Fever, tumor necrosis factor, and interleukin-6 mature, and aged Fischer 344 rats KARA
D. FOSTER,
Department
CAROLE
A. CONN,
of Physiology, University
AND
J. KLUGER
of Michigan Medical School, Ann Arbor, Michigan 48109
Foster, Kara D., Carob A. Conn, and Matthew J. Kluger. Fever, tumor necrosis factor, and interleukin-6 in young, mature, and agedFischer 344 rats. Am. J. PhydoZ. 262 (RegulatoryIntegrative Comp.Physiol. 31): R211-R215,1992.The purposeof this study wasto comparethe febrile responses of Fischer 344 rats of different ages[young (3-5 mo), mature (12-15 mo), and aged (24-27 mo; n = S)] to two psychological stressparadigms, cageswitch and exposureto an open field, as well as to injection of lipopolysaccharide (LPS). In addition, the cytokines tumor necrosisfactor-a (TNF) and interleukin6 were also measuredin the plasma of these rats at 90 min postinjection with LPS. There was no significant difference among groups in febrile responsesto switching their cages. Exposure to an open field for 30 min resulted in a smaller rise in temperature in the agedrats (0.62”C) than in the young rats (1.26”C). This difference disappearedif rats were exposedto an open field for 60 min. Injection of LPS led to fevers that developed at a slower rate in aged rats than in the mature groups. The peak fevers, however, were not different. The activity of interleukin-6 90 min after injection of LPS was higher in aged rats (297,358U/ml) than in young (17,462 U/ ml) and mature rats (28,819U/ml). TNF levelswere alsohigher in aged rats (16,380 U/ml) compared with young (574 U/ml) and mature rats (36 U/ml). We conclude that although the magnitude of the febrile responseis not different among rats of different ages,the rise in body temperature occursslower in agedrats. The finding that agedrats had significantly elevated TNF levels in their plasma during the time when their body temperatureswere lower than that of young and mature rats is consistent with the hypothesis that endogenously produced TNF is an antipyretic (or an endogenouscryogen) in the rat. stress-hyperthermia;cytokines; lipopolysaccharide
become older, they are known to experience a greater number of bacterial infections and a higher mortality rate from these infections than do younger persons (7,38). Retrospective clinical data suggest a diminished febrile response in elderly individuals with infections (5, 35) and an attenuated acute phase response. Refinetti et al. (25) have shown that older rats developed fevers in response to an injection of yeast of similar magnitude to those of younger rats; however, it took the older rats longer for the fever to reach its peak. Because there is considerable evidence that fever is a host defense response (13, 14, 22, 27) this might partly explain the greater susceptibility and higher mortality rate from infections seen among elderly individuals. The development of a fever is but one component of the acute phase response. This stereotyped reaction to an infection or injury also generally included leukocytosis, changes in circulating protein levels (i.e., acute phase proteins), an increase in slow-wave sleep, and loss of food appetite. Since the 194Os, it has been thought that during injury or infection fever is generated by the release of endogenous pyrogens. These mediators are thought to AS INDIVIDUALS
MATTHEW
in young,
be cytokines such as interleukin (IL)-1, tumor necrosis factor (TNF), IL-6, and others (reviewed in Ref. 14). There is some evidence that the cytokine IL-6 is responsible both for fever (17) and for many of the other acute phase responses (6, 11, 24, 28, 36). Although TNF is another cytokine that is often considered to be an endogenous pyrogen, administration of antiserum to TNF has been found to cause larger fevers in rabbits or rats injected with LPS (19, 20, 23). Thus TNF may actually be an endogenous antipyretic or endogenous cryogen (see Ref. 14 for review). Psychological stress also causes an increase in the body temperature of rats (2, 3, 8, 9, 15, 29, 30, 34, 37) and human beings (10, 26). The stress-induced rises in body temperature induced by exposure of rats to an “open field” are attenuated by inhibitors of prostaglandin synthesis (3, 15, 29). In addition, the stress-induced rises in body temperature occur in both thermoneutral and cool ambient temperatures (21). Thus stress-induced rises in body temperature share many of the characteristics of true fevers, which are often induced experimentally by injections of bacterial lipopolysaccharide (LPS). Psychological stress induced by exposure to an open field has also been shown to cause an increase in the plasma concentration of IL-6 (18). The purpose of our studies was to determine the effects of age on body temperature rhythms, stress-induced fever, LPS-induced fever, and LPS-induced changes in plasma concentrations of IL-6 and TNF in the Fischer 344 rat. METHODS
Animals. Specific-pathogen-free young adult (3-5 mo old, 310-345g body wt), mature (12-14 mo old, 410-475 g body wt), and aged (24-27 mo old, 340-410 g body wt) male Fischer 344 rats were housedin individual cagesand maintained on a 12:12 h light-dark photoperiod at an ambient temperature of -2324°C. Tap water and rodent chow were provided ad libitum. Determination of body temperature. Core temperature was measuredby biotelemetry using transmitters implanted intraperitoneally (Mini-Mitter, Sunriver, OR) at least 1 wk before experiments. Each Mini-Mitter was calibrated before implantation. Output (frequency in Hz) was monitored by a mounted antenna placed under each rat’s cageand fed into a peripheral processor(Dataquest III system,Mini-Mitter) connectedto an IBM PC. Temperature was recorded at 2- to 15-min intervals dependingon the experiment. IL-6 bioassay.IL-6 activity in plasma was measuredusing the IL-6 dependentB9 hybridoma cell line (1,33). The B9 cells were cultured in Iscove’smodified Dulbecco’smedium(IMDM; Life Technology) supplementedwith human recombinant IL-6 (8 U/ml; obtained from Dr. Gordon Wong, GeneticsInstitute), 20 PM 2-mercaptoethanol, 10% heat-inactivated fetal calf serum, 100IU/ml penicillin, and 100pg/ml streptomycin. Cells were washed once in the above medium without added IL-6
036306119/92 $2.00 Copyright 0 1992 the American Physiological Society
R211
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R212
AGE AND FEVER,
IL-6, AND TNF IN RATS
