Roles of interleukin in lipopolysaccharide
1p and tumor necrosis factor fever in rats
NANCY C. LONG, IVAN OTTERNESS, STEVEN L. KUNKEL, ARTHUR J. VANDER, AND MATTHEW J. KLUGER of Physiology and Pathology, University of Michigan Medical School, Departments Ann Arbor, Michigan 48109; and Pfizer Corporation, Groton, Connecticut 06340
LONG, NANCY C., IVAN OTTERNESS, STEVEN L. KUNKEL, ARTHUR J, VANDER, AND MATTHEW J. KLUGER. Roles of interleukin l@ and tumor necrosis factor in lipopolysaccharide fever in rats. Am. J. Physiol. 259 (Regulatory Integrative Comp. Physiol. 28): R724-R728, 1990.-The roles of interleukin lp (IL-lp) and tumor necrosis factor-a (TNF) in lipopolysaccharide (LPS)-induced fever were investigated in the rat. We used antisera against IL-lp and TNF to determine whether we could alter the fever by blocking the action of these cytokines. The intravenous injection of antiserum IL-l@ 3.5 days before the intraperitoneal injection of LPS resulted in a mean fever that was significantly lower than that seen in rats that had been injected with control serum (0.36 t 0.11 vs. 0.82 t O.l6”C, P = 0.016). The intravenous injection of antiserum against TNF 3.5 days before the intraperitoneal injection of LPS did not block the fever but significantly enhanced it (1.31 t 0.16 vs. 0.82 t O.l6”C, P = 0.027). These data support the hypotheses that IL-lp is responsible for a significant part of LPS fever and that TNF acts as an endogenous antipyretic to limit the magnitude of LPS fever in the rat. cachectin;
endotoxin;
pyrogen; antipyretic
FEVER HAS LONG BEEN recognized as a symptom of infectious disease. During infection, exogenous pyrogens such as bacterial endotoxin are thought to stimulate macrophages to release an endogenous pyrogen (EP) into the circulation (3). The EP is thought to cause fever by raising the thermoregulatory set point via the release of prostaglandins of the E series in the hypothalamus (32). While this basic model of fever has been in use for many years, the identity of EP continues to elude workers in this field. Because fever is a ubiquitous phenomenon, being found in infected mammals, birds, reptiles, amphibians, fishes, and even many species of invertebrates (16), and because fever is known to have profound effects on immune and host defense responses (16, 23, 30), numerous laboratories have sought to determine the identity of EP. Several years ago, many investigators concluded that the circulating EP is interleukin (IL) 1, a cytokine that occurs in two forms IL-la and IL-l@ (1). Perhaps the strongest evidence consistent with the hypothesis that EP and IL-l are identical is that the injection of either form of recombinant IL-l systematically or intracerebroventricularly causes fever (7, 10, 25, 33). It must be emphasized, however, that almost all the R724
experiments suggesting that EP and IL-l are identical involved the exogenous administration of purified or IL-l, not its endogenous release. To our recombinant knowledge, there is no evidence that nonlethal infection or the injection of a fever-inducing dose of lipopolysaccharide (LPS) causes the release of sufficient IL-lb into the circulation to cause fever. Cannon et al. (4) reported significant, but small, increases in the plasma levels of IL-l& as measured by radioimmunoassay, in humans after the injection of 4 rig/kg of LPS. However, other researchers have failed to detect IL-l in the plasma of humans after bolus injection (14) or infusion (26) of fever-inducing doses of LPS. In our laboratory, we have been unable to detect IL-l in the plasma of rats after the injection of LPS. In studies where IL-l has been detected during infection, rather than after LPS, the subjects have been extremely ill. In a study of 20 patients with meningicoccal meningitis, IL-l was detected in only 3 subjects (34). These subjects were in septic shock and had the most rapidly fatal courses of all the patients who died. IL-l was also detected in the plasma of baboons injected with a lethal dose of Escherichia coZi (14). This dose of E. coli was associated with a drop in mean arterial pressure and shock, and it is likely that body temperature was below normal at the time that the IL-l was measured. It is not clear whether the failure consistently to detect IL-l during fever or infection is due to methodological problems or whether some other cytokine is the circulating EP. Many other cytokines have been found to cause fever when injected (Table 1), and this casts further doubt on the hypothesis that IL-l is the exclusive endogenous mediator of fever. The hypothesis that IL-la, ILlp, or any of the other cytokines listed in Table 1 are EPs can be directly tested by pretreatment with antibodies known to block the biological activity of the cytokine; if the cytokine is an EP, then such pretreatment should attenuate the fever caused by injection of an exogenous pyrogen such as LPS. In an initial experiment (20), we injected rats with antiserum to murine IL-la, demonstrated that this antiserum would attenuate the fever caused by injected IL-la, and then determined whether this antiserum would influence fever caused by injection of LPS. The antiserum had no effect on the magnitude or duration of the LPS-induced fever. This makes it
