Vol. 14, No. 5 Printed in U.S.A.
INFECTION AND IMMUNITY, Nov. 1976, P. 1130-1137 Copyright © 1976 American Society for Microbiology
Mechanism of Fever Induction in Rabbits R. SIEGERT,* W. K. PHILIPP-DORMSTON, K. RADSAK, AND H. MENZEL Hygiene-Institut, Philipps-Universitat, 3550 Marburg/Lahn, Federal Republic of Germany Received for publication 23 February 1976
Three exogenous pyrogens (Escherichia coli lipopolysaccharide, synthetic double-stranded ribonucleic acid, Newcastle disease virus) were compared with respect to their mechanisms of fever induction in rabbits. All inducers stimulated the production of an endogenous pyrogen demonstrated in the blood as well as prostaglandins of the E group, and of cyclic adenosine 3',5'-monophosphate in the cerebrospinal fluid. The concentrations of these compounds were elevated approximately twofold as compared to the controls. Independently of the mode of induction, the fever reaction could be prevented by pretreatment with 5 mg of cycloheximide per kg, although the three fever mediators were induced as in febrile animals. Consequently, at least one additional fever mediator that is sensitive to a 30 to 50% inhibition of protein synthesis by cycloheximide has to be postulated. The comparable reactions of the rabbits after administration of different pyrogens argues for a similar fever mechanism. In contrast to fever induction there was no stimulation of endogenous pyrogen, prostaglandins of the E group, and cyclic adenosine 3',5'-monophosphate in hyperthermia as a consequence of exposure of the animals to exogenous overheating. Furthermore, hyperthermia could not be prevented by cycloheximide. Fever is the most frequent nonspecific symptom of infectious diseases because almost all infectious agents are pyrogenic. However, the question has remained as to whether qualitatively different pyrogens induce fever by analogous or different mechanisms. Thus, three exogenous pyrogens different in origin and chemical makeup were compared with respect to their capacity to induce fever mediators in rabbits. These were Escherichia coli lipopolysaccharide (CLP; 51); a synthetic double-stranded
ribonucleic acid, polyinosinic:polycytidylic acid Lpoly(I:C)], which exhibits effects similar to endotoxin (1, 29); and Newcastle disease virus (NDV), the pyrogen (8, 49) of which is believed to be a lipoprotein (43). These exogenous pyrogens stimulate a secondary "endogenous" pyrogen (EP), which can be demonstrated by transferring blood from febrile to normal animals. EP induces a temperature elevation in recipient animals within 10 min after injection (5, 6, 12, 39, 47). It seems to be identical to the pyrogen that can be extracted from rabbit granulocytes (7) and is released after in vitro incubation at 37°C with an exogenous pyrogen (4, 18, 25). Since macrophages also release EP after in vitro stimulation (23), it would appear that several types of phagocytic cells are capable of producing the secondary pyrogen. The EP is a low-molecularweight protein of unknown structure (27, 38, 42), and the question as to whether EP is syn-
thesized de novo or is activated from an inactive precursor after stimulation by exogenous pyrogens is not settled (15, 30, 46). An additional key function of the fever mechanism has to be attributed to the prostaglandins of the E group (PGE; 13, 14, 48), the concentrations of which are elevated twofold as compared to controls in the cerebrospinal fluid (CSF) of rabbits during endotoxin and virus fever (33, 34). PGE concentration is not only elevated after administration of exogenous pyrogens, but also as a result of intravenous injection of serum containing EP (34, 41). The increase in PGE concentration in the CSF appears to be a consequence of local stimulation of the anterior hypothalamus (31, 35). During endotoxin and virus fever, the concentration of cyclic adenosine 3',5'-monophosphate (AMP) in the CSF and anterior hypothalamus was also elevated twofold compared with the control (36, 41). This observation suggests that PGE may act by stimulation of cyclic AMP (45; W. K. Philipp-Dormston, Tenth Fed. Eur. Biochem. Soc. Meeting, abstr. 1330, 1975). These three fever mediators are, however, apparently not the only prerequisites for the fever reaction. This is inferred from the observation of rabbits during virus-induced fever, which can be prevented by cycloheximide (CH) pretreatment (41). Surprisingly, the afebrile, CH-treated animals had concentrations of the fever mediators similar to those of the un-
1130
VOL. 14, 1976
PYROGENS AND FEVER INDUCTION IN RABBITS
