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IL-12, IL-18 and IFN-gamma in the immune response to bacterial infection

Lauw, F.N.

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Citation for published version (APA):Lauw, F. N. (2000). IL-12, IL-18 and IFN-gamma in the immune response to bacterial infection.

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Download date: 21 Feb 2021

ChapterChapter 14

Epinephrinee inhibit s the production of interferon-y and

interleukin-122 in whole blood stimulated with endotoxin

Fannyy N. Lauw, Peter Speelman, Sander J.H. van Deventer, and Tom van der Poll

Fromm the Laboratory of Experimental Internal Medicine and the Department of Infectious Diseases,

Tropicall Medicine and AIDS, University of Amsterdam, Amsterdam, the Netherlands.

ChapterChapter 14

Abstract t

Systemicc inflammation leads to the activation of multiple host inflammatory responses,

includingg the cytokine network and the release of stress hormones. To determine the effects

off epinephrine on the release of IFN-y and IL-12, two proinflammatory cytokines which

havee been implicated to contribute to the pathogenesis of sepsis and endotoxemia, human

wholee blood was stimulated in vitro with increasing concentrations of epinephrine.

Epinephrinee dose-dependently inhibited IFN-y and IL-12p70 production stimulated by

severall bacterial stimuli, an effect which was mediated by an effect of epinephrine on P

adrenergicc receptors, since it could be prevented by addition of a specific P-adrenergic

antagonist,, while an a-adrenergic antagonist had no effect. In addition, incubation with the

cAMPP analog db-cAMP dose-dependently inhibited IFN-y release stimulated by LPS. In

thee presence of anti-IL-12, epinephrine did not affect IFN-y concentrations in U n-

stimulatedd whole blood, suggesting that epinephrine reduces IFN-y release indirectly

throughh inhibition of IL-12 production. These data suggest that, during acute infection,

epinephrinee may attenuate excessive release of IFN-y and IL-12, but may also contribute to

aa decreased Thl type immune response.

182 2

EpinephrineEpinephrine inhibits IFN-y and IL-12 production

Introductio n n

Interferon-yy (IFN-y) is an important proinflammatory cytokine which is mainly produced by

CD4++ T helper 1 (Thl) cells, CD8+ T cells and natural killer (NK) cells (1). IFN-y is a

potentpotent stimulator of monocyte/macrophage activities, including the production of cytokines

andd phagocytosis (1, 2). IFN-y has been demonstrated to play an important role during

sepsiss and endotoxemia. Elevated plasma concentrations of IFN-y can be found in a subset

off patients with sepsis, and during experimental sepsis in primates (3-6). During

experimentall endotoxemia in mice, neutralization of IFN-y protected against lethality, while

IFN-yy receptor deficient mice were resistant against endotoxin (LPS)-induced shock (7, 8).

Interleukin-122 (IL-12) is a heterodimeric proinflammatory cytokine formed by a p35 and a

p400 subunit, which is produced mainly by antigen-presenting cells. IL-12 plays a central

rolee in both innate immunity by stimulation of T and NK cell activity, and adaptive

immunityy by promoting the differentiation of naive T cells into Thl type cells (9, 10).

Elevatedd levels of IL-12 have been measured in during clinical and experimental sepsis (4-

6).. IL-12 has been demonstrated to be important for IFN-y production during endotoxemia

inn mice, and neutralization of IL-12 protected against LPS-induced lethality (11,12).

Administrationn of LPS to healthy humans leads to the activation of the cytokine

networkk and induces a characteristic stress hormone release (13). Several studies have

demonstratedd that stress hormones can influence the production of cytokines during

experimentall human endotoxemia. Infusion of epinephrine or glucocorticoids have been

shownn to alter the cytokine response elicited by LPS characterized by reduced TNF

productionn and enhanced IL-10 release (14-16). Knowledge of the effects of epinephrine on

IFN-yy and IL-12 release is limited. Such knowledge may contribute to the understanding of

thee endogenous effects of stress hormones during acute systemic infection on the cytokine

network,, and also for the therapeutic use of catecholamines in patients with systemic shock.

Itt is difficult to study the effects of epinephrine on IFN-y and IL-12 release induced by LPS

inn humans in vivo, since in the widely used model of human endotoxemia, IFN-y and IL-12

concentrationss are hardly detectable. Therefore, in the present study, we studied the effect

off epinephrine on IFN-y and IL-12 production by different bacterial stimuli during whole

bloodd stimulation in vitro, a condition which is considered to mimic the human in vivo

conditionn most closely (17). In addition, since IL-12 is known to be important stimulator of

IFN-yy production, we examined whether epinephrine influences IFN-y production directly

orr whether this effect is mediated through effects of epinephrine on IL-12 release.

