Br. 1. exp. Path. (I984) 65, 431-439

Effect of bacterial endotoxin on body temperature, plasmazinc and plasma concentrations of the acute-phase

protein serum amyloid P component in mice

S. Poole, A.H. Gordon, M. Baltz* and B.E. StenningHormones Division, National Institute for Biological Standards and Control, London and *Immunological

Medicine Unit, Royal Postgraduate Medical School, Hammersmith, London

Received for publication 29 September I983

Summary. Bacterial endotoxin and lipid A evoked dose-dependent increases in bodytemperature and plasma SAP concentrations and dose-dependent falls in plasma zincconcentrations in mice. The respective sensitivities of the three variables to lipid A or to wholeendotoxin had the relation SAP> Zinc > body temperature; zinc and SAP responses wereevoked by < 1 ng lipid A/endotoxin. Pretreatment with indomethacin prevented only thetemperature response to lipid A. Pretreatment with dexamethasone did not affect thetemperature response but diminished the zinc response and enhanced the SAP response to lipidA.

Keywords: bacterial endotoxin, body temperature, plasma zinc, acute-phase protein, serumamyloid P component

Parenteral administration of bacterial endo-toxin or the lipid A moiety of endotoxin tomammals elicits a general systemic responsewhich includes fever, decreased plasma zincconcentrations and elevated plasma concen-trations of acute-phase reactants (Bornsteinet al. I 96 3; Pekarek & Beisel I 9 7 I; McAdam& Sipe I976). These responses occur sub-sequent to the synthesis and release of aprotein or proteins by monocytes stimulatedby the lipid A moiety of the endotoxin. Thereleased material may be one protein or anumber of structurally similar proteins andwas often described according to the re-sponse used for its assay, eg. 'endogenouspyrogen' (EP) or 'lymphocyte activating fac-tor' (LAF), but also by the broader term'leucocyte endogenous mediator' (LEM).However, at the second International Lym-

phokine Workshop (Mizel & Farrar I979)EP, LAF and LEM were considered to besynonymous with the monokine interleukini (IL-i); we therefore refer to the releasedmaterial as interleukin i (IL-i).

There is good evidence that IL-i acts onthe pre-optic/anterior hypothalamic area(PO/AH), an area ofthe brain which controlsbody temperature, to cause fever by a path-way that includes production of potent pyro-genic derivatives of arachidonic acid, eg.prostaglandins (PGs; Laburn et al. I977).Thus, fevers evoked by systemic and intra-cranial injections of IL-i are antagonized bydrugs that inhibit PG synthesis, the steroidaland non-steroidal anti-inflammatories(Laburn et al. I977; Willies & Woolf I980).The pathways through which IL-i evokeshypozincaemia and acute-phase reactions

Correspondence: S. Poole, Hormones Division, National Institute for Biological Standards and Control,Hampstead, London NW3 6RB.

43'

are not known, although the latter mayinvolve PGs (Shim I975). Also, since boththe zinc and the acute-phase responses areevoked by intracranial injection of smalldoses of IL-i that are ineffective when in-jected parenterally, it has been suggestedthat the central nervous system also has animportant role in mediating or modifyingthese responses (Turchik & Bornstein I980).The aims of the present study were to

determine in mice the relations among andthe mechanisms underlying endotoxin in-duced changes in body temperature and inplasma concentrations of zinc and of serumamyloid P component (SAP, a major acute-phase reactant in mice: Pepys et al. I979),including the possible involvement of pro-staglandins in the responses.

Materials and methods

Lipid A from the endotoxin of Salmonellaabortus equi and the endotoxins from Shigelladysenteriae and Escherichia coli K23 5 wereinjected intravenously in untreated mice (ofthe Parkes strain), in mice pretreated withsteroidal and non-steroidal anti-inflamma-tory drugs and in mice of the C3H/HeJ strainwhich produce only small amounts of IL-i inresponse to certain endotoxins (McAdam &Sipe I976).

