Ž .Journal of Neuroimmunology 93 1999 149–155

Synergistic interaction of catecholamine hormones and MycobacteriumaÕium results in the induction of interleukin-10 mRNA expression by

murine peritoneal macrophages

Ling Chen, Chad Boomershine, Tianyi Wang, William P. Lafuse, Bruce S. Zwilling )

Departments of Microbiology and Medical Microbiology and Immunology, The Ohio State UniÕersity, 484 West 12th AÕenue, Columbus, OH 43210, USA

Received 20 July 1998; revised 18 September 1998; accepted 18 September 1998

Abstract

The results of this investigation provides evidence that catecholamine hormones interact with macrophages that are infected withMycobacterium aÕium resulting in the induction of IL-10 mRNA and protein. The effect of catecholamine hormones was prevented bytreating the cells with the beta-adrenergic receptor antagonist propranolol but not by alpha-adrenergic antagonist phentolamine. The effectof catecholamine stimulation was mimicked by the addition of beta-2 adrenergic agonists and by the addition of cAMP to the infectedmacrophage cultures. These observations suggest that sympathetic nervous system activation together with microbial infection results in asynergistic interaction that could result in the control of inflammatory processes. q 1999 Elsevier Science B.V. All rights reserved.

Keywords: Catecholamines; Macrophages; Mycobacterium aÕium; Interleukin-10

1. Introduction

Macrophages can control the growth of intracellularpathogens following activation by interferon-gamma. Theproduction of IFN-g, by antigen specific T cells, is con-trolled by the interaction of cytokines produced by NKcells and macrophages. The capacity of macrophages torespond to stimulation with IFN-g is influenced by thepresence of interleukin 10, which itself can be produced by

Žmacrophages Malfeyt et al., 1991a,b; Moore et al., 1993;.Lalani et al., 1997 . Interleukin 10 plays an important role

in regulating not only the ability of the macrophage torespond to stimuli but also plays an important role inregulating the production of pro-inflammatory cytokinesby macrophages, including IL-1a , IL-1b, IL-6, IL-8, G-

ŽCSF and TNF-a Fiorentino et al., 1991; Malfeyt et al.,.1991a; Moore et al., 1993 . In addition, IL-10 suppresses

Žthe production of reactive nitrogen intermediates Gaz-.zininelli et al., 1992 and the expression of MHC class II

) Corresponding author. Tel.: q1-614-292-3310; Fax: q1-614-292-8120; E-mail: zwilling.1@osu.edu

Ž .glycoproteins by macrophages Malfeyt et al., 1991b .IL-10 has also been shown to inhibit its own production by

Ž .macrophages Malfeyt et al., 1991a .Previous studies in our laboratory have shown that

Ž .activation of the hypothalamic–pituitary–adrenal HPAaxis results in an increased susceptibility of mice to thegrowth of Mycobacterium aÕium. Initial studies indicatedthat increased levels of corticosterone correlated with theincreased susceptibility of the mice and a suppression of

Žmacrophage function Brown et al., 1993; Brown and.Zwilling, 1994 . Additional studies also showed that cate-

cholamines produced, by the sympathetic nervous systemor by the adrenal medulla, could also affect macrophagefunction. We have previously reported that catecholaminetreatment of macrophages, via an alpha-2 adrenergic recep-tor, resulted in an increased resistance to mycobacterial

Ž .growth Miles et al., 1996 . In an attempt to furtherunderstand the interaction of catecholamines with M. aÕiuminfected macrophages, we sought to assess the effect ofepinephrine on the production of cytokines that regulatemacrophage function. The results of this investigationindicate that treatment of M. aÕium infected cells with

0165-5728r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0165-5728 98 00220-3

( )L. Chen et al.rJournal of Neuroimmunology 93 1999 149–155150

Fig. 1. Time dependent effect of epinephrine on IL-10 mRNA levels in macrophages infected with M. aÕium. Macrophages were incubated in the presenceŽ . Ž y6 . Ž .of M. aÕium alone lanes 2–6 or M. aÕium and epinephrine 10 M lanes 8–12 for up to 4 h prior to cell extraction and determination of IL-10