treated febrile animals. Thus, one may assume that at least one additional, CH-sensitive mediator is necessary for the onset of a fever reaction. Although "fever" and "hyperthermia" both elevate body temperature, the increase is the result of different mechanisms. Hyperthermia after exposure to an overheated environment was also chosen for study to compare and contrast it with elevation of body temperature induced by fever. MATERIALS AND METHODS Exogenous pyrogens. (i) CLP. Lipopolysaccharide W (E. coli 055:B5; Difco, Detroit, Mich.) was injected intravenously at a dose of 5 pLg/kg. (ii) Poly(I:C). Double-stranded poly(I:C) (1:1; Boehringer Mannheim, W. Germany) was administered intravenously at a dose of 16.5 ,ug/kg. (iii) NDV. NDV strain "Italy" was grown in the allantoic sac of a chicken embryo and injected intravenously at a dose of 2,000 hemagglutination units/ kg. Methodological details of virus growth and purification as well as determination of the hemagglutination titer were described previously (40). Demonstration of EP. Blood was taken by sterile heart puncture from rabbits anesthetized by pentobarbital narcosis 120 min after CLP or poly(I:C) or 240 min after NDV injection because of the difference in lag periods. For determination of EP activity, 6 ml of serum per kg was injected intravenously into rabbits. To exclude the presence of heat-resistant endotoxin in the serum, corresponding samples were heated to 90°C for 30 min. This procedure regularly destroyed EP of all samples. Hyperthermia. Hyperthermia was induced by keeping the rabbits in an incubator at 35°C. Blood and CSF for determination of EP, PGE, and cyclic AMP, respectively, were taken after 2 h. Animals. Young male and female rabbits (cross of Widder and Deutscher Riese), weighing from 2.5 to 3.0 kg and of a consistent breeding, were used. They were obtained from a single local supplier. Animals were kept at room temperature (20°C) and received water and a standard diet ad libitum. Temperature recording. Rectal temperature was examined continuously by thermoelectric recording, as described previously (34). Puncture of CSF. For determination of PGE and cyclic AMP, the cisterna magna was punctured immediately after anesthesia by intravenous barbital injection and bleeding. A 1.5- to 2.0-ml amount of CSF was obtained per animal. Determination of PGE. The concentration of PGE was estimated by radioimmunoassay (24, 33). Determination of cyclic AMP. Determination of cyclic AMP was carried out by a protein-binding assay (19, 36). The standard deviation for the values of PGE and cyclic AMP presented in the tables was calculated as described before (33). Administration of CH. CH was injected intravenously at a dose of 5 mg/kg of body weight 90 min before injection of exogenous pyrogen or exposure to
1131
heat. Control animals received 5 ml of a 0.9% NaCl solution. Inhibition of protein synthesis. Inhibition of protein synthesis was determined after a 3-h pulse with 50 uCi of intravenously injected 14C-labeled protein hydrolysate (specific activity, 57 mCi/matom) for the 3 h preceding bleeding of the animals by comparing the specific radioactivities of liver and brain proteins of CH-treated animals with those of NaCltreated controls. This pulse interval was chosen because CH suppressed the fever reaction during this period. Nucleocytoplasmic protein synthesis in liver and brain was reduced by 30 to 50% at these doses of CH (41). Sterility and apyrogenicity. All glassware used was freed of pyrogen contamination by heating at 180°C for 2 h. For injections, sterile, pyrogen-free disposable syringes and needles were used. Water used for solutions and buffers was first deionized and then made pyrogen-free by distillation. Freshly distilled water was kept for short periods of time at 80°C or stored briefly at 4°C. Care was taken that all buffers prepared were free of pyrogen contamination.
RESULTS Induction of fever by exogenous pyrogen. Rabbits responded to intravenous injection of the exogenous pyrogens, with fever reactions appearing after definite lag periods (Fig. la). The lag phase lasted approximately 15 min after CLP injection at the doses chosen and 60 min after poly(I:C) and 90 min after NDV injection. For the demonstration of EP, blood was taken approximately 120 min after CLP or poly(I:C) and 240 min after NDV injection because of the considerably longer lag period. EP in blood. EP could be demonstrated in all cases by the transfer of serum of the initial fever period into normal animals. This caused an almost immediate temperature rise after intravenous injection (Fig. la). PGE and cyclic AMP in the CSF. Immediately after bleeding for the demonstration of EP, the cisterna magna was punctured and CSF was collected for the estimation of PGE and cyclic AMP concentrations (Table 1, group a). The levels ofboth substances were increased more than twofold (P < 0.001). Increased concentrations were also observed when temperature elevation was induced by EP-containing serum (Table 2, group a). Abolishment of the fever reaction by CH. Intravenous injection of CH at a dose of 5 mg/kg induced temporarily a slight temperature decrease. No fever reaction was observed when animals were injected with an exogenous pyrogen 90 min after CH administration (Fig. lb). Nevertheless, these afebrile animals were also found to have both EP in the blood and elevated concentrations of PGE and cyclic AMP in the
1.
INDUCTION
BY C L P
11.
INDUCTION
BY
ST oc *1.0 *0.5
0
*1.0 * 0.5 0 -0.5
I:C
POLY
SERUM
I
a)
.1.0 .0.5
NaCI
POLY I OC L
I
I/
0
//9
b)
* 1.0 CH
* 0.5
POLY I:C
SERUM
I
0
_
\.L
L
1J
-0.5
-60
0
60
120
INDUCTION
III.
I
I
L
180 240
BY
L
60
NDV I
4
0
L
60 MINUTES
SERUM
°C
a)
.1,0 *0,5 0
NaCI
NDV
I-
\1
I
/
t g
b) CH
.0.5 0
NDV
SERUM
I I NI."