0363-6119/90 $1.50 Copyright 0 1990 the American Physiological Society
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IL-1p
AND
TNF
unlikely that IL-la is responsible for LPS fever in the rat. Nagai et al. (28) and Kawasaki et al. (15) have found that the injection of monoclonal antibodies against tumor necrosis factor-a (TNF), another cytokine capable of causing fever when injected, significantly inhibited the febrile response to LPS in rabbits. In contrast, Mathison et al. (24) found that pretreating rabbits with polyclonal antiserum to TNF did not attenuate the fever produced by LPS; indeed, their data suggest that the fever was actually enhanced (no statistics were reported). We have performed a similar study using antiserum against TNF in rats. We injected antiserum against TNF or control serum intraperitoneally 2 h before the intramuscular injection of LPS. The antiserum-injected rats developed fevers that were not attenuated but were significantly higher than those seen in the rats injected with control serum (P = 0.0005) (21). In the present study, we examined the roles of IL-lp and TNF in LPS fever. We injected antisera against ILlp or TNF into rats intravenously 3.5 days before the intraperitoneal injection of LPS. METHODS
Animals
Specific pathogen-free male Sprague-Dawley rats weighing 200-325 g were obtained from the Charles River Breeding Laboratories, Portage, MI. Rats were housed in individual plastic cages in a room maintained at 26 t l”C, i.e., in the thermoneutral zone for rats, with a photoperiod consisting of 12 h of light and 12 h of dark. Tap water and rodent chow (Purina 5001) were provided ad libitum. Rats were used only once. Measurement of Body Temperature
Body temperature was measured using battery-operated biotelemetry devices (Mini Mitter, Sunriver, OR), implanted intraperitoneally into each rat 4 or more days before experimentation began. Each transmitter was calibrated before implantation. Output (frequency in Hz) was monitored by a mounted antenna placed under each animal’s cage and fed into a peripheral processor (Dataquest III System, Mini Mitter) connected to an IBM-PC (see Ref. 31 for a more detailed description). Temperatures were monitored and recorded at 15-min intervals. TABLE
of Each
of These
Cytokines
Causes
Interleukin la and 10 (IL-la and -1p) Tumor necrosis factor (TNF-(u) Interleukin 6 (IL-6) Interleukin 8 (IL-8) Interferon-a and -y Granulocyte-macrophage colony-stimulating factor (gmCSF) Lymphotoxin (TNFP) Macrophage inhibitory protein-l (MIP-1) * N. tJ. Rothwell,
personal
communication.
FEVER
R725
Antisera and LPS
The antiserum against recombinant mouse IL-lfl (Pfizer Research, Groton, CT) was produced in a rabbit. The resulting antibody was found to be specific for both mouse and rat IL-16 by western blotting. Blocking antibody was titered in vitro against recombinant murine ILlp using the lymphocyte-activating factor assay. The antiserum against TNF was produced by injecting recombinant mouse TNF (Cetus, Emeryville, CA) into a rabbit (5). This antiserum had been shown to neutralize rat TNF in vitro and to block the pulmonary and hepatic injury in rats exposed to hepatic ischemia and reperfusion in vivo (6). The sera were isolated and then stored frozen at -2OOC. Control animals received serum from a nonimmunized rabbit. All sera were kept sterile. The LPS used in these studies was derived from E. coli endotoxin (OllkB4, Sigma lot no. 97F-4089), diluted to 10 pg/ml in 0.9% pyrogen-free sodium chloride (saline), and injected at a dose of 10 pg LPS/kg. Experimental Design Protocol 1. The effect of the intravenous injection of antiserum against IL-l@ 3.5 days before the intraperitoneal injection of LPS. Ten rats that had been implanted
with Mini Mitters were anesthetized with methoxyfluorane (Pitman Moore, Washington Crossing, NJ) and then injected intravenously into the penile vein with 100 ~1 of antiserum against IL-l@ that had been diluted with 400 ~1 of saline. Nine other rats received the same dose of control serum in an identical manner. After 3.5 days, all rats were injected with 10 pg/kg LPS intraperitoneally at 1000 h. In a second experiment, a higher dose of antiserum (400 ~1 of antiserum with 100 ~1 of saline) was injected into seven rats. Six others received the same dose of control serum. Rats from both groups were injected intraperitoneally, 3.5 days later, with 10 pg/kg of LPS at 1000 h. Protocol 2. The effect of the intravenous injection of antiserum against TNF 3.5 days before the intraperitoneal injection of LPS. As a part of the second experiment
described above, nine rats were injected intravenously with 400 ~1 of rabbit-anti-mouse TNF antiserum diluted in saline to a total volume of 0.5 ml. After 3.5 days, these animals received an intraperitoneal injection of 10 pg/kg of LPS. The controls used in this study were the same as those used above, i.e., rats injected with 400 ~1 of control serum. Data Analysis
1. Fever-inducing cytokines Injection
IN
Fever
9 12 13 *
Values reported are means t SE. Comparisons between each experimental and control group were analyzed for statistical significance using Student’s t test. Time intervals for analysis of fever curves were selected in advance based on pilot studies.
27 29
RESULTS
Ref. No.