Ž . Ž . Ž .mRNA by RNase Protection Assay RPA . Control cells were either not infected lane 1 or infected but not treated with epinephrine lane 7 . The cellswere infected with a bacteria to macrophage ratio of 4:1. Assessment of GAPDH mRNA served as a loading control. The data are representative of one ofthree experiments.

epinephrine stimulated IL-10 production via the beta-2adrenergic receptor.

2. Materials and methods

2.1. Animals

ŽMale, BALBrc mice Charles River Laboratories,.Wilmington, MA were housed in groups of 5 and given

food and water ad libitum. The mice were used asmacrophage donors when 6–8 weeks of age.

2.2. Reagents

Catecholamines and adrenergic antagonists and agonistsŽ .were obtained from Sigma St. Louis, MO These included

epinephrine; the beta antagonist, propranolol; phento-lamine, an alpha antagonist; metoprolol, a b1 antagonist;

ICI 118,551, a b2 antagonist; dobutamine, a b1 agonistand terbutaline, a b2 agonist. N 6,2X-O-Dibutryladenosine

X X Ž .3 5 -cyclic monophosphate dibutyryl-cAMP , was also ob-tained from Sigma. The IL-10 ELISA kit was purchased

Ž .from Genzyme Cambridge, MA .

2.3. Macrophage and mycobacterial preparation

Macrophages were obtained by peritoneal lavage 3–4days following the intraperitoneal injection of 4% sterilethioglycollate medium. This operationally definedmacrophage population has recently arrived from the bloodin response to the sterile irritant. The cells were washedwith PBS and resuspended in complete Iscoves Modified

ŽDulbeccos Medium IMDM, Gibco BRL, Grand Island,. ŽNY containing 20% fetal bovine serum HyClone, Logan. Ž .UT and 1% penicillin–streptomycin Gibco BRL . The

macrophages were purified by adding 8=106 cells perwell in 6 well culture plates and culturing the cells for 18 h

Fig. 2. Dose dependent effects of epinephrine on IL-10 mRNA expression by M. aÕium infected cells. Macrophages were treated with 10y8 to 10y5 Mepinephrine or not treated for 2 h prior to extraction and determination of IL-10 mRNA by RPA. Untreated macrophages are in lane 1; macrophagesinfected with M. aÕium alone are in lane 2. GAPDH expression served as a loading control. The data are representative of one of three experiments.

( )L. Chen et al.rJournal of Neuroimmunology 93 1999 149–155 151

Table 1IL-10 produced by M. aÕium and epinephrine stimulated macrophages

Ž .Treatment IL-10 pgrmg cell protein

None 105"44aM. aÕium 1533"109

Epinephrine 248"109bM. aÕiumqEpinephrine 2558"163

Macrophages were infected with M. aÕium at a 4:1 bacteria to macrophageratio and treated with 10y6 M epinephrine. The cultures were incubatedfor 18 h prior to determination of IL-10 in the supernatant fluid byELISA. The data are expressed as the mean"SE.aSignificantly different for uninfected or epinephrine treated, p-0.05.bSignificantly different from epinephrine treated or from M. aÕiuminfected, p-0.05 by Student’s t-test.

at 378C in 5% CO . The monolayers were washed with2

IMDM to remove non-adherent cells and infected with M.aÕium, at a 4:1 bacteria to macrophage ratio, for 2 h or asspecified in each experiment. Infectivity was determined

Žby acid fast staining of the cells. The M. aÕium ATCC.35715, American Type Culture Collection, Rockville, MD

Žwas cultured in Middlebrook 7H9 broth Difco, Detroit,.MI , aliquoted in 1 ml amounts and stored frozen at

y708C until use. Each vial contained 2.8=108 cfu asdetermined by plate count on 7H11 agar plates.