-
-I1/
,~~~~~
-O.S
IV. NORMAL CONTROLS
c) N Cl
*0.5
I
N
I
Ci
SERUM
0
_-
-60
0
60
120
180
240
300
1132
-
60
0
60 MINUTES
PYROGENS AND FEVER INDUCTION IN RABBITS
VOL. 14, 1976
1133
TABLE 1. Induction of PGE and cyclic AMP in CSF by different exogenous pyrogens in untreated or CHpretreated rabbits (see Fig. 1) Cyclic AMP (pmol/ml, x + PGE (ng/ml, i ± SDb) No. of animals Groupa Induction by:
SD) 56.36 ± 7.61 3.97 + 0.75 12 a 57.57 ± 2.73 4.10 ± 1.02 11 b 50.14 ± 8.34 4.10 ± 1.43 12 a Poly (I:C) 56.02 ± 4.88 4.50 ± 0.58 12 b 60.50 ± 12.70 3.98 + 2.41 12 a NDV 57.30 + 11.20 11 7.55 ± 4.19 b 23.70 ± 9.50 1.87 ± 1.55 12 c Normal concn a Group a, exogenous pyrogens in NaCl-treated animals; b, pretreatment with CH and subsequent injection of exogenous pyrogens; c, untreated animals. a - c, P < 0.001; b - c, P < 0.001. b SD, Standard deviation.
CLP
TABLE 2. Induction ofPGE and cyclic AMP in CSF by differently induced EP in untreated or CH-pretreated rabbits (see Fig. 1) Induction by:
Groupa
No. of animals
PGE (ng/ml,
+± SD)
Cyclic AMPSD) (pmol/ml,
+
52.38 ± 4.67 3.61 ± 1.26 59.34 ± 7.79 4.04 ± 0.65 45.13 ± 13.41 3.22 ± 1.16 Poly(I:C) 49.05 ± 14.30 3.78 ± 1.02 62.31 ± 10.72 4.21 ± 1.94 NDV 65.72 ± 13.95 4.63 ± 2.01 23.70 ± 9.50 1.87 ± 1.55 Normal concn a Group a, EP in NaCl-treated animals; b, pretreatment with CH and subsequent injection of EP; c, untreated animals (see Fig. 1). a - c, P < 0.001; b - c, P < 0.001.
CLP
b
a b a b a b c
6 6 6 6 6 6 12
SD, Standard deviation.
CSF (Tables 1 and 2, group b). Furthermore, CH abolished the effect of EP from various sources, as expected (Fig. 2b). Hyperthermia. As a control, the three fever mediators were also examined during exogenously induced hyperthermia (Fig. 3a). No EP could be demonstrated under these conditions (Fig. 3a), nor were the concentrations ofPGE or cyclic AMP elevated in the CSF (Table 3, group a). In contrast to fever, CH did not prevent hyperthermia (Fig. 3b). PGE and cyclic AMP were also within the normal limits of controls kept at +4°C (Table 3). Since exposition to extreme enviromental temperatures was not found to influence the concentrations of PGE and cyclic AMP, these mediators are apparently not involved in normal temperature regulation. DISCUSSION Three exogenous pyrogens were used to induce fever reactions in rabbits after definite lag periods. During the initial phase of fever, EP
could be demonstrated in the serum. Furthermore, the concentrations of PGE and cyclic AMP in the CSF were elevated twofold in all cases. These observations suggest an analogous fever mechanism. All three pyrogens are known to induce EP (5, 6, 12, 39, 47). The maximal elevation of body temperature observed in the animals that had been injected with EP was proportional to the EP content of the corresponding serum obtained from animals treated with exogenous pyrogens (5, 6). One can assume that EP induced by different agents are identical, although the experimental proof is still lacking
(30). The presence of EP in the blood suggests its participation in the pathogenesis of fever. The results of several experimental approaches argue for its direct action on the thermoregulatory center in the anterior hypothalamus.
These results include a fever reaction after administration of EP into the carotid artery and, in particular, after intracisternal injection. In
FIG. 1. Induction offever by exogenous pyrogens in NaCl- or CH-pretreated rabbits and demonstration of EP in serum (see Tables 1 and 2). (a) Fever reaction ofNaCl-pretreated animals; (b) inhibition offever in CHpretreated animals; (c) normal controls. Symbols: J, time of intravenous injection of exogenous pyrogens, CH, NaCl, or serum (EP), respectively; t, heart puncture; I 1, puncture of cisterna magna. Each curve represents the average febrile response of 11 or 12 rabbits, respectively.
1134
INFECT. IMMUN.
SIEGERT ET AL.
eT 10 +
a)
1,0
NaCI
+0,5
BY CLP
EP INDUCED
1.
b) EP
CH
EP
_I
0
-0.5
11.