11 8
Protocol 1. The effect of the intravenous injection of antiserum against IL-l@ 3.5 days before the intraperitoneaZ injection of LPS. Injections of the control serum or
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R726
IL-l@
AND
TNF
antiserum had no effect on body temperature of rats not given LPS (data not shown). The results of protocol 1 are shown in Fig. 1. Figure 1A shows the change in temperature of rats treated with the lower dose (100 ~1) of antiserum against IL-1p before the injection of LPS. The changes in temperature were calculated by subtracting the baseline temperature of each animal (the average of the 0945 and 1000 h temperature readings) from its temperature at each subsequent time point. Before the administration of LPS, the body temperature of the rats that had received antiserum did not differ significantly from that of the rats that had received control serum (37.20 t 0.14 vs. 37.21 t O.O7”C, P = 0.95). During the time period between 1115 and 1530 h, the mean fever developed by rats that received this dose of anti IL-lp was 0.60 t 0.12”C. The rats that had received control serum developed a mean fever of 1.01 t O.lO°C. This difference was significant (P = 0.010). Figure 1B shows the change in temperature of rats that had been injected with the higher dose (400 ~1) of anti-IL-l@ or an equivalent dose of control serum before the injection of LPS. Before the LPS was injected, the rats that had received antiserum had a mean body temperature of 37.21 t O.lO”C, which did not differ significantly from that of the control serum-injected rats (37.17 t O.O6”C, P = 0.75). The rats that had received this dose of antiserum developed a mean fever of 0.36 t O.ll”C, which was significantly lower than that seen in the rats that had been injected with the same dose of control serum (0.82 t O.l6”C, P = 0.016). Protocol 2. The effect of the intravenous injection of antiserum against TNF 3.5 days before the intraperitoneal injection of LPS. Injections of the control serum or
2 0.0~‘“““‘““““““““‘” 6
10
11
1
12
13
+ LPS
14
11
12
13
14
TIME
16 + U
TIME
0s 0.0~~““‘~““““““““““““‘~ 10 4 LPS
15
15 + +
I
I
I
16 17 18 ANTI IL-l BETA N=7 CONTROL N=6
(A)
FIG. 1. Change in body temperature of rats injected with 100 ~1 or ~1 (R) of antiserum against IL-l@ or equal doses of control serum intravenously 3.5 days before intraperitoneal injection of 10 pg/ kg of lipopolysaccharide (LPS).
400
(
17 18 ANTI IL-l BETA N=lO CONTROL N=9
IN
FEVER ~~0.027
LPS FIG. 2. Change antiserum against serum intravenously kg of LPS.
TIME
+ U
ANTITNF N=9 CONTROL N=6
400
in body temperature of rats injected with ~1 of tumor necrosis factor or an equal dose of control 3.5 days before intraperitoneal injection of 10 pg/
antiserum had no effect on body temperature of rats not given LPS (data not shown). The results of protocol 2 are shown in Fig. 2. Before the administration of LPS, the antiserum-injected rats had body temperatures that were not significantly different from those of the controlserum-injected animals (37.33 t 0.08 vs. 37.17 t 0.06, P = 0.19). The mean fever in the rats that had been pretreated with antiserum against TNF was 1.31 t 0.16”C. This was significantly higher than the fevers seen in the control-serum-injected rats (0.82 t O.l6”C, P = 0.027). DISCUSSION
The results of the experiments using anti-IL-1P demonstrate that the action of IL-l@ is necessary for at least half of the febrile response to LPS. From these data it is not clear whether IL-l@ might actually account for 100% of the fever due to LPS, because it is possible that injection of a much higher dose of antiserum would have blocked the entire febrile response. It is also not known whether IL-1p acts directly on the brain to cause fever or whether IL-16 is released locally (for example, in the liver) and acts as a paracrine to cause the release of another pyrogenic cytokine, such as IL-6, which then reaches the brain via the circulation. Consistent with this indirect role for IL-lp is the failure of most investigators to find IL-l@ in the plasma during fever, our observation that injection of antiserum against IL-10 resulted in significantly lower plasma levels of IL-6 (18), and the fact that plasma IL-6 activity correlates highly with the magnitude of LPS-induced fever (19). Because the antiserum against IL-l@ attenuated fever, whereas in our earlier study (21) antiserum against TNF, when injected intraperitoneally 2 h before LPS, had enhanced fever, we felt it essential to repeat the TNF study using the same conditions (iv injection 3.5 days before LPS) as that used for IL-l@ antiserum in the present experiments. The result was essentially the same as in the previous TNF study and adds further evidence that TNF actually functions as an antipyretic during LPS fever in the rat. A similar finding was reported by Mathison et al. (24), who showed that injection of polyclonal antibody against TNF tended to enhance the febrile response to LPS. Mathison et al. also showed that the injection of their antibodies blocked the increase in
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IL-16
AND
TNF
plasma TNF normally seen in rabbits after injection of LPS. This is in contrast to the reports by Nagai et al. (28) and Kawasaki et al. (15), who found that the injection of a monoclonal antibody against TNF attenuated the fever due to LPS. However, neither of these latter studies included data on the plasma concentrations of TNF in their LPS-injected rabbits. It is possible that the monoclonal antibody did not neutralize plasma TNF activity and perhaps resulted in an elevated plasma concentration of this cytokine (e.g., by binding to some site involved in the negative-feedback regulation of TNF production or secretion). The attenuated fever they reported could be the result of an increase in the concentration of TNF itself. Another possible explanation for the elevated fever in the rats that had received antiserum against TNF is that antigen-antibody complexes are forming between the TNF and the antibody, causing a secondary immune response. This is unlikely, however, because the picogram amounts of TNF that are produced in response to LPS are probably not sufficient to trigger an immune reaction that would result in fever. Furthermore, we have observed a similar exaggeration of the thermal response to psychological stress (“stress hyperthermia”) when rats were pretreated with this antiserum against TNF (22). Finally, this antiserum has been shown to attenuate, not enhance, other responses that may be mediated by TNF in the rat (6). In that study, rats were exposed to 90 min of hepatic ischemia followed by reperfusion, a procedure that leads to elevated plasma levels of TNF and to hepatic and pulmonary injury. Pretreatment of rats with this TNF antisera before hepatic ischemia-reperfusion injury blocked the subsequent damage to the liver and the lungs. It is clear from both in vivo (14) and in vitro (2, 17) studies that the cytokines do not act independently but rather participate in complex networks involving synergistic as well as inhibitory interactions. Therefore, it is important to consider the action of each cytokine in terms of how it interacts with others. There is some in vitro evidence that TNF stimulates the release of IL-l and may synergize to cause prostaglandin release (2, 17). However, since IL-l is not detectable in the plasma of animals during moderate fevers, it is not clear whether this interaction occurs in vivo. In any case, our data support the hypothesis that IL-lp, produced in some location, is essential for at least one-half of LPS fever and that this effect of IL-lp does not depend on any secondary release of TNF. This work was supported in part by National Institutes of Health Grant NS-23633 and by grants from the Vice President for Research and the Biomedical Research Council of The University of Michigan. Address for reprint requests: M. J. Kluger, Dept. of Physiology, 7620 Med. Sci. II, Univ. of Michigan Medical School, Ann Arbor, MI 48109.