Mice. Male mice of the Parkes strain(obtained from the National Institute forMedical Research, London) and of theC3H/HeJ strain (obtained from Bantin andKingman Ltd, Hull) weighing 30-40 g wereused for all experiments. They were housed50 mice per cage at 23 ± 20C with water andfood (Diet R. & M. no.i, BP Nutrition) adlibitum and subject to natural light and dark.Twenty-four hours prior to endotoxin injec-tion, mice were housed two per cage at3I i0IC with water and food ad libitum.

Preparation of glassware and solutions. Cleanglassware was baked overnight at 240°C andwhen practicable solutions were autoclavedthree times at I03.5 KPa (I5 lb/in2) for 30

min to destroy bacterial endotoxins. Solu-tions that could not be autoclaved wereshown to be free from bacterial endotoxin bythe Limulus amoebocyte lysate test.

Procedure. Body temperature was measuredfrom a rectal thermistor probe inserted 26mm into the rectum for 30 sec at intervals ofI 5-6o min for 2 h before and 3 h afterendotoxin injection. The probe was con-nected to a bridge circuit and Servoscribe I spen recorder. Endotoxins and indomethacinwere injected intravenously (i.v.) in avolume of 50 ,l via a tail vein and dexameth-asone was injected subcutaneously (s.c.) in avolume of 250 ,ul into the scruff of the neck.At intervals after endotoxin injection (4 h,24 h), mice were bled from the orbital sinus(ioo u1) into glass capillaries coated withheparin (Gelman-Hawksley Ltd) and theblood centrifuged at I 720 g for I 5 min. Zincconcentrations of 40-/A aliquots of plasmadiluted with 500 pl distilled water weremeasured using an atomic absorption spec-trophotometer (Perkin-Elmer model 603)fitted with a micro-sampling device. SAPconcentrations of 2-/A aliquots of undilutedplasma were measured by electro-immuno-assay (Pepys I979).

Results are expressed as the mean+ Istandard error of the mean (s.e. mean)response in at least io mice. Increases inbody temperature are the maximum in-creases that occurred within 3 h ofendotoxininjection. Plasma zinc concentrations weremeasured 4 h after injection of endotoxin orI% HSA (control) and plasma SAP concen-trations were measured 24 h after injectionof endotoxin or i% HSA. (Previous work hadshown that maximum zinc and SAP re-sponses occurred at these time intervalsrespectively after endotoxin injection.)

Drugs. Lipid A from the endotoxin of Sal-monella abortus equi (in ampoules code no.79/522) and the endotoxin from Shigelladysenteriae (ampoule code no. 57/7, FirstInternational Reference Preparation of Pyro-gen) were materials prepared for the Expert

S. Poole et a].432

Effect of bacterial endotoxin

Committee on Biological Standardization ofthe World Health Organization. The endo-toxin from Escherichia coli K2 3 5 (isolated byphenol extraction (Westphal & Jann I965))was kindly donated by Aynchem Ltd (Lon-don). Endotoxins were dissolved in I% pyro-gen-free human serum albumin (HSA, BloodProducts Laboratory, Elstree, Herts). Indo-methacin (milled), kindly donated by Merck,Sharp and Dohme Ltd, was dissolved at 2 7.9mM (Io mg/ml) in 95 mM (8 mg/ml) warmsodium bicarbonate. For injection this solu-tion was diluted with saline (I50 mM) toII.2-22.4 mM (4-8 mg/ml) indomethacin.Dexamethasone (micronized), kindly do-nated by Organon Laboratories, was dis-solved at 5.I0-IO.I9 mM (2.0-4.0 mg/ml)in IO% gum arabic (BDH). For injection thissolution was diluted with saline (I 50 mM) to2.5 5-3. I0 mM (I.o-2.0 mg/ml) dexametha-sone.

Results

Intravenous injection of lipid A (5 ng-o.spg/mouse) and the endotoxins from Shigella

dysenteriae (2.5 Mg/mouse) and E. coli K235(i-io pg/mouse) evoked increases in bodytemperature. Fig. i shows the time course ofthe temperature responses to lipid A (o.5pg/mouse) and to Shigella dysenteriae endo-toxin (2.5 pg/mouse): the maximum in-creases of o.8 ± o. I0C and I.0±0.2°C, re-spectively, occurred 30 min after injection.Also from Fig. i it can be seen that bodytemperature declined during the 2-h controlperiod prior to endotoxin injection. This wasa consistent finding due to the stress ofhandling and insertion of a rectal tempera-ture probe. In control mice body temperaturedid not significantly decline after the 2-hcontrol period. Temperature responses weredose-dependent: the responses to lipid A areshown in Fig. 2. Increases of o.7+ 0.2°C ando.9±o.20C, were evoked by E. coli K235endotoxin: i and I0 Mg/mouse respectively(data not shown).