before the addition of the plasma samplesor known amounts of recombinant human IL-6. To run the IL-6 assay, a 5-111sample to be assayed was combined with 5,000 B9 cells in 195 ~1 IMDM/lO% fetal calf serum in flat-bottom microtiter plates (Corning) for a final volume of 200~1.All sampleswere run in duplicate. The control medium, which contained no IL-6, was run in quadruplicate. In addition to the undiluted plasma samples,a serial dilution of each sample was assayed. Serial dilution was performed becauseour standard curve indicated that high levels of IL-6 result in an inhibition of growth of the B9 cells. Each sample was serially diluted initially to a maximum of 1:62,500. Cellswere pulsedat 68-72 h with 0.5 &i [3H]thymidine and harvested 4 h later onto glass-fiber filter strips (Cambridge Technology), and the radioactivity incorporated into DNA was counted by a /?-scintillation counter (Packard Instruments). For each assay, a standard curve was run with recombinant human IL-6. One unit of IL-6 is equal to the amount that causedhalf-maximum proliferation in the standard curve. From this standard curve, a best-fit regressionwas calculated for the rising portion of the curve in the linear range. The equation for this best-fit line wasusedto calculate IL-6 activity in plasma samples. All sampleswere serially diluted until they became undetectable. All counts per minute that fell within two standard deviations of the baselinecounts per minute were excluded to reduce the potential error resulting when converting from counts per minute to units IL-6 by multiplying by the dilution factor. The largest calculated IL-6 activity that fell outside the two standarddeviations of the baselineand fell well below the peak was taken as the IL-6 value. This ensured that all data usedfell in the steeplinear portion of the standardcurve, which increasedthe accuracy of the value. Z’YVFbioassay.The WEHI 164 subclone 13 cell line (kindly provided by Dr. Anders Waage, University of Trondheim, Norway) wasusedto measureTNF and to determinethe specificity of the TNF antiserum. The WEHI assay is based on the cytotoxic action of TNF on this fibroblast cell line (4). The cellswere grown in RPM1 1640 (GIBCO) containing 10% fetal calf serum (GIBCO), 10 mM glutamic acid, and 20 pg/ml gentomycin (Schering, Kenilworth, NJ) in an incubator at 37°C with 5% COZ.In the assay,cells (5 x lO’/lOO ~1)were incubated in the presenceof 0.5 pg of actinomycin D and 100 ~1of serial dilutions of the test samples.After 20 h, 20 ~1 of MTT tetrazolium (5 mg/ml; Sigma Chemical) was added, and the plates were incubated for an additional 4 h. During this time period, mitochondrial enzymes in the living cells convert the MTT tetrazolium into dark blue MTT formazan crystals. To dissolve these crystals, 150 ~1 of supernatant fluid was removed, and 100 ~1of isoproterenol-0.04N HCl was addedto eachwell. The plates were then wrapped in aluminum foil and left at room temperature overnight. On the following day, the plates were read on a micro-enzyme-linked immunosorbent assay autoreader (Titertek) at 540 nm, and the units of TNF were calculated basedon a recombinant human TNF standard curve run in the sameassay. Characterization of circadian rhythm. The body temperatures of the animals were monitored for three consecutive days. Temperature was recorded at 150minintervals. Hour averages of the 3-day meansof temperatures were calculated and used to determine the circadian patterns of temperature in the three groupsof rats. Stressparadigm. Acute psychological stresswas induced by two methods. Cageswitch involved removing the rat from its homecageand quickly placing it in a cagepreviously occupied by another rat of the sameage. Exposure to the olfactory and visual stimuli associatedwith this new environment results in stress-inducedrise in body temperature. The secondparadigm
employed was exposure to an open field. The open field used in theseexperiments consistedof a 60 X 38 X 81 in. high white acrylic spray temperature-controlled chamber(Warren Sherer) illuminated by two fluorescent lights suspendedfrom above. The temperature in the open field was similar to that in the rat’s home cage. The experimental protocol for this stress involved quickly removing the rat from its own small cageand placing it into the open field. Becauseit has previously been shownthat it requiresat least sevenexposuresto an open field before there is a decreasein temperature response(18), we gave eachrat two exposuresto the open field, one of 30-min duration and the other of 60-min duration. At least 1 wk was allowedto elapse between open field exposures. To minimize possible circadian variability, exposureto the open field occurred only between 9 A.M. and 4 P.M. Temperatures were recorded at 2min intervals during these experiments. Animals were conditioned to handling for 3 days before the first exposureto the open field. Care wastaken not to stressthe animalsbefore the cage-switchor open-field protocol. Blood samplingfrom jugular vein. Each animal was anesthetized with methoxyflurane (Pitman-Moore, Washington Crossing, NJ) and placed on a wooden board in a supine position with legs fully extended and forelimbs perpendicular to the sidesof the body. All appendageswere securedto the wooden board using surgicaltape. A loop of tape waspositionedaround the teeth, and the neck was fully extended at an angleof ~120” from the right shoulder.The left index finger wasplacedagainst the right shoulder as a guide. A 23-gaugeneedle (1 in. length) was placed parallel to the finger ~2 cm from the left finger. The needle was aimed toward either the opposite forearm or the leg diagonally opposedto the shoulder on which the index finger rested. Once the desiredamount of samplewasobtained, the syringe was slowly withdrawn and pressurewas applied to the area for -10 s. Injection of LPS. Bats were left undisturbed for 3 days postexposure to the open field. On day 3 at -10 A.M., the animals were injected intraperitoneally with ~0.3-0.4 ml of LPS (Olll:B4, Sigma Chemical) at a doseof 10 pg/kg. Body temperature was monitored for the next 24 h at lo-min intervals. To avoid the likelihood of the development of tolerance, a minimum of 1 wk wasallowedto elapsebefore rats were once again injected with LPS. In this experiment, at 90 min postinjection blood sampleswere obtained and plasma assayedfor IL-6 and TNF. Data analysis. Values reported are meansit SE. Data were analyzed using the Statview SE+ program. A one-factor analysisof variance (ANOVA) of data wasusedto test whether there were differencesin temperature and cytokine levels in response to cageswitch, open field, and LPS injection among the three agegroups. If significant differences were observed,the Fisher protected least significant difference test wasusedto determine which groupswere significantly different from each other. RESULTS
The 24-h body temperature rhythms of the three age groups are shown in Fig. 1. There was a significant difference among the mean daytime temperatures (ANOVA, F = 4.085, P = 0.0317). The mean daytime temperature was significantly higher in young rats (37.41”C) than in mature (37.13”C) or aged rats (37.19”C). Mean nighttime temperature was not significantly different among groups. Figure 2 shows the temperature response of groups to the switching of cages. The rate of fever development was not different among groups; however, young rats had a tendency to return to baseline temperature faster than
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AGE AND FEVER,
IL-6
36.5 G ” 5 55
37.5.