2.4. RNA preparation and RNase protection assay

Macrophage monolayers were washed with PBS priorŽto lysis with 0.5 ml of lysing solution 4 M guanidium

isothiocyanate, 25 mM sodium citrate, 0.8% 2-mercapto-.ethanol . The total RNA was isolated using the protocol

Ž .described by 5Prime–3Prime Boulder, CO with modifi-cations. The lysate was transferred to a 1.5 ml phase lock

Ž . Ž .gel PLG tube 5 Prime–3Prime , followed by the addi-tion of 33 ml of 3M sodium acetate, 350 ml phenolrH O2

Ž .and 125 ml chloroformrisoamyl alcohol 25:24 . The tubewas mixed well and centrifuged at 12,000=g for 5 min.The top phase was transferred to a new 1.5 ml PLG tubeand 500 ml of phenolrchloroformrisoamyl alcoholŽ .25:24:1 was added. After centrifugation at 12,000=gfor 5 min, the top layer was transferred to a 1.5 mlEppendorf tube, 500 ml of isopropanol was added, thecontents mixed by vortexing, and then incubated at y208Cfor 30 min. The RNA was pelleted by centrifugation at12,000=g for 15 min. The RNA was then washed in 70%ethanol, air dried, resuspended in RNase free water, incu-bated for 15 min at 658C and stored at y208C.

Ž .The RNase protection assay RPA was conducted us-Ž .ing a kit obtained from Pharmingen San Diego, CA .

Briefly, the anti-sense RNA multi-probe set was synthe-sized by in vitro transcription of mouse cytokine template

Ž .set mck2 or 2b using T7 RNA polymerase in the pres-w 32 x Žence of a- P UTP Sp. Act. 3000 CirmM, Amersham,

. Ž 4 .Arlington Heights, IL . The probe set 6=10 cpmrmlŽ .was hybridized with target RNA 2–3 mg at 568C

overnight in a total of 10 ml of hybridization buffer. Thefree probe and single stranded target RNA were digestedwith RNase at 308C for 45 min. The proteins were digestedby treating with proteinase K for 15 min at 378C andextracted with phenol:chloroform:isoamyl alcohol

Fig. 3. Epinephrine induced expression of IL-10 mRNA is blocked by a non-specific beta antagonist. Macrophages were treated with M. aÕium andŽ y6 . Ž y8 y5 .epinephrine 10 M in the presence of increasing concentrations 10 to 10 M of phentolamine, an alpha receptor antagonist or with propranolol, a

Ž .beta receptor antagonist lanes 3–6 . After 2 h the cells were extracted and the levels of IL-10 mRNA determined by RPA. Control cultures wereŽ . y6 Ž .uninfected lane 1 or infected and treated with 10 M epinephrine lane 2 . GAPDH expression served as a loading control. The data are representative

of one of three experiments.

( )L. Chen et al.rJournal of Neuroimmunology 93 1999 149–155152

Ž .25:24:1 . The aqueous phase was removed and the pro-tected RNA precipitated with ethanol. The pellet waswashed with 90% ethanol, air dried and resuspended in 4ml of loading buffer. The sample was then heated for 3

Žmin at 908C and separated on 6% sequencing gel Amresco,.Solon, OH . The gel was then transferred to filter paper

Ž .Whatman , vacuum dried, and exposed to X-ray filmŽ .Kodak, MR film for 3 to 18 h at y708C.

3. Results

Epinephrine stimulates IL-10 production in M. aÕiuminfected macrophages. The results in Fig. 1 show that

Ž y6 .epinephrine 10 M induced the expression of IL-10mRNA within 1 h. The level of IL-10 increased until 2 hand then decreased. While only low levels of IL-10 mRNAwere transiently induced by infection with M. aÕium alone.The effect of treatment with different doses of epinephrineshowed that IL-10 mRNA was induced by as little as 10y7

M epinephrine but required 10y6 M for maximal inductionŽ .Fig. 2 . The results in Table 1 show that treatment ofmacrophages with M. aÕium together with epinephrineresulted in the production of 2558 pgrmg of cell proteinof IL-10. Macrophages infected with M. aÕium aloneproduced 1533 pgrmg cell protein of IL-10.