+ 1,0
NaCI -
EP INDUCED
BY POLY 1I:C
EP
CH
EP
CH
EP
0,5 0
-
0,5
t
1,0
111. EP INDUCED NaCI
BY
NDV
EP
F 0,5
0 -
0.5
-60
I
6
0
60
1 120
2
3
3
180 240 300 360
-
1
6
6
-60
0
60
120
2
30
6
180 240 300 360 MINUTES
FIG. 2. Induction offever by differently stimulated EP in NaCl- or CH-pretreated rabbits (see Tables 1 and 2). (a) Fever reaction ofNaCl-pretreated animals; (b) inhibition of fever by CH pretreatment. Symbols: heart time of intravenous injection of exogenous pyrogens, CH, NaCl, or serum (EP), respectiuely; puncture; a puncture of cisterna magna. Each curve represents the average febrile response of six rabbits. 4,
,
,
the latter case, only 1/500 of the intravenous dose was necessary to induce fever (2, 9, 26). Apparently EP can be transferred from the blood to the CSF and from there exerts its effects on the anterior hypothalamus (3). The original hypothesis suggesting a direct increase in the setting of the thermoregulatory center by EP is no longer supported by the available data. One may assume that EP, as a fever mediator, only initiates mechanisms for stimulation of the neurones regulating body temperature. This is inferred by the activated synthesis of PGE and cyclic AMP after induction of fever by EP-containing serum. This hypothesis is supported by the reaction of rabbits showing complete virus-induced pyrogen tolerance (49). During tolerance, administration of exogenous viral pyrogen fails to induce fever. Furthermore, the concentration of EP in the blood, as well as the concentrations of PGE and cyclic AMP in the CSF, are normal (submitted for publication). After injection of EP, on the other hand, tolerant rabbits exhibit an immediate temperature increase associated with elevated
concentrations of PGE and cyclic AMP in the CSF. It is possible that EP acts via the PGE system; if PGE synthesis from essential precursors is inhibited by antipyretics, there is no fever reaction in spite of the presence of EP (48). Another question that requires consideration is the mechanism by which PGE initiates fever. It has been known for a long time that elevated concentrations of cyclic AMP can be induced in various systems by hormones (45), e.g., epinephrine, as well as by prostaglandins (11). During fever, EP-stimulated prostaglandins may thus exert their effects via a "second messenger," i.e., by altering the concentrations of intracellular cyclic AMP (45). Cyclic AMP and the enzymes for its synthesis and degradation can also be shown to be present in brain tissue, possibly associated with postsynaptic membranes (16, 21). PGE was found to increase cyclic AMP levels in various tissue cultures of the central nervous system and in rat brain homogenates (11, 20). The role of cyclic AMP in fever induction is supported by the finding that cats and rabbits exhibit hyperthermia after in-
PYROGENS AND FEVER INDUCTION IN RABBITS
VOL. 14, 1976 .AT
1135
SERUM
0
C
I
+2,0
+ 1,5
a) 4
I
1,0
+0,5
0
-60
0
60
-60
120
0
+ 1,5
b) + 1,0
+350C
CH + 0,5
0
-0,5
-60
0
60
120
180
240
300
MINUTES
C) +
1,0
SERUM
+roC
I
+ 0.5
-60
0
60
120
-60
, 0
60
MINUTES
FIG. 3. Induction of hyperthermia in rabbits at 35°C (see Table 3). (a) Nonpretreated animals; (b) CHpretreated animals; (c) normal controls. Symbols: I time of intravenous injection ofCH or exposition at +35 or 4°C, respectively; heart puncture; a , puncture of cisterna magna. Each curve represents the average febrile response offour rabbits. ,
,
jection of cyclic AMP into the lateral ventrical or the hypothalamus (10, 17, 37). Its concentration in the CSF may represent its biosynthesis in the brain regions bordering the cerebral ventricle system, since the blood brain barrier is not permeable to cyclic AMP (17). The fever reaction after injection of exogenous pyrogens was abolished when animals were pretreated with CH at a concentration that inhibits nucleocytoplasmic protein synthesis by 30 to 50% (41). It has been anticipated
that production of EP would be prevented. Reports dealing with the effect of metabolic inhibitors like CH on EP production in vitro are inconsistent (30, 46). Our experiments do not verify the inhibitory effect that has been described. On the contrary, they favor the concept that there is no de novo synthesis of EP, but rather a liberation from an inactive propyrogen into an active form which is released by the leukocytes. The effect of CH inducing a temporary decrease in body temperature by up to 0.5°C is
1136
SIEGERT ET AL.
TABLE 3. Concentrations of PGE and cyclic AMP in CSF from hyperthermic and CH-pretreated rabbits (see Fig. 3) Cyclic AMP Groupa No. of an- PGE (ng/ml, imals x ± SDI) (pmol/ml, x ± SD) G Pa a b
4 4 4
1.88 ± 3.75 27.20 ± 3.98 1.84 ± 1.61 30.80 ± 10.65 c 1.86 ± 1.62 25.67 ± 3.21 Group a, Hyperthermia at 35°C; b, pretreatment with CH and subsequent hyperthermia at 35°C; c, controls at 4°C. b
SD, Standard deviation.
not believed to be the consequence of an intoxication. This point of view is supported by the observation that the CH effect is reversible (28, 41). Furthermore, the synthesis of the three fever mediators is not interfered with, compared with nontreated febrile animals. Since hyperthermia cannot be prevented by CH, temperature regulation is apparently not affected by this case. Our results favor the view that the induction of fever by various exogenous pyrogens proceeds via similar mechanisms. They initiate the liberation or synthesis of the same mediators (EP, PGE, cyclic AMP) plus an additional CH-sensitive mediator. Such a protein mediator has been postulated earlier in context with the inhibition of pyrexia by CH in rats that had been treated with monoaminooxidase inhibitors (22). In contrast to fever, hyperthermic animals exhibit neither EP in the blood nor elevated concentrations of PGE and cyclic AMP in the CSF. A further difference is the failure of CH to prevent hyperthermia. This observation implies a basic difference in the mechanisms of fever and hyperthermia. ACKNOWLEDGMENT This investigation was supported by a grant from the Deutsche Forschungsgemeinschaft. LITERATURE CITED 1. Absher, M., and W. R. Stinebring. 1969. Endotoxin-like properties of poly I-poly C, an interferon stimulator. Nature (London) 223:715-717. 2. Adler, R. D., and R. J. T. Joy. 1965. Febrile responses to the intracisternal injection of endogenous (leucocytic) pyrogen in the rabbit. Proc. Soc. Exp. Biol. Med. 119:660-663. 3. Atkins, E. 1960. Pathogenesis of fever. Physiol. Rev. 40:580-646. 4. Atkins, E., M. Cronin, and P. Isacson. 1964. Endogenous pyrogen release from rabbit blood cells incubated in vitro with parainfluenza virus. Science 146:1469-1470. 5. Atkins, E., and W. C. Huang. 1958. Studies on the pathogenesis of fever with influenzal viruses. I. The appearance of an endogenous pyrogen in the blood
INFECT. IMMUN.