P
E F
-
mean young
--0-
mean mature (nd)
(Ike)
-
mean aged (es)
Fig. 1. Daily body temperature patterns of young, mature, and aged rata. Dark bar indicates period of darkness. Mean daytime temperature of young rats (37.41’C) was significantly higher than that of mature (37.13-C) and aged rats (37.19”C). There was no significant difference in mean nighttime temperatures among groups. n, Sample size.
g
1.5
:
1
--+-
mean
young
-
mean
mature
-f-
mean
aged
(n.8) (tk8) (n:8)
R213
AND TNF IN RATS
development in aged rats compared with young and mature rats such that the temperature of aged rats was significantly lower until -160-180 min post-LPS injection (Fig. 4). No significant differences were observed among groups in pattern or amplitude of temperature responses to saline injection (data not shown). Because there were significant differences in body temperatures of rats during the first 2-3 h after injection with LPS, we decided to sample plasma for cytokines at 90 min in our next experiment. Noninjected rats of all ages had baseline values of TNF and IL-6 that were low to nondetectable. Aged rats injected with LPS had significantly higher plasma concentrations of IL-6 (297,858 U/ml; ANOVA, F = 5.64, P = 0.013) than did mature (28,819 U/ml) or young rats (17,462 U/ml; Fig. 5). Aged rats also had significantly higher plasma concentrations of TNF (16,380 U/ml; ANOVA, F = 5.83, P = 0.011) than did young (574 U/ml) or mature rats (36 U/ml; Fig.
6). DISCUSSION
Fig. 2. Effect of cage switch on temperature of young, mature, and aged rats. There were no significant differences among groups in febrile response to switching cages of rats. Body temperature of young rats returned to baseline faster than that observed in older rate. n, Sample size. IS G e
5
W 30 minute 60 minute
l-l-
difference difference
z 1.0 ; 2 I-” .5 0.5
39 0 G e
385
s r;; 390 iiJ
%I i5 6
The data from this study support the hypothesis that older rats have a delayed temperature response when exposed to an open field and to injection of LPS. The fact that the magnitude of fever developed by aged animals after exposure to an open field for 60 min or in response to injection with LPS is not significantly different from that of younger rats suggests that the ability of aged rats to develop high fevers is not compromised.
; I-
375
nn
--o-
mean young (lea) mean mature (n=S)
-
mean aged (ll27)
“”
Young
Mature
Aged
Fig. 3. Effect of exposure to an open field for 30 and 60 min on rise in body temperature of young, mature, and aged rate. Exposure of rats to open tield for 30 min resulted in a smaller rise in temperature in aged rats than in younger rats. This temperature difference disappeared if rats were exposed to open field for 60 min. Numbers in parentheses are sample sizes.
the other age groups. The febrile responses to 30 and 60 min exposure to the open field are shown in Fig. 3. Each bar represents the difference in body temperature for the 6 min after return to the home cage minus the body temperature for the 6 min before exposure to the open field. Exposure to the open field for 30 min led to significant differences among the three groups (ANOVA, F = 6.495, P = 0.0067). The increase in temperature in aged rats (0.62”C) was significantly lower than that of young (1.23”C) and mature rats (0.98”C). There was no significant difference in the temperature changes of aged rats (0.79”C) vs. young (1.22”C) and mature rats (0.93”C) after 60 min in the open field. Injection of LPS resulted in a different pattern of fever
Fig. 4. Effect of injection with lipopolysaccharide (LPS) on temperature of young, mature, and aged rats. After injection with LPS, all 3 groups of rate developed fevers that peaked at similar temperatures. Pattern of fever in aged rats differed from that of mature and young rats such that the average temperature of aged rate was significantly lower than that of other groups of rats until -160-130 min postinjection. There were no significant differences among groups when rats were injected with saline. n, Sample size. l,
W young
(n=Q)
Fig. 5. Plasma interleukin (IL)-6 concentration 90 min after injection with LPS in young, mature, and aged rate. n, Sample sire.
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R214
AGE AND FEVER, IL-6, AND TNF IN RATS Grant AI-27556. Address reprint requests to M. J. Kluger.
*I * -7
(16,360) I
young
(n-9)
Received 20 May 1991; accepted in final form 30 July 1991. REFERENCES 1. Aarden,
L. A.,
Fig. 6. Plasma concentrations of tumor necrosis factor (TNF) 90 min after injection with LPS in young, mature, and aged rats. n, Sample size.
Tocco-Bradley et al. (32) also found that the magnitude of the febrile response to Salmonella typhimurium was not significantly diminished in aged rats exposed to cold stress (15°C). On the other hand, there may be a defect in the kinetics of the biochemical machinery responsible for the development of this fever as manifested in the different pattern of fever development in aged rats after LPS injection as well as the delayed temperature response to exposure to an open field. The levels of TNF and IL-6 were significantly higher in aged rats at 90 min postinjection with LPS even though their body temperatures were lower than those of mature or young rats. We do not know whether these differences in cytokines disappear at a later time (e.g., 4 h after injection with LPS), when body temperatures of all three groups are similar. These findings support the hypothesis first espoused by Long et al. (19, 20) that TNF may be an endogenous antipyretic or cryogen. In these studies, rats were injected with antiserum to TNF or to control serum and after several hours to 3 days were injected with LPS. The rats that received the antiserum to TNF developed larger fevers in response to the injection of LPS. Further support for the hypothesis that TNF is an endogenous antipyretic comes from the study by Mathison et al. (23) showing higher fever in rabbits injected with antibody to TNF and from Holt et al. (12), who showed that injection with small amounts of TNF into the central nervous system led to a decline in metabolic rate and body temperature in rats. If the smaller fevers in the aged rats during the first few hours after injection with LPS are the result of increased production of TNF, this would mean that any pyrogenic action of the increased circulating levels of IL6 was overridden by the elevated TNF levels. Thus TNF’s antipyretic actions might be masking the pyrogenic actions of IL-6 and those of other putative endogenous pyrogens. In light of the evidence that small rises in body temperature enhance host defense responses (13, 14, 22, 27), the presence of a delayed rise in body temperature in older rats may contribute to the increased morbidity and mortality to infection observed in this group.