Fig. 5. The effect of epinephrine is prevented by the beta-2 specificantagonist ICI 118,551. Macrophages were treated with 10y6 Mepinephrine in the presence of 10y8 to 10y5 M metoprolol, a beta 1

Ž . Ž .antagonist lanes 4–7 or with ICI 118,551 lanes 8–11 , a beta 2 specificantagonist. Lanes 1: uninfected; lane 2: infected with M. aÕium alone;lane 3: M. aÕium plus epinephrine. The data are representative of one ofthree experiments.

3.1. Epinephrine stimulates IL-10 Õia beta adrenergicreceptors

Catecholamines can bind to either alpha or beta adren-ergic receptors. In order to identify the ligand-receptor

Fig. 4. A beta-2 receptor agonist but not a beta-1 receptor agonist mimics the effect of epinephrine on IL-10 mRNA. Macrophages were treated with M.Ž . Ž y6 . Ž . y8 y5aÕium alone lane 2 ; M. aÕium and epinephrine 10 M lane 3 or M. aÕium and 10 to 10 M dobutamine, a beta-1 agonists or with terbutaline, aŽ .beta-2 agonist lanes 4–7 . Lane 1 is uninfected macrophages. RNA was extracted 2 h later and the levels of IL-10 mRNA determined by RPA. GAPDH

expression served as a loading control. The data are representative of one of three experiments.

( )L. Chen et al.rJournal of Neuroimmunology 93 1999 149–155 153

Fig. 6. Effect of dibutryl-cAMP on the production of IL-10 mRNA.Macrophages were incubated with M. aÕium in the presence or absence

y4 Ž .of 10 M dibutyryl-cAMP for up to 4 h lanes 2–6 . Lane 1 isuntreated. The level of IL-10 mRNA was determined by RPA. GAPDHexpression served as a loading control. The data are representative of oneof three experiments.

interaction that was responsible for the increase in IL-10mRNA, we treated cells with the beta receptor antagonistpropranolol or the alpha receptor antagonist phentolamine.The results in Fig. 3 show that the stimulatory effect ofepinephrine was inhibited by the addition of the betaantagonist propranolol in a dose dependent manner but notby the alpha antagonist phentolamine.

To identify which b receptor was involved in stimulat-ing IL-10 mRNA, we used the b1 specific agonist dobu-tamine and the b2 specific agonist terbutaline to stimulatethe macrophage cultures. The results in Fig. 4 show thatthe stimulation of IL-10 mRNA is the result of b2 adren-ergic stimulation. The b2 agonist was effective at a con-centration 100 fold less than the b1 agonist.

In order to confirm that the stimulation of IL-10 mRNAwas the result of b2 receptor stimulation we treated theepinephrine treated cells with b1 or b2 specific antago-nists, metoprolol and ICI 118,551, respectively. The result,in Fig. 5 shows that the b2 specific antagonist ICI 118,551prevented the increase in IL-10 mRNA following theaddition of epinephrine. The b1 antagonist was only effec-tive at a concentration 3 logs higher than the b2 antago-nist.

3.2. cAMP Stimulates IL-10 mRNA

Beta adrenergic receptor stimulation results in an in-Ž .crease in intracellular cAMP Stadel, 1991 . The addition

of dibutryl-cAMP to macrophage cultures infected with M.aÕium resulted in an increase in IL-10 mRNA within 1 hŽ .Fig. 6 .