183 3

ChapterChapter 14

Material ss and methods

Reagents s

LPSS (from Escherichia coli serotype Oil 1:B4) and Staphylocoocal enterotoxin B (SEB)

weree obtained from Sigma (St. Louis, MO). Heat-killed Staphylococcus aureus (HKSa) were

preparedd from reference strain 14459B from the National Institute of Public Health and the

Environmentt (Bilthoven, the Netherlands). The isolate was suspended in 50 ml Todd-Hewitt

brothh and cultured overnight in 5% C02 at 37°C. This suspension was diluted in fresh medium

thee next morning and incubated until log-phase growth was obtained. Thereafter, 10-fold

dilutionss of this suspension were made and plated on blood agar plates for colony-forming unit

(CFU)) counts. Bacteria were harvested by centrifugation, washed twice in pyrogen-free 0.9%

NaCl,, resuspended in 20 ml 0.9% NaCl, and heat inactivated for 60 minutes at 80°C. A 500-ul

samplee on a blood agar plate did not show growth of bacteria. Dilutions were made in

pyrogen-freee RPMI 1640 (Bio Wittaker, Verviers, Belgium).

Adrenergicc agents were obtained from the following manufacturers: epinephrine from

Centrafarmm Services (Etten-Leur, the Netherlands), phentolamine from Ciba-Geigy (Basel,

Switzerland),, and propranolol from Zeneca (Ridderkerk, the Netherlands). Dibutyryl-cyclic

AMPP (db-cAMP) was purchased from Sigma. Neutralizing mouse-anti-human IL-12 MAb and

controll mouse IgG were obtained from R&D Systems (Abingdon, UK).

Wholee blood stimulation

Wholee blood was collected aseptically from 6 healthy individuals using a sterile collecting

systemm consisting of a butterfly needle connected to a syringe (Becton Dickinson & Co,

Rutherford,, NJ). Anticoagulation was obtained using endotoxin-free heparin (Leo

Pharmaceuticall Products B.V., Weesp, the Netherlands; final concentration 10 U/ml blood).

Wholee blood, diluted 1:1 in RPMI, was stimulated for 24 h at 37°C with LPS (10 ng/ml),

HKSaa (107 CFU/ml) or anti-CD/anti-CD28 double stimulation (both 1:1000), in the

presencee or absence of increasing concentrations of epinephrine (10"7-10~5 M). To

determinee by which mechanism epinephrine exerts its effects, whole blood was stimulated

withh LPS (10 ng/ml) with or without epinephrine (10"6 M), phentolamine (10"5 M),

propranololl (105 M), or db-cAMP (10"6-10~4 M). In some experiments, neutralizing mAbs

directedd against human IL-12 or isotype-matched control mouse IgG were used (both 10

Hg/ml).. This concentration of anti-IL-12 completely neutralizes activity of recombinant

humann (rh)IL-12 when added at 1-2 log higher concentrations compared with levels found

afterr whole blood stimulation with LPS (information on the neutralizing capacity of the

mAbb was provided by the manufacturer). After the incubation, supernatant was obtained

afterr centrifugation (1600 x g for 15 minutes at 4°C) and stored at -20°C until assays were

performed. .

184 4

EpinephrineEpinephrine inhibits IFN-yand IL-12 production

Assays s

IFNyy and IL-12p70 were measured by specific ELISAs according to the instructions of the

manufacturerr (Central Laboratory of the Netherlands Red Cross Blood Transfusion Service,

Amsterdam,, the Netherlands). The detection limits of the assays were 2.4 pg/ml (IFN-y)

and6.2pg/ml(IL-12p70). .

Statistics s

Al ll data are expressed as mean SE of six donors. Statistical analysis was performed by

Wilcoxonn test. P < 0.05 was considered to represent a significant difference.

Results s

Effectt of epinephrine on IFN-y and IL-1 2 production induced by different bacterial

stimuli i Incubationn of whole blood for 24 h at 37°C without stimulus did not result in detectable

levelss of IFN-y or IL-12p70. Also, epinephrine did not induce cytokine production when

addedd in the absence of a stimulus. Incubation with LPS, HKSa or SEB resulted in high

concentrationss of IFN-y, while low concentrations of IL-12p70 were found (Figure 1).

Additionn of epinephrine induced a strong dose-dependent inhibition of IFN-y and IL-12p70

afterr stimulation with LPS or HKSa, while the inhibitory effect on SEB cytokine

productionn was less strong.

logg 10 [epinephrine! 8 1 0 [epinephrine]

Figuree 1. Epinephrine dose-dependently inhibits LPS-stimulated IFN-y and IL-12p70 release in wholee blood Whole blood from six different donors, diluted 1:1 in RPMI, was incubated for 24 h at 37°C,, with LPS (10 ng/ml), HKSa (107 CFU/ml) or SEB (1 ug/ml) in the presence or absence of increasingg concentrations of epinephrine. Data are expressed as percentage change (mean SE) relativee to incubation with LPS, HKSa or SEB only. IFN-y concentrations were 10.01 2.65 ng/ml (LPSS only) 16 61 7.2 ng/ml (HKSa), and 11.91 4.68 ng/ml (SEB). IL-12p70 concentrations were 73.466 37.'11 pg/ml (LPS), 62.92 23.86 pg/ml (HKSa), and 41.56 26.29 pg/ml (SEB). P < 0.05 vs.. LPS, HKSa or SEB only.