E. R. de Groot,
0.
L.
Schaap,
and
P. M.
of hybridoma growth factors by human monocytea. Rur. J. Immunal. 17: 1411-1416, 1987. 2. Blasig, J., V. Hollt, U. Bauerle, and A. Herz. Involvement of endorphins in emotional hyperthermia of rats. Life Sci. 23: 25252532, 1978. 3. Briese. E., and M. Cabanac. Emotional fever and salicvlates. Prac. Int. Congr. Physivl. Sci. 28th Budapest 1980, p. 161-163. 4. Espevik, T., and J. N&en-Meyer. A highly sensitive cell line, WEHI 164 clone 13, for measuring cytotoxic factor/tumor necrosis factor from human monocytes. J. Immurwl. Methods 95: 99-105, 1986. 5. Fischer, H. C., and H. M. White, Jr. Biliary tract disease in the aged. Arch. Surg. 63: 536-544, 1951. 6. Gauldie, J., C. Richards, D. Harnish, and H. Baumann. Interferon B, is identical to monocyte HSF and regulates the full acute phase protein response in liver cells. In: Progress in Leukocyte Biology, Monokines and Otker Non-Lymphacytic Cytakines, edited by M. C. Powanda, J. J. Oppenheim, M. J. Kluger, and C. A. Dinarello. New York: Liss, 1988, vol. 8, p. 15-20. 7. Gladstone, J. L., and R. Recco. Host factors and infectious diseases in the elderly. Med. Clin. North Am. 60: 1225-1240, 1976. 8. Goldstein, A., and P. Lowery. Effect of the endogenous oaiate naloxoneon body temperature in rats. Life Sci. 17: 927-932, 1975. 9. Gollnick. P. D.. and C. D. Ianuzzo. Colonic temnerature response of rats during exercise. J. Appl. Physiol. 24: 743-750, 1968. 10. Hasan, M. K., and A. C. White. Psychogenic fever, entity or nonentity? Postgrad. Med. J. 66: 152-154,197s. 11. Helle, M., J. P. Brakenhoff, E. R. D. Groot, and L. A. Aarden. Interleukin-6 is involved in interleukin l-induced activities. Eur. J. Immunol. 18: 957-959, 1988. 12. Holt, S. J., R. F. Grimble, and D. A. York. Tumour necrosis factor-a and lymphotoxin have opposite effects on sympathetic efferent nerves to brown adipose tissue by direct action in the central nervous system. Brain Res. 497: 183-186,1989. 13. Kluger, M. J. The adaptive value of fever. In: Fever: Basic Mechanisms and Management, edited by P. Mackowiak. New York: Raven, 1991, p. 105-124. 14. Kluger, M. J. Fever: role of endogenous pyrogens and cryogens. Physiol. Rev. 71: 93-127, 1991. 15. Kluger, M. J., B. O’Reilly, T. R. Shope, and A. J. Vander. Further evidence that stress hyperthermia is a fever. Physial. Landsdorp.
Behau.
16. Lemay,
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39: 763-766, 1987. L. G., I. G. Otterness,
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In vivo evidence that the rise in plasma IL-6 following an injection of fever-inducing dose of LPS is mediated by IL-l& Cytokine 2: 199-204, 1990. 17. Lemay, L., A. J. Vander, and M. J. Kluger. The role of IL-6 in fever in the rat. Am. J. Physiol. 258 (Regulatory Integrative Comp. Physial. 27): R798-R803, 1990. 18. Lemay, L. G., A. J. Vander, and M. J. Kluger. The effects of psychological stress on plasma interleukin-6 activity in rats. Physial. Behau. 47: 957-961, 1990. 19. Long, N. C., S. L. Kunkel, A. J. Vander, and M. J. Kluger. Antiserum against tumor necrosis factor increases stress hyperthermia in rats. Am. J. Physiol. 258 (Regulatory Integrative Comp. Pkysial. 27): R591R595, 1990. Kluger.
