS024 alone at the same infusionrates as above.

At the end of the experiments, rats were deeply anes-thetized. Trunk blood was collected in plastic tubes con-taining EDTA and plasma was separated by centrifugation.Rats were perfused through the left ventricle with ice-coldsaline followed by ice-cold 4% paraformaldehyde. Brains,pituitaries and thymus glands were removed, postfixed for1 h and placed in 20% sucrose overnight at 4 8C. Thefrozen tissues were sectioned at 40 mm in a cryostat,thaw-mounted onto glass slides. The tissue sections and

Žplasma were stored at y70 8C until used Xia and Krukoff,.2001 .

( )Y. Xia et al.rJournal of Neuroimmunology 120 2001 67–77 69

2.4. ACTH and NO assays

ŽPlasma ACTH was measured by RIA ICN Biomedicals.Kit, Costa Mesa, CA, USA in accordance with the manu-

Ž .facturer’s instructions Xia and Krukoff, 2001 . Plasmanitraternitrite concentrations were determined with a col-

Žorimetric assay Alexis Biochemicals Kit, San Diego, CA,. Ž .USA as described Yang and Krukoff, 2000 .

2.5. In situ hybridization histochemistry

35S-labeled cRNA probes were generated from plasmidsŽ .containing cDNA inserts for tyrosine hydroxylase TH ,

Ž .intronic vasopressin AVP , corticotrophin-releasing factorŽ .CRF , or IL-1b. The TH fragment was generated from a

Ž .1.2 kb EcoRI digest of rat TH cDNA Grima et al., 1985 .The fragment containing the q14 to q1165 coding regionwas confirmed by automated sequencing and subclonedinto pGEM-7Zf vector. Antisense probes were transcribedwith SP6 RNA polymerase from plasmids linealized withApa I. The pGEM3 vector containing a 700 bp fragment of

Ž .rat AVP intron I AVPinI was kindly provided by T.G.Ž .Sherman University of Pittsburgh, Pittsburgh, PA, USA .

Antisense probes were transcribed with T7 polymerasefrom plasmids linealized with HindIII. The pGEM-3Z

Ž .vector containing a 770 bp q1283 to q2048 rat CRFŽ .cDNA fragment CRHex 2 was kindly provided by R.C.

Ž .Thompson University of Michigan, Ann Arbor, MI, USA .Antisense probes were transcribed with T7 polymerasefrom plasmids linealized with HindIII. The pGEM2 vectorcontaining a 223 bp fragment of rat IL-1b cDNA was

Žkindly provided by R. Rohan Children’s Hospital, Boston,.MA, USA . Antisense probes were synthesized using T7

polymerase after plasmids were linearized with EcoR1. Tocontrol for the specificity of the probes, sense probes werealso synthesized from related plasmids. 35S-labeled cRNA

35 Žprobes were synthesized using a- S-UTP specific activ-itys800 Cirmmol; New England Nuclear, Boston, MA,

. wUSA , and purified with Bio-spin 30 chromatographyŽ .columns Bio-Rad Laboratories, Hercules, CA, USA .

ŽIn situ hybridization was carried out as described Xia.and Krukoff, 2001 . Sections were then exposed to Kodak

Ž . Ž . Ž .Fig. 1. Effects of g -MSH infusion on mean arterial pressure MAP and heart rate HR in rats with or without LPS challenge. a g -MSH alone induced2 2Ž . Ž . Žincreases in MAP, which began 10 min after initiation of infusion p-0.05 vs. baseline value , and b induced increases in HR only at 10 min p-0.05

. Ž . Ž . Ž .vs. baseline value . c g -MSH eliminated LPS-induced decreases in MAP which occurred 25–55 min after LPS injection p-0.05 , and d g -MSH2 2Ž . Ž .inhibited LPS-induced HR responses during the same period p-0.05 . g -MSH was infused at 0 time open arrows ; LPS injection, open arrowhead;2Ž .HS024 infusion, arrow. Data are represented as mean"SEM nssix rats per group .


X-OMAT, AR films for 2–3 days, dipped in NTB-2Ž .emulsion diluted 1:1 with water , and exposed for an

Žappropriate time at 4 8C CRF and TH probes, 10 days;.AVP and IL-1b, 14 days . Slides were developed in D19

developer and counterstained with cresyl violet.

