Integrative aspects of a human model ofendotoxemia1

C.A. Ottaway, I.W. Fong, B. da Silva, W. Singer, and L. Karrass

Abstract: The production of tumor necrosis factor (TNF)-α is a key step in the response to sepsis and has powerfullocal and systemic effects on the host. These systemic responses include a complex cascade of centrally mediatedendocrine and neural responses. An integrative model of these regulatory cytokine–neuroendocrine interactions inhumans is presented. The rapid kinetics of these responses are illustrated by data showing the response of normalhuman subjects to experimental endotoxemia. Appreciation of the integrative biology of the in vivo response toexperimental endotoxemia can provide a framework for the design of experiments aimed at examining the effects ofphysical training paradigms on particular cytokine and neuroendocrine pathways.

Key words: tumor necrosis factor (TNF)-α, tumor necrosis factor (TNF)-α soluble receptors, hypothalamic–pituitaryaxis, lipopolysaccharide (LPS), integrative biology of human response to LPS, cytokine–neuroendocrine interactions.

Résumé: La production du facteur de nécrose des tumeurs (TNF)-α est une étape clé dans la réponse à uneinflammation généralisée d’origine infectieuse, qui a des effets systémiques et locaux puissants sur l’hôte. Ces réponsessystémiques incluent une chaîne complexe de réactions neurales et endocrines d’origine centrale. On présente unmodèle d’intégration de ces interactions régulatrices impliquant le système des cytokines et le systèmeneuroendocrinien chez les humains. On illustre les cinétiques rapides de ces réponses par des résultats montrant laréponse de sujets humains normaux à une endotoxémie expérimentale. La biologie intégrative de la réponse in vivo àl’endotoxémie expérimentale peut fournir un canevas d’expériences visant à examiner les effets de l’entraînementphysique sur certaines voies spécifiques du système neuroendocrinien et du système des cytokines.

Mots clés: facteur de nécrose des tumeurs (TNF)-α, récepteurs solubles du facteur de nécrose des tumeurs (TNF)-α,axe hypothalamo–hypophysaire, lipopolysaccharide (LPS), biologie intégrative de la réponse humaine au LPS, interac-tions entre système des cytokines et système neuroendocrien.

[Traduit par la Rédaction] Ottaway et al. 478

Cytokines play a critical role in the pathophysiology ofinflammation, infection, and sepsis. Tumor necrosis factor(TNF)-α is the initial cytokine produced in experimentalsepsis models and TNF-α exerts powerful cellular and mo-lecular effects in the intact host. The production and avail-ability of TNF-α is very tightly regulated by complexcytokine, endocrine, and neural pathways. The purpose ofthis report is to examine features that regulate the TNF-α re-sponse to experimental endotoxemic challenge in vivo innormal human subjects and to provide an integrative modelof these responses, which can be useful in exploring host re-sponse to challenges such as exercise and physical training.

The dynamics of the host response to acute infectiouschallenge and its control is illustrated by the network ofcytokine and neuroendocrine signals that can be measured in

the circulation after the experimental administration of smalldoses of lipopolysaccharide (LPS), the separated lipo-carbohydrate component of endotoxin, which is a major cellwall component of most Gram-negative bacteria (Burell 1994).An overview of a number of the processes involved in theregulation of the TNF-α response is represented in Fig. 1.LPS promotes macrophage activation and leads to the re-lease of inflammatory mediators such as tumor necrosis fac-tor (TNF)-α, interleukin (IL)-6, and IL-1 (Kuhns et al. 1995;Martich et al. 1991; Fong et al. 1989). This initial burst ofproinflammatory cytokines is followed by production ofIL-10, which inhibits the further production of TNF-α, IL-6,and IL-1 (de Waal Malefyt et al. 1991; Bogdan et al. 1991;Platzer et al. 1995; Wandiworanum and Strober 1993). Inthe intact host, production of TNF-α and IL-6 also leads toactivation of the hypothalamic–pituitary–adrenal (HPA)axis, the production of adrenocorticotropin (ACTH), andsubsequently increased release of adrenal corticosteroids(Chrousos 1995; Tilders et al. 1994; Turnbull and Rivier1995) (Fig. 1). The ability of corticosteroids such as cortisolto interfere with both the production and the end organ ef-fects of TNF-α provides an important regulatory feedbackloop in the inflammatory response. Regulation is also providedby the central activation of the sympathetic nervous systemand the release of catecholamines into the circulation and atnerve endings (Fig. 1). Epinephrine and norepinephrine can

Can. J. Physiol. Pharmacol.76: 473–478 (1998) © 1998 NRC Canada

473

Received August 1, 1997.