20. Long, M. J.
N. C., Kluger.
I. Otterness,
S. L. Kunkel,
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and
The roles of interleukin-1B and tumor necrosis factor in lipopolysaccharide-fever in rats. Am. J. Pkysiol. 259 (Regulatory Integrative Camp. Physiol. 28): R724-R728, 1990. 21. Long, N. C., A. J. Vander, and M. J. Kluger. Stress-induced rise of body temperature in rats is the same in warm and cool environments. Physiol. Behav. 47: 773-775,199O. 22. Mackowiak, P. A. Direct effects of physiologic variations in temperature on pathogenic microorganisms. In: Feuer: Basic MechWe thank Jennifer McClellan for running the IL-6 and TNF assays anisms and Management, edited by P. Mackowiak. New York: and Dr. Lucien A. Aarden for providing us with the B9 cell line. Raven, 1991, p. 167-182. 23. Mathison. J. C., E. Wolfson, and R. J. Ulevitch. Participation This research was supported by the University of Michigan Geriatof tumor necrosis factor in the mediation of gram negative bacterial rics Center and Institute of Allergy and by Infectious Diseases Downloaded fromNational www.physiology.org/journal/ajpregu ${individualUser.givenNames} ${individualUser.surname} (137.092.098.118) on September 25, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
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N&ten, M. W. N., E. R. DeGroot, H. J. Ten Duis, H. J. Klasen, C. E. Hack, and L. A. Aarden. Serum levels of interleukin-6 and the acute phase response. Lancet 2: 21, 1987. Refinetti, R., H. M. Ma, and E. Satinoff. Body temperature rhythms, cold tolerance, and fever in young and old rats of both genders. Exp. Gerontol. 25: 533-543,199O. Renbourn, E. T. Body temperature and pulse rate in boys and young men prior to sporting contests. A study in emotional hyperthermia, with a review of the literature. J. Psychosom. Res. 4: 149175, 1960. Roberts, N. J., Jr. The immunological consequences of fever. In: Fever: Basic Mechanisms and Management, edited by P. Mackowiak. New York: Raven, 1991, p. 125-142. Santhanam, U., S. B. Tabber, D. C. Helfgott, A. Ray, J. Ghrayeb, L. T. May, and P. B. Seghal. Activation of the human “&-interferon/hepatocyte-stimulating factor/interleukin 6” promoter by cytokines, viruses, and second messenger agonists. Proc. Natl. Acad. Sci. USA 85: 6701-6705,1988. Singer, R., C. T. Harker, A. J. Vander, and M. J. Kluger. Hyperthermia induced by open field stress is blocked by salicylate. Physid. Behuu. 36: 1179-1182,1986. Stewart, J., and R. Eikelboom. Stress masks the hypothermic
effect of naloxone in rats. Life Sci. 25: 1165-1172,1979. Stitt, J. T. Prostaglandin E as the neural mediator response. Yak J. Bid. Med. 59: 137-149,1986. 32 . Tocco-Bradley, R., Kluger, M. J., and C. A. Effects of age on fever and acute phase response of toxin and Salmon& typhimurium. Infect. Immun.
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Van Oers, M. H. J., A. A. P. A. M. Van Der Heyden, and L. A. Aarden. Interleukin-6 (IL-6) in serum and urine of renal transplant patients. Clin. Exp. Immund. 71: 312-318,1988. Vidal, C., C. Suaudeau, and J. Jacob. Hyper- and hypothermia induced by non-noxious stress, effects of naloxone, diazepam, and y-acetylenic GABA. Life Sci. 33: 587-590,1983. Watanakunakorn, C., and J. S. Tan. Diagnostic difficulties of staphylococcal endocarditis in geriatric patients. Geriatrics27: 168173,1983.
36* Wong, G. C., and S. C. Clark. Multiple actions of interleukin-6 with a cytokine network. Immund. Today 9: 137-139,1988. 37. York, J. L., and S. G. Regan. Conditioned and unconditioned influences on body temperature and ethanol hypothermia in laboratory rats. Phurmucol. B&hem. Behuu. 17: 119-124,1982. 38. Yoshikawa, T. T. Geriatric infectious diseases. J. Am. Geriut. Sot. 31: 34-39,1983.
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D. FOSTER,
Department
CAROLE
A. CONN,
of Physiology, University
AND
J. KLUGER
of Michigan Medical School, Ann Arbor, Michigan 48109
Foster, Kara D., Carob A. Conn, and Matthew J. Kluger. Fever, tumor necrosis factor, and interleukin-6 in young, mature, and agedFischer 344 rats. Am. J. PhydoZ. 262 (RegulatoryIntegrative Comp.Physiol. 31): R211-R215,1992.The purposeof this study wasto comparethe febrile responses of Fischer 344 rats of different ages[young (3-5 mo), mature (12-15 mo), and aged (24-27 mo; n = S)] to two psychological stressparadigms, cageswitch and exposureto an open field, as well as to injection of lipopolysaccharide (LPS). In addition, the cytokines tumor necrosisfactor-a (TNF) and interleukin6 were also measuredin the plasma of these rats at 90 min postinjection with LPS. There was no significant difference among groups in febrile responsesto switching their cages. Exposure to an open field for 30 min resulted in a smaller rise in temperature in the agedrats (0.62”C) than in the young rats (1.26”C). This difference disappearedif rats were exposedto an open field for 60 min. Injection of LPS led to fevers that developed at a slower rate in aged rats than in the mature groups. The peak fevers, however, were not different. The activity of interleukin-6 90 min after injection of LPS was higher in aged rats (297,358U/ml) than in young (17,462 U/ ml) and mature rats (28,819U/ml). TNF levelswere alsohigher in aged rats (16,380 U/ml) compared with young (574 U/ml) and mature rats (36 U/ml). We conclude that although the magnitude of the febrile responseis not different among rats of different ages,the rise in body temperature occursslower in agedrats. The finding that agedrats had significantly elevated TNF levels in their plasma during the time when their body temperatureswere lower than that of young and mature rats is consistent with the hypothesis that endogenously produced TNF is an antipyretic (or an endogenouscryogen) in the rat. stress-hyperthermia;cytokines; lipopolysaccharide
become older, they are known to experience a greater number of bacterial infections and a higher mortality rate from these infections than do younger persons (7,38). Retrospective clinical data suggest a diminished febrile response in elderly individuals with infections (5, 35) and an attenuated acute phase response. Refinetti et al. (25) have shown that older rats developed fevers in response to an injection of yeast of similar magnitude to those of younger rats; however, it took the older rats longer for the fever to reach its peak. Because there is considerable evidence that fever is a host defense response (13, 14, 22, 27) this might partly explain the greater susceptibility and higher mortality rate from infections seen among elderly individuals. The development of a fever is but one component of the acute phase response. This stereotyped reaction to an infection or injury also generally included leukocytosis, changes in circulating protein levels (i.e., acute phase proteins), an increase in slow-wave sleep, and loss of food appetite. Since the 194Os, it has been thought that during injury or infection fever is generated by the release of endogenous pyrogens. These mediators are thought to AS INDIVIDUALS