Lipid A (o.s ng-5oo ng/mouse), Shigelladysenteriae endotoxin (i.o ng-ioug/mouse)and E. coli K235 endotoxin (i ng-iopg/mouse) also evoked dose-dependent fallsin plasma zinc concentrations and increases

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Fig. i. Effect ofintravenous injection (arrow) oflipid A (o. pg/mouse) (A), Shigella dysenteriae endotoxin(2.5 Mg/mouse) (M) and HSA (50 p1 of I% HSA/mouse) (0) on body temperature in Parkes mice. Eachpoint is the mean rectal temperature of at least I0 mice, s.e.means (not shown) were typically +o.i0Cand the evoked temperature increases were statistically significant (P<O.OOI). v, non-injected control.

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in plasma SAP concentrations. Maximumchanges in plasma zinc of about - 4O% andin plasma SAP of > + 9O% were evoked byShigella dysenteriae endotoxin (5o ng/mouse)and by E. coli K235 endotoxin (20ng/mouse). Fig. 3 shows the decreases inplasma zinc and increases in plasma SAPconcentrations of mice injected with lipid Arelative to zinc and SAP concentrations of(control) mice injected with I% humanserum albumin (HSA). The respective sensiti-vities of the three variables to lipid A/endo-toxin had the relation SAP> zinc > bodytemperature, zinc and SAP responses beingevoked by <1 ng lipid A.

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C3H/HeJ mice

The maximum changes in body tempera-ture, plasma zinc and plasma SAP concen-trations in Parkes mice and in mice of theC3H/HeJ strain following injection of lipid Aand the endotoxin from E. coli K235 areshown in Table i and in Fig. 4. In Parkesmice lipid A (o. 5 g/mouse) and E. coli K23 5endotoxin (i and I0 jg/mouse) evokedmarked changes in all three variables (asdescribed above). In contrast, in mice of theC3H/HeJ strain lipid A (o.s ,ug/mouse) waswithout effect and E. coli K235 endotoxin(I0 jg/mouse) evoked a zinc response(-3 2 ± 3%) similar to that elicited in Parkesmice (-38 ± 7%) but evoked a much smallerSAP response (+5o±4% compared with+I79± I I%) and no change in body tem-perature.

Pretreatment with anti-inflammatory drugs

The effects of treatment with indomethacinand dexamethasone on body temperature,plasma zinc and SAP concentrations and onlipid A evoked responses of these variablesare summarized in Table 2 and in Fig. 5.

Indomethacin

Indomethacin (200 ,ug/mouse) did not affectbody temperature or plasma zinc but in-creased plasma SAP by 24± i 6%. Indometh-acin (400 ,ug/mouse) lowered body tempera-ture by 0.4±0. i0C, decreased plasma zincby 25± I3% and increased plasma SAP by2I3 ± 29%. Pretreatment with indometha-cin at 200 jg/mouse, 1 h before lipid A (o.sMg/mouse) reversed the lipid A-evoked in-crease in body temperature from+ o.8± 0.2°C to -o.6 ± o.i0C whereas pre-treatment with indomethacin at 400Mg/mouse merely prevented the evoked in-crease. Lipid A-evoked zinc and SAP re-sponses were not affected by pretreatmentwith indomethacin (200 or 400 ,ug/mouse).

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Effect of bacterial endotoxin 435Table i. Effect of lipid A (o.s5 jg/mouse) and the endotoxin from E. coli K235 (i and i0o jg/mouse) onbody temperature (A Tb), plasma zinc concentrations (measured 4 h after endotoxin injection) andplasma SAP concentrations (measured 24 h after endotoxin injection) in Parkes mice and in mice of theC3H/HeJ strain. Results are expressed as themean± i standard error ofthe mean (s.e. mean) temperatureresponse or plasma zinc/SAP concentration in at least I0 mice.