C.A. Ottaway,2 I.W. Fong, B. da Silva, W. Singer, and L.Karrass. Department of Medicine, Room 3-073,St. Michael’s Hospital, 30 Bond Street, University ofToronto, Toronto, ON M5B 1W8, Canada.

1This paper has undergone the Journal’s usual peer review.2Author to whom all correspondence should be addressed(e-mail: ottawayc@smh.toronto.on.ca).

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regulate the response of macrophages to endotoxin in vitro,and recently van der Poll and colleagues showed that epi-nephrine administration to intact humans inhibits the TNFresponse and potentiates the IL-10 response to experimentalLPS challenge (van der Poll et al. 1996).

Here, we will illustrate the time course of circulating con-centrations of cytokines and hormones of normal humansubjects in response to the intravenous administration ofLPS. This data represents a part of a much larger study ofthe effects of HIV infection on cytokine and neuroendocrineresponses to LPS, which is presented elsewhere (B. Da Silva,W. Singer, I. Fong, and C. Ottaway).

Subjects were recruited at St. Michael’s Hospital. A total of 15normal individuals were studied. The protocol for this study wasapproved by the Human Subjects in Research Review Committeeof our Institution. Informed written consent was provided by allsubjects prior to participation. The subjects arrived at the testingarea at 0800 h. An intravenous infusion (3.3% dextrose, 0.3% so-dium chloride) was started and a saline lock for blood taking wasinserted in a second peripheral vein. Baseline blood samples wereobtained for cytokine, cytokine receptor, and endocrine assays, andstandardized endotoxin in the form of purified lipopolysaccharide(LPS) was given by intravenous bolus at a dose of 20 EU/kg(4 ng/kg) body weight. The LPS used in this study was kindly pro-vided to us by Dr. H.D. Hochstein of the Center for BiologicsEvaluation and Research (Washington, D.C.). The blood pressure,heart rate, and tympanic membrane temperature of the subjectswere monitored every 15 min. Blood samples for cytokine,cytokine receptors, and hormone assays were obtained at the timeintervals indicated below.

Cytokine and cytokine receptor measurementsBlood samples were collected in EDTA-containing tubes just

prior to and at 30, 60, 90, 105, 120, 180, 240, and 300 min afterLPS infusion. The samples were immediately separated, and theplasma was aliquoted, coded, and stored at –70°C until assayed.Concentrations of TNF-α, IL-6, IL-8, and IL-10 were quantifiedusing double antigen “sandwich” Elisa immunoassays specific forthe particular cytokine, employing human cytokine specific anti-bodies and human recombinant cytokine standards according to themanufacturers’ instructions (Quantikine (TM), R&D systems, Min-neapolis, Minn.). Concentrations of TNF receptors (R1 (p55) andR2 (p75)) were measured using immunoassays purchased fromR&D Systems.

Hormone measurementsSerum samples for cortisol, dehydroepiandrotestosterone sulfate

(DHEA-S), and plasma samples for adrenocorticotrpic hormone(ACTH) were collected prior to and at 30, 120, 240, and 300 minafter the LPS infusion. Samples were immediately frozen andstored at –70°C until assayed. Concentrations of these hormoneswere determined by radioimmunoassay in the Endocrine PeptideLaboratory at St. Michael’s Hospital. Circulating concentrations ofnorepinephrine were measured using the Katcombi125I-RIA sys-tem (Alpco Ltd, Windham, N.H.).

Following injection of LPS, the normal subjects experi-enced a small temperature rise (mean 1.3 ± 0.2°C), a modestelevation in measured heart rate (mean 17 ± 3 bpm), but nomeasurable change in blood pressure. The circulating con-

centration of TNF-α increased more than two orders of mag-nitude above baseline concentrations with a peak responsebetween 90 and 105 min after the LPS injection (Fig. 2), andthe concentration of IL-6 also showed marked changes afterLPS injection with maximal levels observed at 120 min afterthe challenge (Fig. 2). The response of IL-8 and IL-10showed a slightly delayed response with a plateau of maxi-mal elevation between 120 and 180 min after LPS injection(Fig. 2).