185 5

ChapterChapter 14

Epinephrin ee inhibit s IFN-y production mainly through (3-adrenergic stimulation

Sincee it is known that epinephrine can bind to both a (a, and ct2) and P (P, and p2)

adrenergicc receptors, we sought to determine whether the effect of epinephrine on IFN-y

productionn was mediated through a or p adrenergic stimulation. Therefore, whole blood

wass incubated with LPS and epinephrine (10"6M) in the presence or absence of a specific a

adrenergicc receptor antagonist phentolamine (10"5 M), and /or the specific P receptor

antagonistt propranolol (10~5 M). In the absence of epinephrine, neither phentolamine nor

propranololl affected LPS-induced cytokine production. Epinephrine again strongly

inhibitedd LPS-induced IFN-y production (Figure 2). a receptor blockade with phentolamine

hadd no effect on epinephrine-induced inhibition of LPS-induced IFN-y release. In contrast,

PP receptor blockade with propranolol completely prevented the inhibiting effect of

epinephrine.. The combination of propranolol and phentolamine resulted in IFN-y

concentrationss similar to those found after addition of propranolol alone. These data

suggestt that epinephrine inhibits LPS-induced IFN-y production by an effect on the 0-

adrenergicc receptor.

Figuree 2. Effect of a and/or p adrenergic receptorr blockade on IFN-y production by LPS-stimulatedd whole blood. Whole blood fromm six different donors, diluted 1:1 in RPMI,, was incubated for 24 h at 37°C, with LPSS (10 ng/ml). Epi, with epinephrine (epi, 100 M). A, with epinephrine and the a antagonistt phentolamine (105 M). B with epinephrinee and the P antagonist propranololl (105 M). C, with epinephrine andd phentolamine and propranolol. Data are expressedd as percentage change (meanSE)) relative to incubation with LPS only. * P << 0.05 vs. LPS only.

db-cAM PP inhibit s IFN-y release

P-adrenergicc stimulation is known to result in increased levels of intracellular cAMP (18).

Therefore,, we wanted to determine the effects of db-cAMP on LPS-induced IFN-y

production.. The addition of dbcAMP caused a dose-dependent inhibition of IFN-y release

stimulatedd by LPS (Fig. 3).

Effectt of anti-IL-1 2 on epinephrine-induced inhibitio n ofIFN- y production

Itt has been demonstrated in vitro and in mice that LPS-induced IFN-y production is largely

dependentt on IL-12 (11, 12, 19). Having established that epinephrine inhibits IL-12p70

productionn stimulated by LPS, we were interested whether the inhibiting effect of

50 0

25 5

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** -50

-75 5

-100 0

IFN-y y

TT I :: I

1 1 * *

JL L * *

epi i

186 6

EpinephrineEpinephrine inhibits IFN-yand IL-12 production

epinephrinee on IL-12p70 production contributes to epinephrine-induced inhibition IFN-y.

Therefore,, whole blood was incubated with LPS in the presence or absence of epinephrine

(10"66 M), a neutralizing anti-IL-12 Ab, or an irrelevant control Ab (10 ug/ml). Anti-IL-12

stronglyy reduced LPS-induced IFN-y (Table 1). In the presence of anti-IL-12, addition of

epinephrinee had no effect on LPS-induced IFN-y release.

Figuree 3. dbc-AMP dose-dependently

inhibitss LPS-induced IFN-y release in

wholee blood. Whole blood from six

differentt donors, diluted 1:1 in RPMI, was

incubatedd for 24 h at 37°C, with LPS (10

ng/ml)) in the presence or absence of

increasingg concentrations of db-cAMP.

Dataa are expressed as percentage change

(meann SE) relative to incubation with

LPSS only. * P < 0.05 vs. LPS only.

Tablee 1. Epinephrine does not affect LPS-induced IFN-y production in the presence of anti-IL-12.

IFN-yy (ng/ml)

LPSS 9.39 6

LPSS + epi 4.05 + 2.19* LPSS + anti-IL-12 1.30 * LPSS + anti-IL-12 + epi 1.13 0.58 *#

Valuess are mean SE of six different donors. Whole blood, diluted 1:1 in RPMI, was incubated for 244 h at 37°C with LPS (10 ng/ml) in the presence or absence of epinephrine (epi, 10"6 M) and/or anti-1L-122 (10 ug/ml). * P < 0.05 vs. LPS only. * P < 0.05 vs. LPS + epi.