4. Discussion

The results of this investigation show that cate-cholamine stimulation of murine peritoneal macrophagesinfected with M. aÕium resulted in the stimulation of

IL-10 mRNA expression and cytokine production. Infec-tion with M. aÕium alone or treatment with epinephrine ornorepinephrine alone did not result in the stimulation ofIL-10. Thus, the combination of stimuli acted synergisti-cally to induce IL-10 mRNA and cytokine synthesis.

Our observations are similar to those reported by VanŽ .der Poll and Lowry 1997 who found that epinephrine

increased the level of IL-10 produced by human peripheralblood mononuclear cells that had been stimulated withbacterial lipopolysaccharide. Similarly, Suberville et al.Ž .1996 found that isoproterenol, a non-specific beta ago-nist, stimulated IL-10 production by LPS activated mouseperitoneal macrophages only if the beta agonists was addedprior to the addition of LPS. Others have reported that M.aÕium infection induced IL-10 production by human

Ž .monocytes Muller et al., 1998 . Our observations, thatbeta-2 adrenergic agonists act synergistically with M.aÕium extend these previous observations.

We showed that the stimulatory effect of the catechol-amines was mediated by beta-2 adrenergic stimulation inseveral ways. First, the addition of the beta adrenergicantagonist propranolol prevented the increase in IL-10mRNA while the alpha adrenergic antagonist phentolaminewas without effect. In addition we used beta 1 and beta 2specific agonists to show that the effect of catecholaminestimulation could be mimicked by the beta 2 specificagonist terbutaline. That the beta 2 receptors were mediat-ing the increase in IL-10 mRNA was further confirmedwhen we showed that only the b2 specific antagonist ICI118,551 prevented the epinephrine mediated effect.

The promoter region of mouse IL-10 contains both aŽ .cAMP response element CRE located at y419 to y414

relative to the start codon and an NFk like binding site atBŽ .y553 to y543 Platzer et al., 1994 . Since activation of

cAMP results from beta adrenergic stimulation and NFk B

becomes activated following infection with M. aÕium, thebinding of both sites appears necessary for the up regula-tion of IL-10 mRNA synthesis. We found that the additionof cAMP to M. aÕium infected cells also resulted in the upregulation of IL-10 mRNA.

Catecholamine stimulation of macrophages has beenŽ .reported to both inhibit Van der Poll and Lowry, 1997

Ž .and stimulate Spengler et al., 1994 changes in functionalcapacity. We have previously reported that catecholaminestimulation of interferon-gamma activated macrophagesresulted in an inhibition of MHC class II expressionŽ . Ž .Zwilling et al., 1987 . Similarly, Koff and Dunegan 1985have reported that treatment of macrophages withepinephrine inhibited their anti-tumor activity. We toohave observed that epinephrine inhibited the antimicrobialactivity of interferon gamma activated murine peritoneal

Ž .macrophages unpublished observations . Catecholamineshave also been shown to inhibit the production of TNF-a

Žand IL-1 via beta adrenergic pathways Severn et al.,.1992 . However, treatment of some macrophage popula-

tions with catecholamine hormones results in stimulation

( )L. Chen et al.rJournal of Neuroimmunology 93 1999 149–155154

Žof TNF-a . Thus, Spengler and co-workers Spengler et al.,.1990; Ignatowski et al., 1996 have reported that peritoneal

macrophages, obtained from mice two weeks after aninjection of complete Freund’s adjuvant, produced moreTNF-a following stimulation with LPS and norepineph-rine. The stimulation was shown to be mediated via alphaadrenergic stimulation. Similarly, we have previously re-ported that epinephrine treatment of resident peritonealmacrophages stimulated the capacity of the macrophages

Ž .to inhibit the growth of M. aÕium Miles et al., 1996 . Thestimulation of antimicrobial activity was inhibited by alphaadrenergic antagonists. The antimycobacterial activity wasalso stimulated by the addition of clonidine, an alpha-2adrenergic agonist but not by alpha 1 or beta agonists.Together these results suggest that the functional capacityof macrophages can be stimulated or inhibited by catechol-amines. The different responses are probably mediated bydifferences in adrenergic receptor expression of opera-tionally defined macrophage subpopulations. This possibil-ity is currently being explored by us.