Mousestrain Endotoxin Dose A Tb(WC) Zinc (ug/dl) SAP (Mg/ml)

Parkes LipidA o.5jg +o.6±o.i 102±5 253±12E.coliK235 Ijg +0.7±0.1 io8±io 238±9E.coli K235 I0ojg +0.9±0.1 119±12 248±I9i% HSA (control) 50 Il -0.2±0.2 i9i±6 89±i0

C3H/HeJ Lipid A 0.5 jg -0.2±0.2 119±12 124±8E.coliK235 Ijg +0.1±0.1 92±8 I43±7E.coli K235 10 jig 0.0±0.2 88± 5 i64±6i% HSA (control) 50 PI +O.I ±O.I I30±12 109±17

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Lipid A (0.5 pg) E.Coli K235 (10 ,ig)Fig. 4. The changes (mean + s.e.mean, n= io) in body temperature (0), plasma zinc (0) and plasma SAP(0) concentrations evoked by lipid A (o.jpg/mouse) and by E. coli K235 endotoxin (io jg/mouse) inParkes mice and in C3H/HeJ mice.

Dexamethasone

Dexamethasone injected at 250 jg/mouse,24 h and i h before an injection of 50 j1 I%HSA (control for endotoxin) had no signifi-cant effect on body temperature but loweredplasma zinc by 24± 5% and increasedplasma SAP by 75±8%. This dexametha-sone pretreatment (250 jig X 2/24 h) did not

affect lipid A (o.s jig/mouse) evoked tem-perature and zinc responses but enhancedthe lipid A-evoked increase in plasma SAPfrom +I29+8% to +283+I2%. Dexa-methasone injected at 500 jug/mouse, 72,48, 24 and i h before injection of I% HSAhad no effect on any of the three variables.This dexamethasone pretreatment (500

436 S. Poole et al.Table 2. Effect of pretreatment with indomethacin (INDO) and dexamethasone (DEX) on lipid A-inducedchanges in body temperature (A Tb), plasma zinc concentrations (measured 4 h after lipid A 0.5pg/mouse) and plasma SAP concentrations (measured 24 h after lipid A). Indomethacin was injected i.v.at 200 or 400 pg/mouse i h before lipid A; dexamethasone (in 5% gum arabic) was given chronically ass.c. injections at 2 50 pg/mouse, 24 h and i h before lipid A or at 500 pg/mouse, 72,48,24 and i h beforelipid A. Results are expressed as the mean i standard error of the mean (s.e. mean) temperatureresponse or plasma zinc/SAP concentration in at least I0 mice.

Pretreatment Injection A Tb (°C) Zinc (pg/dl) SAP (pg/ml)

Saline i% HSA -0.1±O.I i82±9 117±13INDO(200 pg) -0.2±0.I i80±9 I45 ±19Saline Lipid A (o.5sg) +0.8+0.2 I03+6 203±10INDO (200 pg) -o.6±o.i I04±5 202±Il

Saline i% HSA 0.0±0.1 217±4 82±9INDO (400 Pg) -0.4±0.I i62±7 257±24Saline Lipid A (o.5 g) + o.8 ±o. I 128±io0 22I±9INDO(400Pg) -0.1±O.I 142±I2 239±8

5% gum arabic i% HSA -0.4±0.I 2I5±11 77±6DEX (250 pgX 2/24 h) -0.3±0.1 i63±9 135±65% gum arabic Lipid A (o. 5 pg) +0.7±0.I 131± II 176±6DEX(250pgx2/24h) +o.8±o.i I37±I5 295±9

5% gum arabic i% HSA -0.4±0.1 143±11 ii8±I7DEX (500 pg x 4/72 h) -0.2±0.2 I35 ± 7 126±I55% gum arabic Lipid A (0.5 pg) +0.5 ±0.1 79 ± 3 223±11DEX (500 pg x 4/72 h) +o.6±o.i 98± 5 298 ± 26

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Fig. 5. Effect of pretreatment with indomethacin (INDO, 200 pg/mouse) and dexamethasone (DEX, 500pg/mouse) on changes (mean±s.e.mean, n= io) in body temperature (U), plasma zinc (m) and plasmaSAP (0) concentrations evoked by lipid A (o.s pg/mouse) in Parkes mice.