IN
2.
3.
4.
5.
6
7.
8.
9. 10.
11. 12.
13.
14.
15.
16. 17.
18. Received
16 January
1990; accepted
in final
form
1 June
1990.
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FEVER
R727
LEFKOWITS, R. I. MISHELL, S. B. MIZEL, J. J. OPPENHEIM, V. PAETKAU, J. PLATE, M. ROLLINGHOFF, D. ROSENSTREICH, A. S. ROSENTHAL, L. J. ROSENWASSER, A. SCHIMPL, H, S. SHIM, P. L. SIMON, K. A. SMITH, H. WAGNER, J. D. WATSON, E. WECKER, AND D. D. WOOD. Revised nomenclature for antigen-non-specific T cell proliferation and helper factors (Letter to Editor). J. Immunol. 123: 2928, 1979. BACKWICH, P. R., S. W. CHENSUE, J. W. LARRICK, AND S. L. KUNKEL. Tumor necrosis factor and prostaglandin E2 production in resting macrophages. Biochem. Biophys. Res. Commun. 136: 94101, 1986. BEESON, P. B. Temperature-elevating effect of a substance obtained from polymorphonuclear leukocytes. J. Clin. Inuest. 27: 524, 1948. CANNON, J. G., R. G. THOMPKINS, J. A. GELFAND, H. R. MICHIE, G. G STANFORD, J. W. M. VAN DER MEER, S. ENDRED, G. LONNEMANN, J. CORSETTI, B. CHERNOW, D. W. WILMORE, S. M. WOLFF, J. F. BURKE, AND C. A. DINARELLO. Circulating interleukin-l and tumor necrosis factor in septic shock and experimental endotoxin fever. J. Infect. Dis. 161: 79-235, 1990. CHENSUE, S. W., D. G. REMICK, C. SHMYR-FORSCH, T. F. BEALS, AND S. L. KUNKEL. Immunochemical demonstration of cytoplasmic and membrane associated tumor necrosis factor in murine macrophages. Am. J. Pathol. 133: 564-572, 1988. COLLETTI, L. M., D. G. REMICK, G. D. BURTCH, S. L. KUNKEL, R. M. STREITER, AND D. A. CAMPBELL, JR. The role of TNF in pathophysiologic alterations following hepatic ischemia/reperfurion (Abstract). J. Clin. Inuest. 85: 1936-1943, 1990. DASCOMBE, M. J., N. J. ROTHWELL, B. 0. SAGAY, AND M. J. STOCK. Pyrogenic and thermogenic effects of interleukin-10 in the rat. Am. J. Physiol. 256 (Endocrinol. Metab. Physiol. 25): E7-Eli, 1989. DAVATELIS, G., S. D. WOLFE, B. SHERRY, J.-M. DAYER, R. CHICHEPORTICHE, AND A. CERAMI. Macrophage inflammatory protein-l: a prostaglandin-independent endogenous pyrogen. Science Wash. DC 243: 1066-1068,1989. DINARELLO, C. A. Interleukin-1. Reu. Inject. Dis. 6: 51-95, 1984. DINARELLO, C. A., J. G. CANNON, J. W. MEIR, H. A. BERNHEIM, G. LOPRESTE, D. L. LYNN, R. N. LOVE, A. C. WEBB, P. E. AURON, R. C. REUBEN, A. RICH, S. M. WOLFF, AND S. D. PUTNEY. Multiple biological activities of human recombinant interleukin1. J. Clin. Invest. 77: 1734-1739, 1986. DINARELLO, D. A., J. G. CANNON, AND S. M. WOLFF. New concepts on the pathogenesis of fever. Reu. Inject. Dis. 10: 168-189, 1988. DINARELLO, C. A., J. G. CANNON, S. M. WOLFF, H. A. BERNHEIM, B. BEUTLER, A. CERAMI, I. S. FIGARI, M. A. PALLADINO, JR., AND J. V. O’CONNOR. Tumor necrosis factor (cachectin) is an endogenous pyrogen and induces the production of interleukin-1. J. Exp. Med. 163: 1433-1450,1986. HELLE, M., J. P. J. BRAKENHOFF, E. R. DEGROOT, AND L. A. AARDEN. Interleukin-6 is involved in interleukin-l-induced activities. Eur. J. Immunol. 