MATTHEW
in young,
be cytokines such as interleukin (IL)-1, tumor necrosis factor (TNF), IL-6, and others (reviewed in Ref. 14). There is some evidence that the cytokine IL-6 is responsible both for fever (17) and for many of the other acute phase responses (6, 11, 24, 28, 36). Although TNF is another cytokine that is often considered to be an endogenous pyrogen, administration of antiserum to TNF has been found to cause larger fevers in rabbits or rats injected with LPS (19, 20, 23). Thus TNF may actually be an endogenous antipyretic or endogenous cryogen (see Ref. 14 for review). Psychological stress also causes an increase in the body temperature of rats (2, 3, 8, 9, 15, 29, 30, 34, 37) and human beings (10, 26). The stress-induced rises in body temperature induced by exposure of rats to an “open field” are attenuated by inhibitors of prostaglandin synthesis (3, 15, 29). In addition, the stress-induced rises in body temperature occur in both thermoneutral and cool ambient temperatures (21). Thus stress-induced rises in body temperature share many of the characteristics of true fevers, which are often induced experimentally by injections of bacterial lipopolysaccharide (LPS). Psychological stress induced by exposure to an open field has also been shown to cause an increase in the plasma concentration of IL-6 (18). The purpose of our studies was to determine the effects of age on body temperature rhythms, stress-induced fever, LPS-induced fever, and LPS-induced changes in plasma concentrations of IL-6 and TNF in the Fischer 344 rat. METHODS
Animals. Specific-pathogen-free young adult (3-5 mo old, 310-345g body wt), mature (12-14 mo old, 410-475 g body wt), and aged (24-27 mo old, 340-410 g body wt) male Fischer 344 rats were housedin individual cagesand maintained on a 12:12 h light-dark photoperiod at an ambient temperature of -2324°C. Tap water and rodent chow were provided ad libitum. Determination of body temperature. Core temperature was measuredby biotelemetry using transmitters implanted intraperitoneally (Mini-Mitter, Sunriver, OR) at least 1 wk before experiments. Each Mini-Mitter was calibrated before implantation. Output (frequency in Hz) was monitored by a mounted antenna placed under each rat’s cageand fed into a peripheral processor(Dataquest III system,Mini-Mitter) connectedto an IBM PC. Temperature was recorded at 2- to 15-min intervals dependingon the experiment. IL-6 bioassay.IL-6 activity in plasma was measuredusing the IL-6 dependentB9 hybridoma cell line (1,33). The B9 cells were cultured in Iscove’smodified Dulbecco’smedium(IMDM; Life Technology) supplementedwith human recombinant IL-6 (8 U/ml; obtained from Dr. Gordon Wong, GeneticsInstitute), 20 PM 2-mercaptoethanol, 10% heat-inactivated fetal calf serum, 100IU/ml penicillin, and 100pg/ml streptomycin. Cells were washed once in the above medium without added IL-6
036306119/92 $2.00 Copyright 0 1992 the American Physiological Society
R211
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R212
AGE AND FEVER,
IL-6, AND TNF IN RATS
before the addition of the plasma samplesor known amounts of recombinant human IL-6. To run the IL-6 assay, a 5-111sample to be assayed was combined with 5,000 B9 cells in 195 ~1 IMDM/lO% fetal calf serum in flat-bottom microtiter plates (Corning) for a final volume of 200~1.All sampleswere run in duplicate. The control medium, which contained no IL-6, was run in quadruplicate. In addition to the undiluted plasma samples,a serial dilution of each sample was assayed. Serial dilution was performed becauseour standard curve indicated that high levels of IL-6 result in an inhibition of growth of the B9 cells. Each sample was serially diluted initially to a maximum of 1:62,500. Cellswere pulsedat 68-72 h with 0.5 &i [3H]thymidine and harvested 4 h later onto glass-fiber filter strips (Cambridge Technology), and the radioactivity incorporated into DNA was counted by a /?-scintillation counter (Packard Instruments). For each assay, a standard curve was run with recombinant human IL-6. One unit of IL-6 is equal to the amount that causedhalf-maximum proliferation in the standard curve. From this standard curve, a best-fit regressionwas calculated for the rising portion of the curve in the linear range. The equation for this best-fit line wasusedto calculate IL-6 activity in plasma samples. All sampleswere serially diluted until they became undetectable. All counts per minute that fell within two standard deviations of the baselinecounts per minute were excluded to reduce the potential error resulting when converting from counts per minute to units IL-6 by multiplying by the dilution factor. The largest calculated IL-6 activity that fell outside the two standarddeviations of the baselineand fell well below the peak was taken as the IL-6 value. This ensured that all data usedfell in the steeplinear portion of the standardcurve, which increasedthe accuracy of the value. Z’YVFbioassay.The WEHI 164 subclone 13 cell line (kindly provided by Dr. Anders Waage, University of Trondheim, Norway) wasusedto measureTNF and to determinethe specificity of the TNF antiserum. The WEHI assay is based on the cytotoxic action of TNF on this fibroblast cell line (4). The cellswere grown in RPM1 1640 (GIBCO) containing 10% fetal calf serum (GIBCO), 10 mM glutamic acid, and 20 pg/ml gentomycin (Schering, Kenilworth, NJ) in an incubator at 37°C with 5% COZ.In the assay,cells (5 x lO’/lOO ~1)were incubated in the presenceof 0.5 pg of actinomycin D and 100 ~1of serial dilutions of the test samples.After 20 h, 20 ~1 of MTT tetrazolium (5 mg/ml; Sigma Chemical) was added, and the plates were incubated for an additional 4 h. During this time period, mitochondrial enzymes in the living cells convert the MTT tetrazolium into dark blue MTT formazan crystals. To dissolve these crystals, 150 ~1 of supernatant fluid was removed, and 100 ~1of isoproterenol-0.04N HCl was addedto eachwell. The plates were then wrapped in aluminum foil and left at room temperature overnight. On the following day, the plates were read on a micro-enzyme-linked immunosorbent assay autoreader (Titertek) at 540 nm, and the units of TNF were calculated basedon a recombinant human TNF standard curve run in the sameassay. Characterization of circadian rhythm. The body temperatures of the animals were monitored for three consecutive days. Temperature was recorded at 150minintervals. Hour averages of the 3-day meansof temperatures were calculated and used to determine the circadian patterns of temperature in the three groupsof rats. Stressparadigm. Acute psychological stresswas induced by two methods. Cageswitch involved removing the rat from its homecageand quickly placing it in a cagepreviously occupied by another rat of the sameage. Exposure to the olfactory and visual stimuli associatedwith this new environment results in stress-inducedrise in body temperature. The secondparadigm