Infection of macrophages with an intracellular microbialpathogen such as Mycobacteria or stimulation with bacte-rial lipopolysaccharide results in the production of pro-in-flammatory cytokines such as IL-1, IL-6 and TNF-aŽ .Malfeyt et al., 1991a . These cytokines have been shownto affect the hypothalamus, probably via the vagal nerve,and stimulate the production of corticotrophin releasing

Žfactors that result in activation of the HPA axis Goehler et.al., 1997; Maier et al., 1998 . IL-1 and TNF-a have also

been shown to stimulate the sympathetic nervous systemresulting in the release of adrenal medullary epinephrineand the stimulation of peripheral adrenergic neurons that

Žrelease norepinephrine into the micro environment Be-.sedovsky et al., 1983; Felten et al., 1987 . The interaction

of these catecholamine hormones with macrophages in-fected with M. aÕium results in the stimulation of IL-10, acytokine that has been shown to have anti-inflammatoryeffects by virtue of its capacity to inhibit the production ofcytokines that are stimulated by bacterial infection of themacrophages. Thus, sympathetic nervous system activationresulting from cytokine production by infectedmacrophages together with sympathetic neurotransmitterscan act together to control inflammatory processes.

Acknowledgements

Supported by NIH grants AI42901, HL59795 andMH54966.

References

Besedovsky, H., Del Ray, A., Sorkin, E., Da Prada, M., Barri, R.,Honegger, C., 1983. The immune response evokes changes in brainnoradrenergic neurons. Science 221, 564–566.

Brown, D.H., Sheridan, J., Pearl, D., Zwilling, B.S., 1993. Regulation ofmycobacterial growth by the hypothalamic–pituitary–adrenal axis:differential responses of Mycobacterium boÕis BCG-resistant and-susceptible mice. Infect. and Immun. 61, 4793–4800.

Brown, D.H., Zwilling, B.S., 1994. Activation of the hypothalamic–pitui-tary–adrenal axis differentially affects the anti-mycobacterial activityof macrophages from BCG-resistant and susceptible mice. J. Neu-roimmunol. 53, 181–187.

Felten, D.L., Ackerman, K.D., Wiegand, S.J., Felten, S.Y., 1987. Nora-drenergic sympathetic innervation of the spleen: I. Nerve fibersassociate with lymphocytes and macrophages in specific compart-ments of the splenic white pulp. J. Neurosci. Res. 18, 28–36.

Fiorentino, D.F., Zlotnik, A., Mosmann, T.R., Howard, M., O’Garra, A.,1991. IL-10 inhibits cytokine production by activated macrophages. J.Immunol. 147, 3815–3822.

Gazzininelli, R.T., Oswald, I.P., James, S.L., Sher, A., 1992. IL-10inhibits parasite killing and nitrogen oxide production by IFN-gammaactivated macrophages. J. Immunol. 148, 1792–1796.

Goehler, L.E., Relton, J.K., Dripps, D., Kiechle, R., Tartaglina, N.,Maier, S.F., Watkins, L.R., 1997. Vagal paraganglia bind biotinylatedinterleukin-1 receptor antagonist: a possible mechanism for immuneto brain communication. Brain Res. Bull. 43, 357–364.

Ignatowski, T.A., Gallant, S., Spengler, R.N., 1996. Temporal regulationŽ .by adrenergic receptor stimulation of macrophage Mf derived

Ž .tumor necrosis factor TNF production post-LPS challenge. J. Neu-roimmunol. 65, 107–117.

Koff, W.C., Dunegan, M.A., 1985. Modulation of macrophage mediatedtumoricidal activity by neuropeptides and neurohormones. J. Im-munol. 135, 350–354.

Lalani, I., Bhol, K., Ahmed, A.R., 1997. Interleukin-10: biology, role ininflammation and autoimmunity. Ann Allergy, Asthma and Immunol.79, 469–484.