2.6. Data and image analysis

Hybridized tissue sections were examined using a Leitzmicroscope and levels of signal were quantified with im-

Ž .age analysis as described Xia and Krukoff, 2001 . Briefly,optical density was obtained as the percentage of the areacovered by silver grains per section. Background signalwas subtracted from the signal in the target region. Toaid analysis of the hypothalamic paraventricular nucleusŽ . ŽPVN , the middle region between y1.85 and y1.70 mm

.caudal to bregma was subdivided into its functional subdi-Ž .visions: posterior magnocellular pm , dorsal parvocellular

Ž . Ž .dp , ventral medial parvocellular mp , and dorsal me-vŽ . Ž .dial parvocellular mp Yang et al., 1999 .d

Time-dependent changes in mean arterial pressureŽ .MAP and HR were calculated every 5 min. The baselinewas calculated as the average of values during 15 min

before the start of infusion. ANOVA repeated measure-ments were used for analysis of variance within groups.Data among groups were analyzed with ANOVA, followedby the Student–Newman–Keuls test. The unpaired t-testwas used to test for differences in IL-1b gene expressionin tissues before and after g-MSH. Data are expressed asmeans"SEM and p-0.05 was considered significant.

3. Results

3.1. ReÕersal of LPS-induced hypotension and tachycardia

Baseline MAP and HR values were not significantlyŽ . Ž .different among the groups Fig. 1 and vehicle saline

infusion did not alter MAPs and HRs. Infusion of g -MSH2Ž .50 nmolrkgrh led to significant increases in MAPs at

Ž .10 min 4"1 mmHg after initiation of infusion, andMAPs remained significantly elevated for the remainder of

Ž .the experiment Fig. 1a . HRs were briefly increased at 10Ž .min 12"6 bpm and a significant decrease occurred 105

Ž .min after initiation of g -MSH infusion Fig. 1b .2

Ž . Ž . Ž .Fig. 2. Effects of g -MSH infusion on TH gene expression in the locus coeruleus LC . a TH mRNA levels in vehicle control rats. b LPS injections2Ž . Ž .stimulated TH gene expression and c g -MSH prevented the LPS-induced increase in TH mRNA. d Comparison of TH mRNA levels in the four groups2

Ž ) )) .ns five rats per group; p-0.05 vs. LPS-treated rats; p-0.01 vs. vehicle controls . Abbreviation: 4V, fourth cerebroventricle. Scale bars100 mm.


Ž .Rats receiving LPS 50 mgrkg showed decreases inŽMAPs that occurred between 25 and 55 min y5"2 to

.y9"3 mmHg after LPS injections; MAPs returned tobaseline after 60 min and no significant changes occurred

Ž .thereafter Fig. 1c . Significant increases in HRs occurredŽ .10 min 21"5 bpm after LPS injections and lasted for 80

Ž .min, with a peak response at 50 min 58"3 bpm afterŽ .LPS injections Fig. 1d . Infusion of g -MSH prevented2

Ž .the LPS-induced decreases in MAPs Fig. 1c and attenu-Ž .ated the LPS-induced HR responses Fig. 1d .

Infusion of HS024 alone had no significant effects onŽ .baseline MAPs and HRs ns4; data not shown . HS024

had no significant effects on MAPs or HRs of g -MSHq2Ž .LPS rats Fig. 1c and d .

3.2. Inhibition of TH, but not AVP, gene expression

It has been reported that LPS administration leads toincreased norepinephrine content and turnover in the brain-

Ž .stem locus coeruleus LC as part of the central responseŽto an immune stressor Lacosta et al., 1999; Sabban and