18: 957-959, 1988. HESSE, D. G., K. J. TRACEY, Y. FONG, K. R. MANOGUE, M. A. PALLADINO, JR., A. CERAMI, G. T. SHIRES, AND S. F. LOWRY. Cytokine appearance in human endotoxemia and primate bacteremia. Surg. Gynecol. Obstet. 166: 147-153, 1988. KAWASAKI, H., M. MOYIYAMA, Y. OHTANI, M. NAITOH, A. TANAKA, AND H. NARIUCHI. Analysis of endotoxin fever in rabbits using a monoclonal antibody to tumor necrosis factor (cachectin). Infect. Immun. 57: 3131-3135, 1989. KLUGER, M. J. Fever: Its Biology, Evolution, and Function. Princeton, NJ: Princeton Univ. Press, 1979. LAST-BARNEY, K., C. A. HOMON, R. B. FAANES, AND V. J. MERLUZZI. Synergistic and overlapping effects of tumor necrosis factor cy and IL-l. J. Immunol. 141: 527-530, 1988. LEMAY, L. G., A. J. VANDER, AND M. J. KLUGER. In vivo evidence that the rise in plasma IL-6 following injection of fever-inducing dose of LPS is mediated by IL-l@. Cytokine 2: 199-204, 1990. LEMAY, L. G., A. J. VANDER, AND M. J. KLUGER. The role of interleukin-6 in fever in the rat. Am. J. Physiol. 258 (Regulatory Integrative Comp. Physiol. 27): R798-R803, 1990. LONG, N. C., S. L. KUNKEL, A. J. VANDER, AND M. J. KLUGER. Antiserum against mouse IL-1 alpha does not block stress hyperthermia or LPS fever in the rat. In: Thermoregulation: Research
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AND
and Clinical Applications, edited by P. Lomax and E. Schonbaum. Basel: Karger, 1989, p. 78-84. LONG, N. C., S. L. KUNKEL, A. J. VANDER, AND M. J. KLUGER. Antiserum against TNF enhances LPS fever in the rat. Am. J. Physiol. 258 (Regulatory Integrative Comp. Physiol. 27): R332R337, 1990. LONG, N. C., A. J. VANDER, S. L. KUNKEL, AND M. J. KLUGER. Antiserum against tumor necrosis factor increases stress hyperthermia in rats. Am. J. Physiol. 258 (Regulatory Integrative Camp. Physiol. 27): R591-R595, 1990. MAKOWIAK, P. A. Direct effects of hyperthermia on pathogenic microorganisms: teleologic implications with regard to fever. Rev. Infect. Dis. 3: 508-520, 1981. MATHISON, J. C., E. WOLFSON, AND R. J. ULEVITCH. Participation of tumor necrosis factor in the mediation of gram negative bacterial lipopolysaccharide-induced injury in rabbits. J. Clin. Invest. 81: 1925-1937,1988. MCCARTHY, D. O., M. J. KLUGER, AND A. J. VANDER. Supression of food intake during infection: is interleukin-1 involved? Am. J. Clin. Nutr. 42: 1179-1182, 1985. MICHIE, H. R., D. R. SPRIGGS, K. R. MANOGUE, M. L. SHERMAN, A. REVHAUG, S. T. O’DWYER, K. ARTHUR, C. A. DINARELLO, A. CERAMI, S. M. WOLFF, D. W. KUFE, AND D. W. WILMORE. Tumor necrosis factor and endotoxin induce similar metabolic responses in human beings. Surgery St. Louis 104: 280-286, 1988.