following intravenous injection of virus. J. Exp. Med. 107:383-401.
6.
Atkins, E., and W. B. Wood. 1955. Studies on the pathogenesis of fever. I. The presence of transferable
pyrogen in the blood stream following the injection of
typhoid vaccine. J. Exp. Med. 101:519-528. 7. Beeson, P. B. 1948. Temperature elevating effect of substances obtained from polymorphonuclear leuco-
cytes. J. Clin. Invest. 27:524. 8. Bennett, I. L., R. R. Wagner, and V. S. Le Quire. 1949. Pyrogenicity of influenza virus in rabbits. Proc. Soc. Exp. Med. Biol. 71:132-133. 9. Bornstein, D. L., C. Bredenberg, and W. B. Wood. 1963. Studies on the pathogenesis of fever. XI. Quantitative features of the febrile response to leucocytic pyrogen. J. Exp. Med. 117:349-364. 10. Clark, W. G., H. R. Cumby, and H. R. Davies. 1974. IV. The hyperthermic effect of intraventricular cholera enterotoxin in the unanaesthetized cat. J. Physiol. 240:493-504.
Collier, H. O., and A. C. Roy. 1974. Morphine-like drugs inhibit the stimulation by E prostaglandins of cyclic AMP formation by rat brain homogenate. Nature (London) 248:24-27. 12. Cox, C. G., and G. W. Rafter. 1971. Pyrogen and enzyme release from rabbit blood leucocytes promoted by endotoxin and polyinosinic polycytidylic acid. Bio11.
chem. Med. 5:227-236. 13. Feldberg, W., and K. P. Gupta. 1973. Pyrogen fever and prostaglandin-like activity in cerebrospinal fluid. J. Physiol. 228:41-53. 14. Feldberg, W., K. P. Gutpa, A. S. Milton, and S. Wendlandt. 1972. Effect of bacterial pyrogen and antipyretics on prostaglandin activity in cerebrospinal fluid of unanaesthetized cats. Br. J. Pharmacol. 46:550551. 15. Fleetwood, M. K., G. W. Gander, and F. Goodale. 1975. Effect of metabolic inhibitors on pyrogen production by rabbit leucocytes. Proc. Soc. Exp. Biol. Med.
149:336-339.
16. Florenco, N. T., R. J. Barrnett, and P. Greengard. 1971. Cyclic 3',5'-nucleotide phosphodiesterase: cytochemical localization in cerebral cortex. Science
173:745-747. 17. Gessa, G. L., G. Krishna, J. Forn, A. Tagliamonte, and B. B. Brodie. 1970. Behavioral and vegetative effects produced by dibutyryl cyclic AMP injected into different areas of the brain, p. 371-381. In P. Greengard and E. Costa (ed.), Role of cyclic AMP in cell function. Raven Press, New York. 18. Gillman, S. M., D. L. Bornstein, and W. B. Wood. 1961. Studies on the pathogenesis of fever. VIII. Further observations on the role of endogenous pyrogen in endotoxin fever. J. Exp. Med. 114:729-739. 19. Gilman, A. G. 1970. A protein binding assay for adenosine 3',5'-cyclic monophosphate. Proc. Natl. Acad. Sci. U.S.A. 67:305-312. 20. Gilman, A. G. 1972. Regulation of cyclic AMP metabolism in cultured cells of the nervous system, p. 389410. In P. Greengard, R. Paoletti, and G. A. Robison (ed.), Advances in cyclic nucleotide research. Raven Press, New York. 21. Gilman, A. G., and M. Nirenberg. 1971. Regulation of adenosine 3',5'-cyclic monophosphate metabolism in cultured neuroblastoma cells. Nature (London) 234:356-357. 22. Graham-Smith, D. G. 1972. The prevention by inhibitors of brain protein synthesis of the hyperactivity and hyperpyrexia produced in rats by monoamine oxidase inhibition and the administration of L-tryptophan or 5-methoxy-N,N-dimethyl-tryptophan. J. Neurochem. 19:2409-2422. 23. Hahn, H. H., D. C. Char, W. B. Postel, and W. B.