The biological effects of TNF-α are mediated through theinteraction of the cytokine with its cellular receptors(Fig. 1). Two classes of TNF receptors have been identified:TNF-R1 (type 1 or p55) and TNF-R2 (type 2 or p75), whichmediate a range of activities including apoptosis, cyto-toxicity response, and proliferative responses of thymocytes,lymphocytes, and stromal cells (Flier and Underhill 1996).One factor that can limit the availability of TNF-α in vivo isthe processing of these cellular TNF receptors into solublecirculating receptors that can maintain their affinity forTNF-α (Spinas et al. 1992; van Zee et al. 1992). When ex-amined in our subjects, the circulating concentrations of sol-uble TNF-R1 and TNF-R2 were both significantly elevatedafter stimulation with LPS (Fig. 3). This elevation was par-ticularly marked with TNF-R2 and was sustained for a moreprolonged period than the elevation of TNF-α itself in thesesubjects (Figs. 2 and 3).

Significant central neuroendocrine activation was alsodemonstrated by our subjects (Fig. 4). There were substan-tial elevations in the circulating concentrations of ACTH,cortisol, and norepinephrine consistent with the longer rangeneuroendocrine regulatory loops illustrated in Fig. 1. In con-trast, there was no significant elevation or alteration in thecirculating levels of the androgen precursor dehydroxy-androsterone-sulphate (DHEA-S), a steroid molecule thathas been shown to inhibit the in vitro and in vivo production of

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Fig. 1. Scheme representing several regulatory pathways thatcontrol the production and availability of TNF-α in vivo. APC,antigen presenting cell; LPS, lipopolysaccharide; CNS, centralnervous system; CRH, corticotrophin-releasing factor; AVP,arginine vasopressin; ACTH, adrenocorticotrophin; MIF,macrophage migration inhibition factor; SNS, sympatheticnervous system; TNF(3), the trimeric form of TNF-α; TNFR(c),cellular TNF-α receptors.

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TNF in response to LPS by a mechanism that is independentof glucocorticoid receptor occupancy (Di Santo et al. 1996).

TNF is a central mediator of the host response toendotoxemia, and the control of this cytokine is of major im-portance for the host. A great deal is now known about themolecular biology of TNF biosynthesis and its regulation. Inthe intact host there are a number of short range and longrange regulatory pathways that contribute to the integrativebiology of TNF, but that are not always apparent in in vitrostudies. The observations presented here for the response ofnormal subjects to an experimental challenge with LPS(Figs. 2, 3, and 4) illustrate the rapid time course for a num-ber of regulatory pathways illustrated in Fig. 1.

LPS activation of human macrophages in vitro leads tothe induction of TNF-α and IL-6, and later, the induction of

IL-10. TNF-α is a major upregulator of the expression ofmRNA for IL-10 in human monocytes and the principaldriver of IL-10 production (Wandiworanum and Strober1993; Platzer et al. 1995). In mice challenged in vivo withLPS, neutralization of IL-10 production in response to LPSby means of passive immunization with anti-IL-10 serumenhances the production of TNF-α, and leads to significantlygreater mortality after LPS challenge (Standiford et al.1995). In primates, the infusion of IL-10 markedly decreasesthe production of proinflammatory cytokines in response toexperimental LPS challenge (van der Poll et al. 1997). Thus,TNF-α production promotes IL-10 production, and IL-10, inturn, decreases TNF-α production. Since IL-10 can alsodownregulate its own production in an autocrine fashion, areciprocal regulatory loop results (Platzer et al. 1995).

TNF-α and IL-6 produced peripherally act synergisticallyto activate the HPA axis (Tilders et al. 1994; Perlstein at al.1993) and promote the systemic release of ACTH and

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Ottaway et al. 475

Fig. 2. Time course of the circulating concentrations of various cytokines after intravenous challenge of normal subjects with LPS.Results are means ± sem.

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corticosteroids. Corticosteroids decrease the transcriptionrate and the stability of mRNA for IL-6 and TNF-α (Lesset al. 1988; Zanker et al. 1990), and the administration ofcorticosteroids to humans severely blunts their cytokine re-sponse to LPS challenge (Barber et al. 1993). In experimen-tal animals, interruption of the ability to producecorticosteroids enhances the production of TNF-α in re-sponse to in vivo challenge with LPS, and the administrationof ACTH decreases the in vivo production of TNF-α in re-sponse to LPS (Fantuzzi et al. 1995). Thus, the HPA re-sponse constitutes a second regulatory loop operating inparallel with that involving IL-10 (Fig. 1).