.Kvetnansky, 2001 . To determine if g -MSH infusion2

affects the LPS-induced activation of the LC cate-cholamine system, we measured gene expression for TH,the rate-limiting enzyme in catecholamine synthesis, in theLC 2 h after treatments. TH mRNA levels were not alteredby g -MSH infusion alone, whereas LPS-treated rats2

showed significant increases in TH mRNA levels com-Ž .pared to control rats Fig. 2a and b . This latter increase

was abolished in LPS-treated rats that received g -MSH2Ž .infusions Fig. 2c . Quantitative results in the four groups

are shown in Fig. 2d.To determine if g -MSH infusions affect the response2

of the AVP system in the PVN to LPS, we measured AVPŽ .heteronuclear RNA AVP hnRNA levels in the functional

subdivisions of the PVN 1 h after treatments. Compared toŽ .vehicle controls Fig. 3a , rats receiving LPS displayed

Žincreases in AVP hnRNA levels throughout the PVN Fig..3b , and these increases were particularly evident in the

dorsal parvocellular and posterior magnocellular subdivi-Ž .sions Fig. 3b and d . g -MSH infusion alone did not alter2

basal levels of AVP hnRNA in the PVN, except in the dpŽ .division Fig. 3d . LPS-induced increases in AVP hnRNA

Ž .were not affected by infusions of g -MSH Fig. 3c and d .2

Ž . Ž . Ž .Fig. 3. Effects of g -MSH infusion on AVP hnRNA in the paraventricular nucleus PVN . a AVP hnRNA levels in vehicle control rats. b LPS2Ž . Ž .injections induced increases in AVP hnRNA levels but c g -MSH did not affect the LPS-induced increase. d Changes in AVP hnRNA levels in PVN2

Ž ) )) a .subdivisions ns five rats per group; p-0.05 or p-0.01 vs. vehicle controls; ps0.09 vs. LPS-treated rats . Abbreviations: pm, posteriormagnocellular division; dp, dorsal parvocellular division; mp , dorsal medial parvocellular division; mp , ventral medial parvocellular division; 3V, thirdd v

cerebroventricle. Scale bars100 mm.


3.3. Modulation of ACTH response and CRF gene expres-sion.

We next examined effects of g -MSH infusion on the2Ž .LPS-induced ACTH response Fig. 4a . g -MSH infusion2

alone did not influence plasma ACTH levels 1 h after LPSinjections, but significantly reduced levels of ACTH 2 hafter treatment. LPS stimulated significant increases inplasma ACTH levels at both 1 and 2 h after injections,although the ACTH level was reduced at 2 h compared tothat at 1 h. In LPS-treated rats that also received g -MSH2

infusions, ACTH levels were significantly reduced at 2 hŽ .from those in LPS-treated rats Fig. 4a . In rats receiving

HS024qg -MSHqLPS, levels of plasma ACTH were2

not significantly different from those in g -MSHqLPS2

rats.To evaluate the mechanism of g -MSH on modulation2

of the LPS-induced ACTH response, we measured CRFgene expression in the PVN 2 h after treatments. g -MSH2

Žinfusion did not alter basal CRF mRNA levels Fig. 4b and. Ž .c . Compared with control rats Fig. 4b and d , rats

receiving LPS displayed marked increases in CRF mRNA

Ž .levels Fig. 4b and e ; these increases occurred primarily inthe dorsal parvocellular and medial dorsal parvocellular

Ž .subdivisions of the PVN Fig. 4c and e . Increases inlevels of CRF mRNA in the PVN were blocked with

Ž .g -MSH infusion Fig. 4b and f . Further analysis of PVN2

subdivisions indicated that the greatest inhibitory effect ofg -MSH on LPS-induced CRF mRNA occurred in the2

Ž .dorsal parvocellular division Fig. 4c .

3.4. Inhibition of IL-1b gene expression

It has been established that IL-1b mediates both im-mune and CNS responses during the early stages of infec-

Ž .tionrinflammation Rothwell and Luheshi, 2000 . We haveŽpreviously shown that i.v. administration of LPS 50

.mgrkg induced a marked increase in IL-1b gene expres-Ž .sion in the circumventricular organs CVOs of the brainŽand pituitary 1 h after LPS injection Xia and Krukoff,

.2001 . To determine whether g -MSH infusion affects2

LPS-induced pro-inflammatory cytokine production, weŽmeasured levels of IL-1b mRNA in three CVOs organum

vasculosum of the lamina terminalis, subfornical organ,

Ž . Ž) ))Fig. 4. Effect of g -MSH infusion on the HPA response. a LPS increased plasma ACTH levels at 1 and 2 h p-0.05 or p-0.01 vs. vehicle2. Ž) . Žacontrols . At 2 h, g -MSH alone inhibited plasma ACTH levels p-0.05 vs. vehicle controls and attenuated LPS-induced ACTH levels p-0.05 vs.2