employed was exposure to an open field. The open field used in theseexperiments consistedof a 60 X 38 X 81 in. high white acrylic spray temperature-controlled chamber(Warren Sherer) illuminated by two fluorescent lights suspendedfrom above. The temperature in the open field was similar to that in the rat’s home cage. The experimental protocol for this stress involved quickly removing the rat from its own small cageand placing it into the open field. Becauseit has previously been shownthat it requiresat least sevenexposuresto an open field before there is a decreasein temperature response(18), we gave eachrat two exposuresto the open field, one of 30-min duration and the other of 60-min duration. At least 1 wk was allowedto elapse between open field exposures. To minimize possible circadian variability, exposureto the open field occurred only between 9 A.M. and 4 P.M. Temperatures were recorded at 2min intervals during these experiments. Animals were conditioned to handling for 3 days before the first exposureto the open field. Care wastaken not to stressthe animalsbefore the cage-switchor open-field protocol. Blood samplingfrom jugular vein. Each animal was anesthetized with methoxyflurane (Pitman-Moore, Washington Crossing, NJ) and placed on a wooden board in a supine position with legs fully extended and forelimbs perpendicular to the sidesof the body. All appendageswere securedto the wooden board using surgicaltape. A loop of tape waspositionedaround the teeth, and the neck was fully extended at an angleof ~120” from the right shoulder.The left index finger wasplacedagainst the right shoulder as a guide. A 23-gaugeneedle (1 in. length) was placed parallel to the finger ~2 cm from the left finger. The needle was aimed toward either the opposite forearm or the leg diagonally opposedto the shoulder on which the index finger rested. Once the desiredamount of samplewasobtained, the syringe was slowly withdrawn and pressurewas applied to the area for -10 s. Injection of LPS. Bats were left undisturbed for 3 days postexposure to the open field. On day 3 at -10 A.M., the animals were injected intraperitoneally with ~0.3-0.4 ml of LPS (Olll:B4, Sigma Chemical) at a doseof 10 pg/kg. Body temperature was monitored for the next 24 h at lo-min intervals. To avoid the likelihood of the development of tolerance, a minimum of 1 wk wasallowedto elapsebefore rats were once again injected with LPS. In this experiment, at 90 min postinjection blood sampleswere obtained and plasma assayedfor IL-6 and TNF. Data analysis. Values reported are meansit SE. Data were analyzed using the Statview SE+ program. A one-factor analysisof variance (ANOVA) of data wasusedto test whether there were differencesin temperature and cytokine levels in response to cageswitch, open field, and LPS injection among the three agegroups. If significant differences were observed,the Fisher protected least significant difference test wasusedto determine which groupswere significantly different from each other. RESULTS
The 24-h body temperature rhythms of the three age groups are shown in Fig. 1. There was a significant difference among the mean daytime temperatures (ANOVA, F = 4.085, P = 0.0317). The mean daytime temperature was significantly higher in young rats (37.41”C) than in mature (37.13”C) or aged rats (37.19”C). Mean nighttime temperature was not significantly different among groups. Figure 2 shows the temperature response of groups to the switching of cages. The rate of fever development was not different among groups; however, young rats had a tendency to return to baseline temperature faster than
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AGE AND FEVER,
IL-6
36.5 G ” 5 55
37.5.
P
E F
-
mean young
--0-
mean mature (nd)
(Ike)
-
mean aged (es)
Fig. 1. Daily body temperature patterns of young, mature, and aged rata. Dark bar indicates period of darkness. Mean daytime temperature of young rats (37.41’C) was significantly higher than that of mature (37.13-C) and aged rats (37.19”C). There was no significant difference in mean nighttime temperatures among groups. n, Sample size.
g
1.5
:
1
--+-
mean
young
-
mean
mature
-f-
mean
aged
(n.8) (tk8) (n:8)
R213
AND TNF IN RATS
development in aged rats compared with young and mature rats such that the temperature of aged rats was significantly lower until -160-180 min post-LPS injection (Fig. 4). No significant differences were observed among groups in pattern or amplitude of temperature responses to saline injection (data not shown). Because there were significant differences in body temperatures of rats during the first 2-3 h after injection with LPS, we decided to sample plasma for cytokines at 90 min in our next experiment. Noninjected rats of all ages had baseline values of TNF and IL-6 that were low to nondetectable. Aged rats injected with LPS had significantly higher plasma concentrations of IL-6 (297,858 U/ml; ANOVA, F = 5.64, P = 0.013) than did mature (28,819 U/ml) or young rats (17,462 U/ml; Fig. 5). Aged rats also had significantly higher plasma concentrations of TNF (16,380 U/ml; ANOVA, F = 5.83, P = 0.011) than did young (574 U/ml) or mature rats (36 U/ml; Fig.
6). DISCUSSION
Fig. 2. Effect of cage switch on temperature of young, mature, and aged rats. There were no significant differences among groups in febrile response to switching cages of rats. Body temperature of young rats returned to baseline faster than that observed in older rate. n, Sample size. IS G e
5
W 30 minute 60 minute
l-l-
difference difference
z 1.0 ; 2 I-” .5 0.5
39 0 G e
385
s r;; 390 iiJ
%I i5 6
The data from this study support the hypothesis that older rats have a delayed temperature response when exposed to an open field and to injection of LPS. The fact that the magnitude of fever developed by aged animals after exposure to an open field for 60 min or in response to injection with LPS is not significantly different from that of younger rats suggests that the ability of aged rats to develop high fevers is not compromised.