VOL. 14, 1976
PYROGENS AND FEVER INDUCTION IN RABBITS
Wood. 1967. Studies on the pathogenesis of fever. XV. The production of endogenous pyrogen by peritoneal macrophages. J. Exp. Med. 126:385-394. 24. Jaffe, B. M., H. R. Behrmann, and C. W. Parker. 1973. Radioimmunoassay measurement of prostaglandins E, A, and F in human plasma. J. Clin. Invest. 52:398405. 25. Kanoh, S., and H. Kawasaki. 1966. Studies on myxovirus pyrogen (I). Interaction of myxovirus and rabbit polymorphonuclear leucocytes. Biken J. 9:177-184. 26. King, M. K., and W. B. Wood. 1958. Studies of the pathogenesis of fever. IV. The site of action of leucocytic and circulating endogenous pyrogen. J. Exp.
Med. 107:291-303. 27. Kozak, M. S., H. H. Hahn, W. J. Lennarz, and W. B. Wood. 1968. Studies on the pathogenesis of fever. XVI. Purification and further chemical characterization of granulocytic pyrogen. J. Exp. Med. 127:341357. 28. Lau, R. Y., D. Van Alstyne, R. Berckmans, and A. F. Graham. 1975. Synthesis of reovirus-specific polypeptides in cells pretreated with cycloheximide. J. Virol. 16:470-478. 29. Lindsay, H. L., P. W. Trown, J. Brandt, and M. Forbes. 1969. Pyrogenicity of poly I * poly C in rabbits. Nature (London) 223:717-718. 30. Moore, D. M., P. A. Murphy, P. J. Chesney, and W. B. Wood. 1973. Synthesis of endogenous pyrogen by rabbit leucocytes. J. Exp. Med. 137:1263-1274. 31. Orczyk, G. P., and H. R. Behrmann. 1972. Ovulation blockade by aspirin or indomethacin-in vivo evidence for a role of prostaglandin in gonadotropin secretion. Prostaglandins 1:3-20. 32. Philipp-Dormston, W. K. 1975. Prostaglandine in Zentralnervensystem des Kaninchens. Hoppe-Seyler's Z. Physiol. Chem. 356:263-263. 33. Philipp-Dormston, W. K., and R. Siegert. 1974. Identification of prostaglandin E by radioimmunoassay in cerebrospinal fluid during endotoxin fever. Naturwissenschaften 61:134-135. 34. Philipp-Dormston, W. K., and R. Siegert. 1974. Prostaglandins of the E and F series in rabbit cerebrospinal fluid during fever induced by Newcastle disease virus, E. coli-endotoxin, or endogenous pyrogen. Med. Microbiol. Immunol. 159:279-284. 35. Philipp-Dormston, W. K., and R. Siegert. 1974. Plasma prostaglandins of the E and F series in rabbits during fever induced by Newcastle disease virus, E. coliendotoxin, or endogenous pyrogen. Z. Naturforsch. Teil C 29:773-776. 36. Philipp-Dormston, W. K., and R. Siegert. 1975. Adenosine 3',5'-cyclic monophosphate in rabbit cerebrospinal fluid during fever induced by E. coli-endotoxin. Med. Microbiol. Immunol. 161:11-13. 37. Philipp-Dormston, W. K., and R. Siegert. 1975. Fever produced in rabbits by N6,O2'-dibutyryl adenosine
1137
3',5'-cyclic monophosphate. Experientia 31:471-472. 38. Rafter, G. W., S. F. Cheuk, D. W. Krause, and W. B. Wood. 1966. Studies on the pathogenesis of fever. XIV. Further observations on the chemistry of leukocytic pyrogen. J. Exp. Med. 123:433-444. 39. Siegert, R. 1957. Natur und Wirkungsmechanismus der Virus-pyrogene. Klin. Wochenschr. 35:652-652. 40. Siegert, R., and P. Braune. 1964. The pyrogens of myxoviruses. I. Induction of hyperthermia and its tolerance. Virology 24:209-217. 41. Siegert, R., W. K. Philipp-Dormston, K. Radsak, and H. Menzel. 1975. Inhibition of Newcastle disease virus-induced fever in rabbits by cycloheximide. Arch. Virol. 48:367-373. 42. Siegert, R., W. Pollmann, and H. L. Shu. 1967. Zur chemischen Natur des endogenen, durch Myxoviren induzierten Pyrogens. Z. Naturforsch. Teil B 22:320323. 43. Siegert, R., H. L. Shu, W. Pollmann, and H. Menzel. 1967. Untersuchungen uber Viruspyrogene. I. Mitteilung: Versuche zur Abtrennung des pyrogenen Prinzips von Myxoviren. Z. Naturforsch. Teil B 22:155158. 44. Steinberg, D., M. Vaughan, P. J. Nestel, 0. Strand, and S. Bergstrom. 1964. Effects of prostaglandins on hormone-induced mobilization of free fatty acids. J. Clin. Invest. 43:1533-1540. 45. Sutherland, E. W., I. 0ye, and R. W. Butcher. 1965. The action of epinephrine and the role of the adenyl cyclase system in hormone action. Recent Prog. Horm. Res. 21:623-646. 46. Taborsky, I. 1972. Effect of cycloheximide on in vitro formation of rabbit granulocyte pyrogen induced with influenza A/PR& virus or endotoxin. Acta Virol. 16:376-381. 47. Taborsky, I., J. Doskocil, and V. Zajicova. 1974. Pyrogenic activity of double-stranded RNA from Escherichia coli f2 phage. Acta Virol. 18:106-112. 48. Vane, J. R. 1971. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature (London) New Biol. 231:232-235. 49. Wagner, R. R., I. L. Bennett, and V. S. Lequire. 1949. The production of fever by influenzal viruses. I. Factors influencing the febrile response to single injections of virus. J. Exp. Med. 90:321-334. 50. Weiss, B., and E. Costa. 1968. Regional and subcellular distribution of adenyl cyclase and 3',5'-cyclic nucleotide phosphodiesterase in brain and pineal gland. Biochem. Pharmacol. 17:2107-2116. 51. Westphal, O., A. Nowotny, 0. Loderitz, H. Hurni, E. Eichenberger, and G. Schonholzer. 1958. Die Bedeutung der Lipid-Komponente (Lipoid A) fur die biologischen Wirkungen bakterieller Endotoxine (Lipopolysaccharide). Pharm. Acta Helv. 33:401-411. 52. Wood, W. B. 1955. The pathogenesis of fever. Am. J. Med. 18:351-353.