. Ž . Ž .LPS-treated rats . HS024 infusion had no significant effect on the response to g -MSHqLPS ns five to seven rats per group . b LPS increased CRF2Ž)) . Ža . Ž .mRNA levels p-0.01 vs. vehicle controls and g -MSH inhibited the LPS-induced increase p-0.05 vs. LPS-treated rats . c CRF mRNA levels2

Ž . Ž .in PVN subdivisions. The LPS-induced increase in CRF mRNA was found primarily in the dorsal dp and dorsal medial parvocellular mpd divisionsŽ) . Ža . Ž . Ž .p-0.05 vs. vehicle controls , and g -MSH inhibited this increase in the dp p-0.05 vs. LPS-treated rats ns five rats per group . d–f Dark-field2

Ž . Ž . Ž .photomicrographs show CRF mRNA levels in rats receiving vehicle d , LPS e or g -MSHqLPS f . Abbreviations as in Fig. 3. Scale bars100 mm.2


.and area postrema , pituitary gland, and thymus 1 h afterrats received LPS injections or treatment with g -MSHq2

LPS. Levels of IL-1b mRNA were undetectable afterinfusion with saline or with g-MSH alone. LPS injectioninduced IL-1b mRNA levels in all tissues studied, andthese increases were significantly reduced by infusions of

Ž .g -MSH except for the subfornical organ Fig. 5 . After2

treatment with HS024, the level of IL-1b mRNA in thethymus was similar to that in g -MSHqLPS-treated rats,2

suggesting that HS024 did not block inhibitory effects ofŽ .g -MSH data not shown .2

3.5. Reduction of plasma NO production

NO levels in plasma have been shown to be closelyassociated with hemodynamic disturbances during devel-opment of septic shock. To assess effects of g -MSH on2

plasma NO content, we measured plasma nitraternitriteŽ .levels 2 h after treatments Fig. 6 . Infusion of g -MSH2

Ž .Fig. 5. Effect of g -MSH infusion on IL-1b gene expression in the brain and peripheral tissues. a and b g -MSH inhibited the LPS-induced increase in2 2Ž . Ž . Ž . ) )) aIL-1b mRNA ns five to six rats per group in three circumventricular organs a , pituitary gland and thymus b . p-0.05, p-0.01 and ps0.06.

Ž . Ž . Ž . Ž .c–f Photomicrographs show LPS-induced IL-1b mRNA c and e and inhibition of expression by g -MSH d and f in the area postrema c and d and2Ž .posterior lobe of the pituitary e and f . Abbreviations: CVOs, circumventricular organs; OVLT, organum vasculosum of the lamina terminalis; SFO,

subfornical organ; AP, area postrema; CC, central canal; Pit-PL, posterior pituitary lobe; Pit-AL, anterior lobe; Pit-IL, intermediate lobe. THY, thymus.Scale bars100 mm.


Ž ))Fig. 6. Effect of g -MSH infusion on plasma NO production. LPS led to increased plasma nitraternitrite levels ns five rats per group; p-0.01 vs.2. Ž) .vehicle controls and g -MSH attenuated this increase p-0.05 vs. LPS-treated rats . Infusion with HS024 had no significant effect on the response to2

g -MSHqLPS.2

alone did not influence plasma levels of nitraternitrite.LPS injections induced marked increases in plasma ni-traternitrite levels, and g -MSH infusions inhibited these2

LPS-induced increases. In HS024 q g -MSH q LPS-2

treated rats, levels of nitraternitrite were not significantlyŽdifferent from those in rats receiving g -MSHqLPS Fig.2

.6 .