Effect of bacterial endotoxinxgX 4/72 h) did not affect the lipid A-evoked

temperature response but diminished thelipid A-evoked decrease in plasma zinc from-45±2% to -3I+4% (P<0.002) andenhanced the lipid A-evoked increase inplasma SAP from + 89± 9% to + I 5 3 ± 22%(P<O.OI).

Relation between temperature increases andplasma concentrations of zinc and of SAP inindividual mice

Correlation coefficients for each pair of vari-ables, ie. temperature increases vs zinc con-centrations, temperature increases vs SAPconcentrations, and zinc concentrations vsSAP concentrations were calculated for thedifferent treatment groups in each experi-ment.

Temperature increases vs zinc concentrations.There was no correlation between theseresponses in control mice or in endotoxin-treated mice.

Temperature increases vs SAP concentrations.Values of correlation coefficients appeared todepend upon treatment. The combined valuefor control mice of + 0.37 differed signifi-cantly (standard normal deviate= 3.5 I,P<o.ooi) from the combined values of-0.50 for endotoxin-treated mice. Withineither treatment group considered separatelythere was no evidence of heterogeneityamong the different experiments.

Zinc concentrations vs SAP concentrations.There was no evidence of heterogeneityamong the correlation coefficients, whichwere almost entirely negative. The combinedvalue for control mice was -0.42, slightlylarger (P>o.os) than that of -0.2 3 forendotoxin-treated mice.

Discussion

The time-course of endotoxin-induced feverin rabbits and in man, with an onset after40-60 min and maximum temperature in-

creases within 2-4 h of injection, is consis-tent with a fever process in which macro-phages stimulated by endotoxin synthesizeand release IL-i which circulates, enters thebrain and stimulates synthesis and release ofproducts of the arachidonic acid cascadewithin the PO/AH (Rosendorff & WoolfI9 79). The markedly different time-course inmice, with an onset within I5 min andmaximum increases within 30 min of injec-tion, suggests a more direct mechanism,perhaps independent of IL-i but requiringPGs since the temperature increases wereprevented by pretreatment with indometha-cin. Since purified IL-i evoked maximumtemperature increases in mice within I 5 minof injection (Duff & Durum I983), if theendotoxin-induced temperature increasesdescribed above were mediated by IL-i theIL-i must have been synthesized andreleased within IS min of endotoxin injec-tion because Ir-i is not preformed withinmacrophages (Moore et al. I973).

Temperature, plasma zinc and plasmaSAP responses of groups of io mice to lipidA/endotoxin were dose-dependent but therelations among the three responses in indi-vidual mice were complex. Body tempera-ture and plasma zinc concentrations in indi-vidual mice were not related and changedindependently following endotoxin injection.In contrast, in untreated mice plasma zincand SAP concentrations were negativelycorrelated (low zinc concentrations wereassociated with high SAP concentrations)but the correlation was reduced rather thanenhanced by endotoxin injection. Also,endotoxin evoked substantial zinc responsesin C3H/HeJ mice in the absence of markedSAP responses. Since C3H/HeJ mice produceonly small amounts of IL-i in response toendotoxin, the zinc responses of mice of thisstrain may have been elicited by a pathwaythat was independent ofIL-i or required onlyvery small amounts of IL-i. Taken together,the above findings indicate that zinc and SAPconcentrations vary independently despitethe reported common role of IL-i in themediation of zinc and SAP responses to

437

438 S. Poole et al.endotoxin (Pekarek & Beisel I97I; Wanne-macher et al. I975). This independence isconsistent with a report that human serumconcentrations of zinc and the acute-phaseprotein, C-reactive protein, were not corre-lated (Rice I962).