Activation of the sympathetic nervous system also regulatesTNF-α production. Short-term in vitro exposure of humanmonocyte or macrophages to the catecholamines epinephrineand norepinephrine has been shown to inhibit the productionof TNF-α in response to LPS challenge (Severn et al. 1992;Spengler et al. 1994), infusions of epinephrine in normal hu-man subjects challenged with LPS in similar doses to thatused here inhibited the circulating TNF-α response, but en-

hanced the availability of IL-10 (van der Poll et al. 1996). Inseparate experiments, those investigators showed that the ef-fect of epinephrine on IL-10 production was mediatedthrough bothα andβ adrenergic receptors, whereas the ef-fect of epinephrine on TNF-α production appeared to be me-diated solely through b adrenergic receptors (van der Pollet al. 1996). Thus, central activation of sympathetic nervoussystem provides another pathway for regulating both TNF-αand its counter-regulator, IL-10 (Fig. 1).

There are at least two other regulator pathways of poten-tial relevance to the integrative biology of TNF-α that werenot studied here. One of these is migration inhibitory factor(MIF), a cytokine produced by macrophages and the pitu-itary gland in response to LPS stimulation (Calandra et al.1994; Bucala 1994) that when co-injected with LPS in micepotentiates the lethal effect of LPS (Bernhagen et al. 1993).Furthermore, an anti-MIF antibody can protect mice againstexperimental endotoxemia. (Bernhagen et al. 1993) Thus,MIF, which can be available both locally and centrally, is anupregulator of TNF-α production by macrophages, and MIFand TNF-α can act together in a proinflammatory regulatoryloop (Fig. 1). A further potential pathway is provided by thepro-opiomelanocortin derived peptideα-MSH. This peptidehas well-established immunomodulating and anti-inflammatory properties (Catanna and Lipton 1993) and re-cently has been shown to be a potent inducer of IL-10 byhuman monocytes in vitro (Bhardwaj et al. 1996). Thus invivo, α-MSH may exert control over IL-10 production(Fig. 1). Neither of these pathways were investigated herebecause, as yet, reagents suitable for studies of circulatinglevels of MIF andα-MSH in humans are not readily avail-able.

The host response to experimental LPS challenge is amodel of the response to infection and inflammation. Inwhat ways can physical exertion, physical training, andoverwork paradigms alter the integrative regulatory interac-tions between these cytokine and neuroendocrine pathways(Fig. 1)? Varying effects of exercise on circulating concen-trations of TNF-α in vivo have been reported. For example,TNF-α concentrations were increased 2 h after a 5-km run(Espersen et al. 1990), and 1 h after a 30-km run (Dufauxand Order 1989), increased circulating concentrations ofTNF-α have been reported 2 h after a 250-km competitivecycle (Gannon et al. 1997). It is important to note that theabsolute TNF-α concentrations achieved after these exertionswere only two- to three-fold greater than resting levels. Al-though these changes are smaller than those found duringexperimental LPS challenge (Fig. 2), it is likely that theregulatory interactions between the different pathways describedhere are similar. With this in mind, future studies of cytokineproduction during exercise paradigms (e.g., TNF-α) wouldbenefit from a more kinetic approach in which the timecourse of various regulatory and counter-regulatory signals(e.g., IL-10, catecholamines, cortisol) are studied in parallel.

Another implication of this approach is the question of theextent to which overtraining paradigms might modulatethese cytokine–neuroendocrine pathways. For example, it hasbeen reported that the TNF-α response to LPS challenge wassignificantly reduced in rats subjected to exhaustive exercise.What pathways mediate this blunted response to LPS aftersevere exertion? Although such an effect of overtraining has

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Fig. 3. Time course of circulating soluble TNF receptorsconcentrations after intravenous challenge of normal subjectswith LPS. Results are means ± sem.

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not been studied in human subjects, it could be of clinicalimportance, and the scheme outlined here (Fig. 1) offers aframework for the design of studies to determine the particularcontributions of several cytokine and neuroendocrine path-ways that might be affected by physical training paradigms.

This work was supported in part by the CanadianFoundation for Aids Research and the Crohn’s and ColitisFoundation of Canada.

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