4. Discussion

The regulation of autonomic activity is a fundamentalmechanism influencing the host response to infectionrin-flammation. We demonstrate here that intravenous infusionof g -MSH, a peptide which has potent effects on auto-2

nomic function, attenuates systemic inflammatory re-sponses to endotoxin in rats. We describe four major

Ž .findings: a g -MSH reverses LPS-induced acute hypoten-2Ž .sion and tachycardia; b g -MSH modulates the ACTH2

Ž .response to LPS challenge; c g -MSH inhibits LPS-in-2

duced IL-1b gene expression in the brain and peripheralŽ .tissues; and d g -MSH inhibits LPS-induced increases in2

plasma NO levels. These results suggest that pharmaco-logical manipulation of autonomic activity by g -MSH2

may offer a new approach for controlling the systemicinflammatory response.

The effect of g -MSH in preventing LPS-induced tran-2

sient hypotension and tachycardia is likely due to itspressor and chronotropic activities, which are dependenton sympathetic tone, as previous studies have shown thatpharmacologic suppression of sympathetic drive alters car-diovascular responses to g -MSH. For example, g -MSH2 2

induced increases in arterial pressure and heart rate inŽconscious rats Gruber and Callahan, 1989; De Wildt et al.,

.1993 , and these responses were markedly reduced bypre-treatment with prazosin, an a -adrenoceptor antagonist1Ž .Callahan et al., 1985 . In pithed rats or animals treated

with ganglionic blockers, the pressorrtachycardia action ofŽg -MSH was blunted Sun et al., 1992; Gruber and Calla-2

.han, 1989 . These studies suggest that an intact centrallymediated sympathetic drive is essential for the pressor andcardioexcitatory effects of g -MSH. In addition, the cen-2

tral arginine vasopressin system may also contribute to thepressor response to g -MSH, as i.v. administration of2

g -MSH caused activation of vasopressinergic neurons in2Ž .the hypothalamic supraoptic nucleus Mitchell et al., 1989 .

Blockade of melanocortin receptors using HS024 didnot affect the cardiovascular effects of g -MSH. This2

Ž .result is consistent with the observation Li et al., 1996that the g -MSH-induced pressor response was not blocked2

with intracarotid injection of SHU9119, a MC rMC4 3

receptor antagonist. These data support the idea thatcardiovascular effects of g -MSH are not mediated by any2

of the known melanocortin receptors. Indeed, studies onthe relationship between structure and function of g -MSH2

have shown that its cardiovascular effects are determinedŽby the C-terminal Arg–Phe sequence Klein et al., 1985;

.Gruber and Callahan, 1989; Nijsen et al., 2000 . FMRFrNPFFa peptides, which contain the Arg–Phe sequence,have similar cardiovascular effects as g -MSH, leading to2

the suggestion that the receptor for FMRFrNPFFa pep-tides is a candidate for mediating g -MSH-induced cardio-2

Žvascular responses Versteeg et al., 1998; Nijsen et al.,.2000 .

The reversal of hypotension may have a significantimpact on the process of inflammatory responses, as LPS-induced hypotension leads to reduced tissue perfusion

Ž .during the acute phase response Suffredini et al., 1999 . Ifallowed to progress, reductions in arterial pressure result in

Ž .hypoxia, circulatory collapse septic shock , and multi-organ failure. Improvement of hemodynamic states bypressor and chronotropic actions of g -MSH may block2

these processes, and attenuate inflammatory responses.Ž .Since activation of the central nervous system CNS plays


a key regulatory role in developing the systemic inflamma-tory response, and we have previously demonstrated thatLPS-induced activation of the CNS is related to both

Žimmune input and cardiovascular responses Xia and.Krukoff, 2001 , it is interesting to examine the central

responses after g -MSH treatment. Previous studies have2

shown that systemic administration of LPS augments sym-pathetic output by increasing turnover of norepinephrine,dopamine, and serotonin in several regions of the brainŽ . ŽLavicky and Dunn, 1995 , including the LC Lacosta et

.al., 1999 , a major brainstem noradrenergic center. Wedemonstrate that g -MSH inhibits the LPS-induced in-2

crease in tyrosine hydroxylase gene expression in the LC,suggesting that g -MSH may decrease LPS-enhanced sym-2

pathetic drive through reduction of central catecholamineproduction.