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1p and tumor necrosis factor fever in rats
NANCY C. LONG, IVAN OTTERNESS, STEVEN L. KUNKEL, ARTHUR J. VANDER, AND MATTHEW J. KLUGER of Physiology and Pathology, University of Michigan Medical School, Departments Ann Arbor, Michigan 48109; and Pfizer Corporation, Groton, Connecticut 06340
LONG, NANCY C., IVAN OTTERNESS, STEVEN L. KUNKEL, ARTHUR J, VANDER, AND MATTHEW J. KLUGER. Roles of interleukin l@ and tumor necrosis factor in lipopolysaccharide fever in rats. Am. J. Physiol. 259 (Regulatory Integrative Comp. Physiol. 28): R724-R728, 1990.-The roles of interleukin lp (IL-lp) and tumor necrosis factor-a (TNF) in lipopolysaccharide (LPS)-induced fever were investigated in the rat. We used antisera against IL-lp and TNF to determine whether we could alter the fever by blocking the action of these cytokines. The intravenous injection of antiserum IL-l@ 3.5 days before the intraperitoneal injection of LPS resulted in a mean fever that was significantly lower than that seen in rats that had been injected with control serum (0.36 t 0.11 vs. 0.82 t O.l6”C, P = 0.016). The intravenous injection of antiserum against TNF 3.5 days before the intraperitoneal injection of LPS did not block the fever but significantly enhanced it (1.31 t 0.16 vs. 0.82 t O.l6”C, P = 0.027). These data support the hypotheses that IL-lp is responsible for a significant part of LPS fever and that TNF acts as an endogenous antipyretic to limit the magnitude of LPS fever in the rat. cachectin;
endotoxin;
pyrogen; antipyretic
FEVER HAS LONG BEEN recognized as a symptom of infectious disease. During infection, exogenous pyrogens such as bacterial endotoxin are thought to stimulate macrophages to release an endogenous pyrogen (EP) into the circulation (3). The EP is thought to cause fever by raising the thermoregulatory set point via the release of prostaglandins of the E series in the hypothalamus (32). While this basic model of fever has been in use for many years, the identity of EP continues to elude workers in this field. Because fever is a ubiquitous phenomenon, being found in infected mammals, birds, reptiles, amphibians, fishes, and even many species of invertebrates (16), and because fever is known to have profound effects on immune and host defense responses (16, 23, 30), numerous laboratories have sought to determine the identity of EP. Several years ago, many investigators concluded that the circulating EP is interleukin (IL) 1, a cytokine that occurs in two forms IL-la and IL-l@ (1). Perhaps the strongest evidence consistent with the hypothesis that EP and IL-l are identical is that the injection of either form of recombinant IL-l systematically or intracerebroventricularly causes fever (7, 10, 25, 33). It must be emphasized, however, that almost all the R724
experiments suggesting that EP and IL-l are identical involved the exogenous administration of purified or IL-l, not its endogenous release. To our recombinant knowledge, there is no evidence that nonlethal infection or the injection of a fever-inducing dose of lipopolysaccharide (LPS) causes the release of sufficient IL-lb into the circulation to cause fever. Cannon et al. (4) reported significant, but small, increases in the plasma levels of IL-l& as measured by radioimmunoassay, in humans after the injection of 4 rig/kg of LPS. However, other researchers have failed to detect IL-l in the plasma of humans after bolus injection (14) or infusion (26) of fever-inducing doses of LPS. In our laboratory, we have been unable to detect IL-l in the plasma of rats after the injection of LPS. In studies where IL-l has been detected during infection, rather than after LPS, the subjects have been extremely ill. In a study of 20 patients with meningicoccal meningitis, IL-l was detected in only 3 subjects (34). These subjects were in septic shock and had the most rapidly fatal courses of all the patients who died. IL-l was also detected in the plasma of baboons injected with a lethal dose of Escherichia coZi (14). This dose of E. coli was associated with a drop in mean arterial pressure and shock, and it is likely that body temperature was below normal at the time that the IL-l was measured. It is not clear whether the failure consistently to detect IL-l during fever or infection is due to methodological problems or whether some other cytokine is the circulating EP. Many other cytokines have been found to cause fever when injected (Table 1), and this casts further doubt on the hypothesis that IL-l is the exclusive endogenous mediator of fever. The hypothesis that IL-la, ILlp, or any of the other cytokines listed in Table 1 are EPs can be directly tested by pretreatment with antibodies known to block the biological activity of the cytokine; if the cytokine is an EP, then such pretreatment should attenuate the fever caused by injection of an exogenous pyrogen such as LPS. In an initial experiment (20), we injected rats with antiserum to murine IL-la, demonstrated that this antiserum would attenuate the fever caused by injected IL-la, and then determined whether this antiserum would influence fever caused by injection of LPS. The antiserum had no effect on the magnitude or duration of the LPS-induced fever. This makes it
0363-6119/90 $1.50 Copyright 0 1990 the American Physiological Society
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IL-1p
AND
TNF
unlikely that IL-la is responsible for LPS fever in the rat. Nagai et al. (28) and Kawasaki et al. (15) have found that the injection of monoclonal antibodies against tumor necrosis factor-a (TNF), another cytokine capable of causing fever when injected, significantly inhibited the febrile response to LPS in rabbits. In contrast, Mathison et al. (24) found that pretreating rabbits with polyclonal antiserum to TNF did not attenuate the fever produced by LPS; indeed, their data suggest that the fever was actually enhanced (no statistics were reported). We have performed a similar study using antiserum against TNF in rats. We injected antiserum against TNF or control serum intraperitoneally 2 h before the intramuscular injection of LPS. The antiserum-injected rats developed fevers that were not attenuated but were significantly higher than those seen in the rats injected with control serum (P = 0.0005) (21). In the present study, we examined the roles of IL-lp and TNF in LPS fever. We injected antisera against ILlp or TNF into rats intravenously 3.5 days before the intraperitoneal injection of LPS. METHODS
Animals
Specific pathogen-free male Sprague-Dawley rats weighing 200-325 g were obtained from the Charles River Breeding Laboratories, Portage, MI. Rats were housed in individual plastic cages in a room maintained at 26 t l”C, i.e., in the thermoneutral zone for rats, with a photoperiod consisting of 12 h of light and 12 h of dark. Tap water and rodent chow (Purina 5001) were provided ad libitum. Rats were used only once. Measurement of Body Temperature
Body temperature was measured using battery-operated biotelemetry devices (Mini Mitter, Sunriver, OR), implanted intraperitoneally into each rat 4 or more days before experimentation began. Each transmitter was calibrated before implantation. Output (frequency in Hz) was monitored by a mounted antenna placed under each animal’s cage and fed into a peripheral processor (Dataquest III System, Mini Mitter) connected to an IBM-PC (see Ref. 31 for a more detailed description). Temperatures were monitored and recorded at 15-min intervals. TABLE
of Each
of These
Cytokines
Causes
Interleukin la and 10 (IL-la and -1p) Tumor necrosis factor (TNF-(u) Interleukin 6 (IL-6) Interleukin 8 (IL-8) Interferon-a and -y Granulocyte-macrophage colony-stimulating factor (gmCSF) Lymphotoxin (TNFP) Macrophage inhibitory protein-l (MIP-1) * N. tJ. Rothwell,
personal
communication.