Since lipid A/endotoxin evokes fever by apathway that includes production of arachi-donic acid derivatives, e.g. prostaglandins,and since indomethacin and dexamethasoneboth inhibit PG synthesis but by differentmechanisms (Vane I97I; Flower & Black-well I979), it is surprising that only indo-methacin antagonized the endotoxin-induced temperature increases in mice, afinding that contrasts with the antipyreticeffect of corticosteroids in rabbits (Willies &Woolf I980). Although the dose of lipid A(o.0 g/mouse) injected after indomethacinor dexamethasone pretreatment was neces-sarily supramaximal with respect to the zincand SAP responses-to ensure lipidA evokedtemperature responses of sufficient magni-tude to reveal antagonism by the anti-inflammatory drugs-the lack of effect ofindomethacin on either zinc or SAP re-sponses to lipid A suggests that these re-sponses did not require synthesis and releaseof PGs. Consistent with this suggestion is thefailure of pretreatment with an inhibitor ofboth cyclo-oxygenase and lipoxygenase, BW755C (I.5 mg/mouse x 4/72 h), to affect zincand SAP responses to a submaximal dose (I0ng/mouse) of the endotoxin from E. coli05 5:B5 (data not presented).The reduced zinc response and enhanced

SAP response to endotoxin observed in micetreated with dexamethasone (but not withindomethacin) were therefore due to actionsof dexamethasone other than inhibition ofPG synthesis. This conclusion is consistentwith a recent report that dexamethasone(but not indomethacin) exacerbated Sal-monella typhimurium infection in mice andStaphylococcus aureus infection in rabbits(Adlam et al. I983), and with reports thatvarious acute-phase proteins are corticoster-oid-dependent (Weimer & Coggshall I967;Feinberg et al. I983).

Reduced plasma zinc concentrations fol-lowing treatment with dexamethasone for24 h accords with the role of this drug as aprimary inducer of the zinc binding proteinhepatic zinc thionein (Brady I 982) althoughthe induction was transient since plasmazinc concentrations were not lowered by thethird day of dexamethasone treatment.Reduced plasma zinc concentrations evokedby the larger dose of indomethacin (400pg/mouse) are consistent with the proposedrole ofPGE2 in zinc absorption in rats (Song &Adham I978) although it is possible that theindomethacin represented a sufficient stressto itself induce synthesis of hepatic zincthionein (Brady I982).

In summary, the present study confirmsthe sensitivity to endotoxin of plasma zincconcentrations in mice (Boobis & HartleyI98 i) and demonstrates that concentra-tions of another constituent of mouseplasma, serum amyloid P component (SAP),are even more sensitive to endotoxin. Also,evidence is presented that endotoxin in-creases body temperature in mice by apathway that is independent of IL-i butrequires PGs, whereas endotoxin evokesplasma zinc and SAP responses by pathwaysthat do not involve PGs.

Acknowledgements

The authors are very grateful to Dr Rose E.Gaines Das for performing the statisticalanalysis, and thank Dr D.R. Bangham and DrM.B. Pepys for their interest and advice inthis study.

ReferencesADLAM C., EARMAN C.S.F., HIGGS G.A. & PLANT J.E.

(I983) Anti-inflammatory drugs and bacterialinfection. Proc. Br. pharmac. Soc. 79, 220P.

BOOBIS A. & HARTLEY R.E. (I 98 I) Measurement ofhypozincaemia in mice: a sensitive test fordetection of pyrogens. Clin. Sci. 6I, 445.

BORNSTEIN D.L., BRENDENBERG C. & WOOD W.B.(I963) Studies on the pathogenesis of fever. XI.Quantitative features of the febrile response toleucoyte pyrogen. J. exp. Med. Il7, 349.

Effect of bacterial endotoxin 439BRADY F.O. (I982) The physiological function of

metallothionein. TIBS April, 143.DUFF G.W. & DURAM S.K. (I983) The pyrogenicand mitogenic actions of interleukin-i arerelated. Nature 304, 449.

FEINBERG R.F., SUN L.-H.K., ORDAHL C.P. & FRAN-KEL F.R. (I983) Identification of glucocorticoid-induced genes in rat hepatoma cells by isolationofcloned cDNA sequences. Proc. Natn. Acad. Sci.U.S.A. 80, 5042.