INFECTION AND IMMUNITY, Nov. 1976, P. 1130-1137 Copyright © 1976 American Society for Microbiology
Mechanism of Fever Induction in Rabbits R. SIEGERT,* W. K. PHILIPP-DORMSTON, K. RADSAK, AND H. MENZEL Hygiene-Institut, Philipps-Universitat, 3550 Marburg/Lahn, Federal Republic of Germany Received for publication 23 February 1976
Three exogenous pyrogens (Escherichia coli lipopolysaccharide, synthetic double-stranded ribonucleic acid, Newcastle disease virus) were compared with respect to their mechanisms of fever induction in rabbits. All inducers stimulated the production of an endogenous pyrogen demonstrated in the blood as well as prostaglandins of the E group, and of cyclic adenosine 3',5'-monophosphate in the cerebrospinal fluid. The concentrations of these compounds were elevated approximately twofold as compared to the controls. Independently of the mode of induction, the fever reaction could be prevented by pretreatment with 5 mg of cycloheximide per kg, although the three fever mediators were induced as in febrile animals. Consequently, at least one additional fever mediator that is sensitive to a 30 to 50% inhibition of protein synthesis by cycloheximide has to be postulated. The comparable reactions of the rabbits after administration of different pyrogens argues for a similar fever mechanism. In contrast to fever induction there was no stimulation of endogenous pyrogen, prostaglandins of the E group, and cyclic adenosine 3',5'-monophosphate in hyperthermia as a consequence of exposure of the animals to exogenous overheating. Furthermore, hyperthermia could not be prevented by cycloheximide. Fever is the most frequent nonspecific symptom of infectious diseases because almost all infectious agents are pyrogenic. However, the question has remained as to whether qualitatively different pyrogens induce fever by analogous or different mechanisms. Thus, three exogenous pyrogens different in origin and chemical makeup were compared with respect to their capacity to induce fever mediators in rabbits. These were Escherichia coli lipopolysaccharide (CLP; 51); a synthetic double-stranded
ribonucleic acid, polyinosinic:polycytidylic acid Lpoly(I:C)], which exhibits effects similar to endotoxin (1, 29); and Newcastle disease virus (NDV), the pyrogen (8, 49) of which is believed to be a lipoprotein (43). These exogenous pyrogens stimulate a secondary "endogenous" pyrogen (EP), which can be demonstrated by transferring blood from febrile to normal animals. EP induces a temperature elevation in recipient animals within 10 min after injection (5, 6, 12, 39, 47). It seems to be identical to the pyrogen that can be extracted from rabbit granulocytes (7) and is released after in vitro incubation at 37°C with an exogenous pyrogen (4, 18, 25). Since macrophages also release EP after in vitro stimulation (23), it would appear that several types of phagocytic cells are capable of producing the secondary pyrogen. The EP is a low-molecularweight protein of unknown structure (27, 38, 42), and the question as to whether EP is syn-
thesized de novo or is activated from an inactive precursor after stimulation by exogenous pyrogens is not settled (15, 30, 46). An additional key function of the fever mechanism has to be attributed to the prostaglandins of the E group (PGE; 13, 14, 48), the concentrations of which are elevated twofold as compared to controls in the cerebrospinal fluid (CSF) of rabbits during endotoxin and virus fever (33, 34). PGE concentration is not only elevated after administration of exogenous pyrogens, but also as a result of intravenous injection of serum containing EP (34, 41). The increase in PGE concentration in the CSF appears to be a consequence of local stimulation of the anterior hypothalamus (31, 35). During endotoxin and virus fever, the concentration of cyclic adenosine 3',5'-monophosphate (AMP) in the CSF and anterior hypothalamus was also elevated twofold compared with the control (36, 41). This observation suggests that PGE may act by stimulation of cyclic AMP (45; W. K. Philipp-Dormston, Tenth Fed. Eur. Biochem. Soc. Meeting, abstr. 1330, 1975). These three fever mediators are, however, apparently not the only prerequisites for the fever reaction. This is inferred from the observation of rabbits during virus-induced fever, which can be prevented by cycloheximide (CH) pretreatment (41). Surprisingly, the afebrile, CH-treated animals had concentrations of the fever mediators similar to those of the un-
1130
VOL. 14, 1976
PYROGENS AND FEVER INDUCTION IN RABBITS