FEVER
R725
Antisera and LPS
The antiserum against recombinant mouse IL-lfl (Pfizer Research, Groton, CT) was produced in a rabbit. The resulting antibody was found to be specific for both mouse and rat IL-16 by western blotting. Blocking antibody was titered in vitro against recombinant murine ILlp using the lymphocyte-activating factor assay. The antiserum against TNF was produced by injecting recombinant mouse TNF (Cetus, Emeryville, CA) into a rabbit (5). This antiserum had been shown to neutralize rat TNF in vitro and to block the pulmonary and hepatic injury in rats exposed to hepatic ischemia and reperfusion in vivo (6). The sera were isolated and then stored frozen at -2OOC. Control animals received serum from a nonimmunized rabbit. All sera were kept sterile. The LPS used in these studies was derived from E. coli endotoxin (OllkB4, Sigma lot no. 97F-4089), diluted to 10 pg/ml in 0.9% pyrogen-free sodium chloride (saline), and injected at a dose of 10 pg LPS/kg. Experimental Design Protocol 1. The effect of the intravenous injection of antiserum against IL-l@ 3.5 days before the intraperitoneal injection of LPS. Ten rats that had been implanted
with Mini Mitters were anesthetized with methoxyfluorane (Pitman Moore, Washington Crossing, NJ) and then injected intravenously into the penile vein with 100 ~1 of antiserum against IL-l@ that had been diluted with 400 ~1 of saline. Nine other rats received the same dose of control serum in an identical manner. After 3.5 days, all rats were injected with 10 pg/kg LPS intraperitoneally at 1000 h. In a second experiment, a higher dose of antiserum (400 ~1 of antiserum with 100 ~1 of saline) was injected into seven rats. Six others received the same dose of control serum. Rats from both groups were injected intraperitoneally, 3.5 days later, with 10 pg/kg of LPS at 1000 h. Protocol 2. The effect of the intravenous injection of antiserum against TNF 3.5 days before the intraperitoneal injection of LPS. As a part of the second experiment
described above, nine rats were injected intravenously with 400 ~1 of rabbit-anti-mouse TNF antiserum diluted in saline to a total volume of 0.5 ml. After 3.5 days, these animals received an intraperitoneal injection of 10 pg/kg of LPS. The controls used in this study were the same as those used above, i.e., rats injected with 400 ~1 of control serum. Data Analysis
1. Fever-inducing cytokines Injection
IN
Fever
9 12 13 *
Values reported are means t SE. Comparisons between each experimental and control group were analyzed for statistical significance using Student’s t test. Time intervals for analysis of fever curves were selected in advance based on pilot studies.
27 29
RESULTS
Ref. No.
11 8
Protocol 1. The effect of the intravenous injection of antiserum against IL-l@ 3.5 days before the intraperitoneaZ injection of LPS. Injections of the control serum or
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R726
IL-l@
AND
TNF
antiserum had no effect on body temperature of rats not given LPS (data not shown). The results of protocol 1 are shown in Fig. 1. Figure 1A shows the change in temperature of rats treated with the lower dose (100 ~1) of antiserum against IL-1p before the injection of LPS. The changes in temperature were calculated by subtracting the baseline temperature of each animal (the average of the 0945 and 1000 h temperature readings) from its temperature at each subsequent time point. Before the administration of LPS, the body temperature of the rats that had received antiserum did not differ significantly from that of the rats that had received control serum (37.20 t 0.14 vs. 37.21 t O.O7”C, P = 0.95). During the time period between 1115 and 1530 h, the mean fever developed by rats that received this dose of anti IL-lp was 0.60 t 0.12”C. The rats that had received control serum developed a mean fever of 1.01 t O.lO°C. This difference was significant (P = 0.010). Figure 1B shows the change in temperature of rats that had been injected with the higher dose (400 ~1) of anti-IL-l@ or an equivalent dose of control serum before the injection of LPS. Before the LPS was injected, the rats that had received antiserum had a mean body temperature of 37.21 t O.lO”C, which did not differ significantly from that of the control serum-injected rats (37.17 t O.O6”C, P = 0.75). The rats that had received this dose of antiserum developed a mean fever of 0.36 t O.ll”C, which was significantly lower than that seen in the rats that had been injected with the same dose of control serum (0.82 t O.l6”C, P = 0.016). Protocol 2. The effect of the intravenous injection of antiserum against TNF 3.5 days before the intraperitoneal injection of LPS. Injections of the control serum or
2 0.0~‘“““‘““““““““‘” 6
10
11
1
12
13
+ LPS
14
11
12
13
14
TIME
16 + U
TIME
0s 0.0~~““‘~““““““““““““‘~ 10 4 LPS
15
15 + +
I
I
I
16 17 18 ANTI IL-l BETA N=7 CONTROL N=6
(A)
FIG. 1. Change in body temperature of rats injected with 100 ~1 or ~1 (R) of antiserum against IL-l@ or equal doses of control serum intravenously 3.5 days before intraperitoneal injection of 10 pg/ kg of lipopolysaccharide (LPS).