; I-
375
nn
--o-
mean young (lea) mean mature (n=S)
-
mean aged (ll27)
“”
Young
Mature
Aged
Fig. 3. Effect of exposure to an open field for 30 and 60 min on rise in body temperature of young, mature, and aged rate. Exposure of rats to open tield for 30 min resulted in a smaller rise in temperature in aged rats than in younger rats. This temperature difference disappeared if rats were exposed to open field for 60 min. Numbers in parentheses are sample sizes.
the other age groups. The febrile responses to 30 and 60 min exposure to the open field are shown in Fig. 3. Each bar represents the difference in body temperature for the 6 min after return to the home cage minus the body temperature for the 6 min before exposure to the open field. Exposure to the open field for 30 min led to significant differences among the three groups (ANOVA, F = 6.495, P = 0.0067). The increase in temperature in aged rats (0.62”C) was significantly lower than that of young (1.23”C) and mature rats (0.98”C). There was no significant difference in the temperature changes of aged rats (0.79”C) vs. young (1.22”C) and mature rats (0.93”C) after 60 min in the open field. Injection of LPS resulted in a different pattern of fever
Fig. 4. Effect of injection with lipopolysaccharide (LPS) on temperature of young, mature, and aged rats. After injection with LPS, all 3 groups of rate developed fevers that peaked at similar temperatures. Pattern of fever in aged rats differed from that of mature and young rats such that the average temperature of aged rate was significantly lower than that of other groups of rats until -160-130 min postinjection. There were no significant differences among groups when rats were injected with saline. n, Sample size. l,
W young
(n=Q)
Fig. 5. Plasma interleukin (IL)-6 concentration 90 min after injection with LPS in young, mature, and aged rate. n, Sample sire.
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R214
AGE AND FEVER, IL-6, AND TNF IN RATS Grant AI-27556. Address reprint requests to M. J. Kluger.
*I * -7
(16,360) I
young
(n-9)
Received 20 May 1991; accepted in final form 30 July 1991. REFERENCES 1. Aarden,
L. A.,
Fig. 6. Plasma concentrations of tumor necrosis factor (TNF) 90 min after injection with LPS in young, mature, and aged rats. n, Sample size.
Tocco-Bradley et al. (32) also found that the magnitude of the febrile response to Salmonella typhimurium was not significantly diminished in aged rats exposed to cold stress (15°C). On the other hand, there may be a defect in the kinetics of the biochemical machinery responsible for the development of this fever as manifested in the different pattern of fever development in aged rats after LPS injection as well as the delayed temperature response to exposure to an open field. The levels of TNF and IL-6 were significantly higher in aged rats at 90 min postinjection with LPS even though their body temperatures were lower than those of mature or young rats. We do not know whether these differences in cytokines disappear at a later time (e.g., 4 h after injection with LPS), when body temperatures of all three groups are similar. These findings support the hypothesis first espoused by Long et al. (19, 20) that TNF may be an endogenous antipyretic or cryogen. In these studies, rats were injected with antiserum to TNF or to control serum and after several hours to 3 days were injected with LPS. The rats that received the antiserum to TNF developed larger fevers in response to the injection of LPS. Further support for the hypothesis that TNF is an endogenous antipyretic comes from the study by Mathison et al. (23) showing higher fever in rabbits injected with antibody to TNF and from Holt et al. (12), who showed that injection with small amounts of TNF into the central nervous system led to a decline in metabolic rate and body temperature in rats. If the smaller fevers in the aged rats during the first few hours after injection with LPS are the result of increased production of TNF, this would mean that any pyrogenic action of the increased circulating levels of IL6 was overridden by the elevated TNF levels. Thus TNF’s antipyretic actions might be masking the pyrogenic actions of IL-6 and those of other putative endogenous pyrogens. In light of the evidence that small rises in body temperature enhance host defense responses (13, 14, 22, 27), the presence of a delayed rise in body temperature in older rats may contribute to the increased morbidity and mortality to infection observed in this group.
E. R. de Groot,
0.
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Schaap,
and
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of hybridoma growth factors by human monocytea. Rur. J. Immunal. 17: 1411-1416, 1987. 2. Blasig, J., V. Hollt, U. Bauerle, and A. Herz. Involvement of endorphins in emotional hyperthermia of rats. Life Sci. 23: 25252532, 1978. 3. Briese. E., and M. Cabanac. Emotional fever and salicvlates. Prac. Int. Congr. Physivl. Sci. 28th Budapest 1980, p. 161-163. 4. Espevik, T., and J. N&en-Meyer. A highly sensitive cell line, WEHI 164 clone 13, for measuring cytotoxic factor/tumor necrosis factor from human monocytes. J. Immurwl. Methods 95: 99-105, 1986. 5. Fischer, H. C., and H. M. White, Jr. Biliary tract disease in the aged. Arch. Surg. 63: 536-544, 1951. 6. Gauldie, J., C. Richards, D. Harnish, and H. Baumann. Interferon B, is identical to monocyte HSF and regulates the full acute phase protein response in liver cells. In: Progress in Leukocyte Biology, Monokines and Otker Non-Lymphacytic Cytakines, edited by M. C. Powanda, J. J. Oppenheim, M. J. Kluger, and C. A. Dinarello. New York: Liss, 1988, vol. 8, p. 15-20. 7. Gladstone, J. L., and R. Recco. Host factors and infectious diseases in the elderly. Med. Clin. North Am. 60: 1225-1240, 1976. 8. Goldstein, A., and P. Lowery. Effect of the endogenous oaiate naloxoneon body temperature in rats. Life Sci. 17: 927-932, 1975. 9. Gollnick. P. D.. and C. D. Ianuzzo. Colonic temnerature response of rats during exercise. J. Appl. Physiol. 24: 743-750, 1968. 10. Hasan, M. K., and A. C. White. Psychogenic fever, entity or nonentity? Postgrad. Med. J. 66: 152-154,197s. 11. Helle, M., J. P. Brakenhoff, E. R. D. Groot, and L. A. Aarden. Interleukin-6 is involved in interleukin l-induced activities. Eur. J. Immunol. 18: 957-959, 1988. 12. Holt, S. J., R. F. Grimble, and D. A. York. Tumour necrosis factor-a and lymphotoxin have opposite effects on sympathetic efferent nerves to brown adipose tissue by direct action in the central nervous system. Brain Res. 497: 183-186,1989. 13. Kluger, M. J. The adaptive value of fever. In: Fever: Basic Mechanisms and Management, edited by P. Mackowiak. New York: Raven, 1991, p. 105-124. 14. Kluger, M. J. Fever: role of endogenous pyrogens and cryogens. Physiol. Rev. 71: 93-127, 1991. 15. Kluger, M. J., B. O’Reilly, T. R. Shope, and A. J. Vander. Further evidence that stress hyperthermia is a fever. Physial. Landsdorp.
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