The change in sympathetic tone may also occur throughthe PVN, a major neuroendocrine and autonomic center, asthe LC is known to be functionally associated with the

Ž .PVN Elenkov et al., 2000 . The increase or decrease inthe activity of the LCrsympathetic nervous system by LPSor g -MSH treatment may correspond to altered activity of2

ascending adrenergic projections from the LC to the hypo-thalamus. In the PVN, many neurotransmitters, including

Ž .CRF Jezova et al., 1999 , are found in neurons of theŽ .dorsal parvocellular dp division which project to auto-

Žnomic neurons in the brainstem and spinal cord Valentino.et al., 1993 , and which mediate changes in sympathetic

outflow. We show that g -MSH treatment reduced the2

LPS-induced increases in CRF mRNA levels in the dpdivision, supporting the idea that CRF may be involved inmodulation of autonomic activity during inflammation.

The finding that g -MSH inhibits the LPS-induced2

ACTH response suggests that g -MSH modulates activity2

of the HPA axis during inflammation. Furthermore, reduc-tion of CRF mRNA levels in the parvocellular PVN afterg -MSHqLPS treatment indicates a primary action at the2

transcriptional level. However, while g -MSH alone de-2

creased plasma ACTH levels 2 h after initiation of infu-sion, it had no effect on basal CRF transcription rates in

Ž .the dorsal medial parvocellular mpd division, which con-trols anterior pituitary function. Taken together, these ob-servations suggest that early inhibition of ACTH responsesby g -MSH may be mediated through other mechanisms2

such as inhibition of CRF release. Indeed, melanocortinshave been shown to inhibit CRF release from the hypo-

Ž .thalamus in vitro Suda et al., 1986 and a-MSH inhibitedthe LPSrIL-1b-induced ACTH response and CRF release

Žin vivo and in vitro Rivier et al., 1989; Lyson and.McCann, 1993 .

It has recently been shown that g -MSH inhibits release2Žof IL-1b in a murine model of peritonitis Getting et al.,

.2001 , and we demonstrate here that g -MSH inhibits2

LPS-induced IL-1b gene expression in CVOs in the brain,in the pituitary, and in the thymus gland. These resultssuggest that g -MSH has the ability to modulate IL-1b, an2

important pro-inflammatory cytokine in inflammation. Itshould be noted, however, that g -MSH did not com-2

pletely inhibit IL-1b gene expression and its effectivenessvaried among tissues.

The mechanism of the inhibitory action of g -MSH on2

cytokine production is unknown. g -MSH, like other2

melanocortin peptides, contains a MSH core sequenceŽ .His–Phe–Arg–Arp essential for binding G protein-cou-pled melanocortin receptors. Activation of melanocortinreceptors by receptor–ligand interaction increases intra-

Žcellular cAMP levels Mountjoy et al., 1992; Wikberg et.al., 2000 , a response which is thought to mediate some of

the inhibitory actions of a-MSH and related peptides onpro-inflammatory cytokines and NO production during

Žinflammation Star et al., 1995; Delgado et al., 1998;.Wikberg et al., 2000 . Five melanocortin receptors have

been identified to date, some of which are present onŽ .immune cells: monocytes Bhardwaj et al., 1997 ,

Ž . Žmacrophages Chiao et al., 1996 , and microglia Delgado.et al., 1998 possess MC receptors; mouse macrophages1

Ž .Getting et al., 1999 express MC receptors; and B-3Ž .lymphocytes Buggy, 1998 express MC receptors. Based5

on the evidence that MC receptor has a high affinity for3Ž .g -MSH Roselli-Rehfuss et al., 1993; Wikberg, 1999 , the2

inhibitory effect of g -MSH on cytokine production may2

be mediated through this receptor. However, i.v. adminis-tration of HS024 did not block the inhibitory effects ofg -MSH on LPS-induced IL-1b mRNA levels in the thy-2

mus and on plasma NO production, indicating that theaction via HS024-sensitive melanocortin receptors may notplay a major role in vivo.

In conclusion, i.v. administration of g -MSH attenuates2

LPS-induced systemic inflammatory responses. These ef-fects are associated with changes in central autonomic andneuroendocrine activities, suggesting that modulation ofthese systems contributes to the anti-inflammatory effectsof g -MSH.2

Acknowledgements

This study was supported by the Canadian Institutes ofHealth Research.

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