Discussion n

Duringg acute infection, both the production of cytokines and the release of stress hormones

aree increased. Previous studies have established that complex interactions exist between

sfresss hormones and the cytokine network. Administration of proinflammatory cytokines

includingg TNF, IL-1 and IL-6 induce the release of stress hormones in humans and primates

(20-22).. Further, the catecholamines epinephrine and norepinephrine have been shown to

inhibitt the production of LPS-stimulated TNF, IL-6 and IL-i p in vitro, while increasing the

releasee of IL-10 (15, 18, 23). Also, infusion of epinephrine or glucocorticoids in humans

e " 2 5 5

O) ) c c TO TO

55 -50

-75 5 IFN-y y * *

* * -66 -5 -4

logg 10 [db-cAMP]

187 7

ChapterChapter 14

inhibitedd TNF release during experimental endotoxemia, while enhancing IL-10 secretion (14-16). .

Wee demonstrate here that epinephrine inhibits production of IFN-y and IL-12 induced

byy several bacterial stimuli during whole blood stimulation in vitro. Similar concentrations

off epinephrine have been used in previous in vitro studies to determine the effect of

epinephrinee on cytokine release (15, 23, 24). The inhibitory effects of epinephrine was

moree pronounced on LPS and HKSa-stimulated IFN-y and IL-12 production compared to

thee release induced by SEB. This discrepancy may reflect the different mechanism of cell

activationn involved with the stimuli, i.e. LPS and HKSa mainly activate

monocytes/macrophages,, while the bacterial superantigen SEB, after simultaneously

bindingg to MHC class II molecules of an APC and the specific V(3 domain of the T cell

receptor,, activates large fractions of the T cell population (25). Epinephrine inhibited IFN-y

releasee by an effect on p-adrenergic receptors. Indeed, |3-adrenergic blockade by

propranololl completely prevented the inhibition of epinephrine on LPS-induced IFN-y

production.. In contrast, a-adrenergic blockade by phentolamine did not influence IFN-y

release. .

Elevationn of intracellular cAMP levels is a well-known postreceptor effect of [3-adrenergicc stimulation (18). Incubation with the cAMP analog db-cAMP dose-dependently inhibitedd IFN-y release stimulated by LPS. In previous studies, prostaglandin̂ (PGE2), ann inflammatory mediator known to increase intracellular levels of cAMP, was reported to alsoo inhibit LPS-induced IL-12 production, an effect which could be mimicked by other cAMPP inducers (26, 27). Also, (32-agonists have been reported to inhibit IL-12 production fromm monocytes and dendritic cells, which was associated with increased concentrations of intracellularr cAMP (28). Together, these data suggest that changes in intracellular concentrationss of cAMP are involved in the suppression of IFN-y and IL-12 production by epinephrine. .

IL-122 is a important for the regulation of IFN-y in vitro and in mice during endotoxemiaa (11, 12, 19) and the present study). Since epinephrine potently inhibited LPS-inducedd IL-12 production, the reduction of IFN-y release could in part be the result of the inhibitingg effect on IL-12. To eliminate the effect of reduced IL-12 concentrations, experimentss with a neutralizing Ab against IL-12 were performed. In the presence of anti-IL-12,, epinephrine did not affect IFN-y concentrations in LPS-stimulated whole blood. This suggestss that epinephrine reduces IFN-y release during whole blood stimulation with LPS indirectlyy through inhibition of IL-12 production.

Wee used the whole blood assay to study the effects of epinephrine, rather than cultures

off isolated cells. The use of whole blood eliminates possible artifacts that may result from

isolationn of cells, such as an alteration in cytokine-producing capacity (29). In addition, the

effectt of a hormone on cytokine release in whole blood can be studied under conditions

188 8

EpinephrineEpinephrine inhibits IFN-y and IL-12 production

withh a physiological endocrine background and in the presence of all blood components,

whichh is likely to be of more relevance for the in vivo situation.

Systemicc inflammation leads to the activation of multiple host inflammatory responses,

includingg the cytokine network and the release of stress hormones (13). We here report that

epinephrinee strongly inhibits LPS-stimulated IL-12 production and, indirectly of IFN-y, in

wholee blood in vitro mainly through (3-adrenergic stimulation. IL-12 plays an important

rolee in the regulation of Thl/Th2 balance by promoting the differentiation of Thl type cells

(9,, 10). A Thl-mediated immune response, accompanied by the production of Thl type

cytokiness including IFN-y, is associated with cell-mediated immunity (30). Therefore,

releasee of epinephrine during the acute phase of inflammation attenuates excessive

proinflammatoryy cytokine release, but may also contribute to a decreased Thl type immune

response,, which may render the host more susceptible to concurrent infection.

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