400
(
17 18 ANTI IL-l BETA N=lO CONTROL N=9
IN
FEVER ~~0.027
LPS FIG. 2. Change antiserum against serum intravenously kg of LPS.
TIME
+ U
ANTITNF N=9 CONTROL N=6
400
in body temperature of rats injected with ~1 of tumor necrosis factor or an equal dose of control 3.5 days before intraperitoneal injection of 10 pg/
antiserum had no effect on body temperature of rats not given LPS (data not shown). The results of protocol 2 are shown in Fig. 2. Before the administration of LPS, the antiserum-injected rats had body temperatures that were not significantly different from those of the controlserum-injected animals (37.33 t 0.08 vs. 37.17 t 0.06, P = 0.19). The mean fever in the rats that had been pretreated with antiserum against TNF was 1.31 t 0.16”C. This was significantly higher than the fevers seen in the control-serum-injected rats (0.82 t O.l6”C, P = 0.027). DISCUSSION
The results of the experiments using anti-IL-1P demonstrate that the action of IL-l@ is necessary for at least half of the febrile response to LPS. From these data it is not clear whether IL-l@ might actually account for 100% of the fever due to LPS, because it is possible that injection of a much higher dose of antiserum would have blocked the entire febrile response. It is also not known whether IL-1p acts directly on the brain to cause fever or whether IL-16 is released locally (for example, in the liver) and acts as a paracrine to cause the release of another pyrogenic cytokine, such as IL-6, which then reaches the brain via the circulation. Consistent with this indirect role for IL-lp is the failure of most investigators to find IL-l@ in the plasma during fever, our observation that injection of antiserum against IL-10 resulted in significantly lower plasma levels of IL-6 (18), and the fact that plasma IL-6 activity correlates highly with the magnitude of LPS-induced fever (19). Because the antiserum against IL-l@ attenuated fever, whereas in our earlier study (21) antiserum against TNF, when injected intraperitoneally 2 h before LPS, had enhanced fever, we felt it essential to repeat the TNF study using the same conditions (iv injection 3.5 days before LPS) as that used for IL-l@ antiserum in the present experiments. The result was essentially the same as in the previous TNF study and adds further evidence that TNF actually functions as an antipyretic during LPS fever in the rat. A similar finding was reported by Mathison et al. (24), who showed that injection of polyclonal antibody against TNF tended to enhance the febrile response to LPS. Mathison et al. also showed that the injection of their antibodies blocked the increase in
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IL-16
AND
TNF
plasma TNF normally seen in rabbits after injection of LPS. This is in contrast to the reports by Nagai et al. (28) and Kawasaki et al. (15), who found that the injection of a monoclonal antibody against TNF attenuated the fever due to LPS. However, neither of these latter studies included data on the plasma concentrations of TNF in their LPS-injected rabbits. It is possible that the monoclonal antibody did not neutralize plasma TNF activity and perhaps resulted in an elevated plasma concentration of this cytokine (e.g., by binding to some site involved in the negative-feedback regulation of TNF production or secretion). The attenuated fever they reported could be the result of an increase in the concentration of TNF itself. Another possible explanation for the elevated fever in the rats that had received antiserum against TNF is that antigen-antibody complexes are forming between the TNF and the antibody, causing a secondary immune response. This is unlikely, however, because the picogram amounts of TNF that are produced in response to LPS are probably not sufficient to trigger an immune reaction that would result in fever. Furthermore, we have observed a similar exaggeration of the thermal response to psychological stress (“stress hyperthermia”) when rats were pretreated with this antiserum against TNF (22). Finally, this antiserum has been shown to attenuate, not enhance, other responses that may be mediated by TNF in the rat (6). In that study, rats were exposed to 90 min of hepatic ischemia followed by reperfusion, a procedure that leads to elevated plasma levels of TNF and to hepatic and pulmonary injury. Pretreatment of rats with this TNF antisera before hepatic ischemia-reperfusion injury blocked the subsequent damage to the liver and the lungs. It is clear from both in vivo (14) and in vitro (2, 17) studies that the cytokines do not act independently but rather participate in complex networks involving synergistic as well as inhibitory interactions. Therefore, it is important to consider the action of each cytokine in terms of how it interacts with others. There is some in vitro evidence that TNF stimulates the release of IL-l and may synergize to cause prostaglandin release (2, 17). However, since IL-l is not detectable in the plasma of animals during moderate fevers, it is not clear whether this interaction occurs in vivo. In any case, our data support the hypothesis that IL-lp, produced in some location, is essential for at least one-half of LPS fever and that this effect of IL-lp does not depend on any secondary release of TNF. This work was supported in part by National Institutes of Health Grant NS-23633 and by grants from the Vice President for Research and the Biomedical Research Council of The University of Michigan. Address for reprint requests: M. J. Kluger, Dept. of Physiology, 7620 Med. Sci. II, Univ. of Michigan Medical School, Ann Arbor, MI 48109.
IN
2.
3.
4.
5.
6
7.
8.
9. 10.
11. 12.
13.
14.
15.
16. 17.
18. Received
16 January
1990; accepted
in final
form
1 June
1990.
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FEVER
R727
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AND
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