FLOWER R.J. & BLACKWELL G.J. (I 9 79) Anti-inflammatory steroids induce biosynthesis of aphospholipase A2 inhibitor which preventsprostaglandin generation. Nature 278, 456.

LABURN H., MITCHELL D. & ROSENDORFF C. (I977)Effects of prostaglandin antagonism on sodiumarachidonate fever in rabbits. J. Physiol. 267,559.

McADAM K.P.W.J. & SIPE J.D. (1976) Murinemodel for human secondary amyloidosis: gene-tic variability of the acute phase serum proteinSAA response to endotoxins and casein. J. exp.Med. 144, 1121.

MIZEL S.B. & FARRAR J.J. (I 9 79) Revised nomen-clature for antigen-non-specific T cell prolifer-ation and helper factors. Cell. Immun. 48, 433.

MOORE D.M., MURPHY P.A., CHESNEY J.P. & WOODW.B. (I973) Synthesis of endogenous pyrogenby rabbit leukocytes. 1. exp. Med. 137, I263.

PEKAREK R.S. & BEISEL W.R. (I 9 7 I) Characteriza-tion of the endogenous mediator(s) of serumzinc and iron depression during infection andother stresses. Proc. Soc. exp. Biol. Med. 138,728.

PEPYs M.B. (I979) Isolation of serum amyloidP-component (protein SAP) in the mouse.Immunology, 37, 637.

PEPYS M.B., BALTZ M., GOMER K., DAVIEs A.J.S. &DOENHOFF M. (I979) Serum amyloid P-com-ponent is an acute-phase reactant in themouse. Nature 278, 259.

RICE E.W. (I962) Lack of correlation betweenzinc, copper and C-reactive protein in humanserum. Proc. Soc. exp. Biol. Med. 110, 450.

ROSENDORFF C. & WOOLF C.J. (I979) Inhibition offever. In Anti-inflammatory Drugs. (Eds J.R.Vane & S.H. Ferreira). Berlin: Springer-Verlag.

SHIM B.S. (I975) Increase in serum haptoglobinstimulated by prostaglandins. Nature 259, 326.

SONG M.K. & ADHAM N.F. (I978) Role of prostag-landin E2 in zinc absorption in the rat. Am. J.Physiol. 3, Egg.

TURCHIK J.B. & BoRNsTEIN D.L. (I980) Role of thecentral nervous system in acute-phase re-sponses to leukocytic pyrogen. Infect. Immun.30, 439.

VANE J.R. (1971) Inhibition of prostaglandinsynthesis as a mechanism of action for aspirin-like drugs. Nature 231, 232.

WANNERMACHER R.W., PEKAREK R.S., THOMPSONW.L., CuRNow R.T., BEALL F.A., ZENSER T.V.,DERUBERTIs F.R. & BEISE W.R. (I975) A pro-tein from polymorphonuclear leukocytes (LEM)which affects the rate of hepatic amino acidtransport and synthesis of acute phase globu-lins. Endocrinology 96, 6 5 I-6 5 9.

WEIMER H.E. & COGGSHALL V. (196 7) a2 macroglo-bulin ofrat is cortisol dependent. Can. J. Physiol.Pharm. 45, 767.

WESTPHAL 0. & JANN K. (I965) In Methods inCarbohydrate Chemistry. Vol. 5. (Ed R.L.Whistler). New York: Academic Press. p. 83.

WILLIES G.H. & WOOLF C.J. (I980) The site ofaction ofcorticosteroid antipyresis in the rabbit.J. Physiol 300, I.

Note added in proof

The following papers have appeared subsequentto submission of the preceding article and containdata that are consistent with our findings.SOBRADO J., MOLDAWER L.L., BISTRIAN B.R., DINAR-

ELLO C.A. & BLACKBURN G.L. (I983) Effect ofibuprofen on fever and metabolic changesinduced by continuous infusion of leukocyticpyrogen (interleukin i) or endotoxin. Infect.Immun. 42, 997.

Tocco R.J., KAHN L.L., KLUGER M.J. & VANDER A.J.(1983) Relationship of trace metals duringinfection: are prostaglandins involved? Am. J.Physiol. 244, R368.