Maier, S.F., Goehler, L.E., Fleshner, M., Watkins, L.R., 1998. The roleof the vagus nerve in cytokine-to-brain communication. Ann. N.Y.Acad. Sci. 840, 289–300.

Malfeyt, R.W., Abrams, J.M., Bennett, B., Figdor, C.G., Vries, J.E.,Ž .1991a. Interleukin 10 IL10 inhibits cytokine synthesis by human

monocytes: an autoregulatory role of Il-10 produced by monocytes. J.Exp. Med. 174, 1209–1220.

Malfeyt, R.W., Hannen, J., Spits, H., Roncarolo, M.-G., Velde, A.,Figdor, C., Johnson, K., Kastelein, R., Yssel, H., Vries, J.E., 1991b.Interleukin 10 and viral IL-10 strongly reduce antigen specific humanT cell proliferation by diminishing the antigen-presenting capacity ofmonocytes via down regulation of class II major histocompatibilitycomplex expression. J. Exp. Med. 174, 915–924.

Miles, B.A., Lafuse, W.P., Zwilling, B.S., 1996. Binding of a-adrenergicreceptors stimulates the anti-mycobacterial activity of murine peri-toneal macrophages. J. Neuroimmunol. 71, 19–24.

Moore, K.W., O’Garra, A., Malfeyt, R.W., Vieira, P., Mosmann, T.R.,1993. Interleukin-10. Ann. Rev. Immunol. 11, 165–190.

Muller, F., Aukrust, P., Lien, E., Haug, C.J., Froland, S.S., 1998.Enhanced interleukin-10 production in response to MycobacteriumaÕium products in mononuclear cells from patients with humanimmunodeficiency virus infection. J. Infect. Dis. 177, 586–594.

Platzer, C., Volk, H.-D., Platzer, M., 1994. 5X Noncoding sequence ofhuman IL-10 gene obtained by oligo-cassette PCR walking. DNAsequence 4, 399–401.

Severn, A., Rapson, N.T., Hunter, C.A., Liew, F.Y., 1992. Regulation oftumor necrosis factor production by adrenaline and b-adrenergicagonists. J. Immunol. 148, 3441–3445.

Spengler, R.N., Allen, R.M., Remick, D.G., Strieter, R.M., Kunkel, S.L.,1990. Stimulation of a-adrenergic receptor augments the productionof macrophage derived tumor necrosis factor. J. Immunol. 145,1430–1434.

Spengler, R.N., Chensue, S.W., Giacherio, D.A., Blenk, N., Kunkel, S.L.,1994. Endogenous norepinephrine regulates tumor necrosis factor-aproduction from macrophages in vitro. J. Immunol. 152, 3024–3031.

( )L. Chen et al.rJournal of Neuroimmunology 93 1999 149–155 155

Stadel, J.M., 1991. b-Adrenoceptor Signal Transduction. In: Ruffolo,Ž .R.R., Jr. Ed. , b-Adrenoceptors: Molecular Biology, Biochemistry

and Pharmacology, Prog. Basic Clin. Pharmacol. Karger, Basel,Switzerland, 7, 67–104.

Suberville, S., Bellocq, A., Fouqueray, B., Philippe, C., Lantz, O., Perez,J., Baud, L., 1996. Regulation of interleukin-10 production by betaadrenergic agonists. Eur. J. Immunol. 26, 2601–2605.

Van der Poll, T., Lowry, S.F., 1997. Epinephrine inhibits endotoxin-in-duced IL-1b production: roles of tumor necrosis factor-a and IL-10.Am. J. Physiol. 273, R1885–R1890, Regulatory Integrative CompPhysiol., 42.

Zwilling, B.S., Vespa, L., Massie, M., 1987. Regulation of I-A expres-sion by murine peritoneal macrophages: differences linked to the Bcggene. J. Immunol. 138, 1372–1376.