treated febrile animals. Thus, one may assume that at least one additional, CH-sensitive mediator is necessary for the onset of a fever reaction. Although "fever" and "hyperthermia" both elevate body temperature, the increase is the result of different mechanisms. Hyperthermia after exposure to an overheated environment was also chosen for study to compare and contrast it with elevation of body temperature induced by fever. MATERIALS AND METHODS Exogenous pyrogens. (i) CLP. Lipopolysaccharide W (E. coli 055:B5; Difco, Detroit, Mich.) was injected intravenously at a dose of 5 pLg/kg. (ii) Poly(I:C). Double-stranded poly(I:C) (1:1; Boehringer Mannheim, W. Germany) was administered intravenously at a dose of 16.5 ,ug/kg. (iii) NDV. NDV strain "Italy" was grown in the allantoic sac of a chicken embryo and injected intravenously at a dose of 2,000 hemagglutination units/ kg. Methodological details of virus growth and purification as well as determination of the hemagglutination titer were described previously (40). Demonstration of EP. Blood was taken by sterile heart puncture from rabbits anesthetized by pentobarbital narcosis 120 min after CLP or poly(I:C) or 240 min after NDV injection because of the difference in lag periods. For determination of EP activity, 6 ml of serum per kg was injected intravenously into rabbits. To exclude the presence of heat-resistant endotoxin in the serum, corresponding samples were heated to 90°C for 30 min. This procedure regularly destroyed EP of all samples. Hyperthermia. Hyperthermia was induced by keeping the rabbits in an incubator at 35°C. Blood and CSF for determination of EP, PGE, and cyclic AMP, respectively, were taken after 2 h. Animals. Young male and female rabbits (cross of Widder and Deutscher Riese), weighing from 2.5 to 3.0 kg and of a consistent breeding, were used. They were obtained from a single local supplier. Animals were kept at room temperature (20°C) and received water and a standard diet ad libitum. Temperature recording. Rectal temperature was examined continuously by thermoelectric recording, as described previously (34). Puncture of CSF. For determination of PGE and cyclic AMP, the cisterna magna was punctured immediately after anesthesia by intravenous barbital injection and bleeding. A 1.5- to 2.0-ml amount of CSF was obtained per animal. Determination of PGE. The concentration of PGE was estimated by radioimmunoassay (24, 33). Determination of cyclic AMP. Determination of cyclic AMP was carried out by a protein-binding assay (19, 36). The standard deviation for the values of PGE and cyclic AMP presented in the tables was calculated as described before (33). Administration of CH. CH was injected intravenously at a dose of 5 mg/kg of body weight 90 min before injection of exogenous pyrogen or exposure to
1131
heat. Control animals received 5 ml of a 0.9% NaCl solution. Inhibition of protein synthesis. Inhibition of protein synthesis was determined after a 3-h pulse with 50 uCi of intravenously injected 14C-labeled protein hydrolysate (specific activity, 57 mCi/matom) for the 3 h preceding bleeding of the animals by comparing the specific radioactivities of liver and brain proteins of CH-treated animals with those of NaCltreated controls. This pulse interval was chosen because CH suppressed the fever reaction during this period. Nucleocytoplasmic protein synthesis in liver and brain was reduced by 30 to 50% at these doses of CH (41). Sterility and apyrogenicity. All glassware used was freed of pyrogen contamination by heating at 180°C for 2 h. For injections, sterile, pyrogen-free disposable syringes and needles were used. Water used for solutions and buffers was first deionized and then made pyrogen-free by distillation. Freshly distilled water was kept for short periods of time at 80°C or stored briefly at 4°C. Care was taken that all buffers prepared were free of pyrogen contamination.
RESULTS Induction of fever by exogenous pyrogen. Rabbits responded to intravenous injection of the exogenous pyrogens, with fever reactions appearing after definite lag periods (Fig. la). The lag phase lasted approximately 15 min after CLP injection at the doses chosen and 60 min after poly(I:C) and 90 min after NDV injection. For the demonstration of EP, blood was taken approximately 120 min after CLP or poly(I:C) and 240 min after NDV injection because of the considerably longer lag period. EP in blood. EP could be demonstrated in all cases by the transfer of serum of the initial fever period into normal animals. This caused an almost immediate temperature rise after intravenous injection (Fig. la). PGE and cyclic AMP in the CSF. Immediately after bleeding for the demonstration of EP, the cisterna magna was punctured and CSF was collected for the estimation of PGE and cyclic AMP concentrations (Table 1, group a). The levels ofboth substances were increased more than twofold (P < 0.001). Increased concentrations were also observed when temperature elevation was induced by EP-containing serum (Table 2, group a). Abolishment of the fever reaction by CH. Intravenous injection of CH at a dose of 5 mg/kg induced temporarily a slight temperature decrease. No fever reaction was observed when animals were injected with an exogenous pyrogen 90 min after CH administration (Fig. lb). Nevertheless, these afebrile animals were also found to have both EP in the blood and elevated concentrations of PGE and cyclic AMP in the
1.
INDUCTION
BY C L P
11.
INDUCTION
BY
ST oc *1.0 *0.5
0
*1.0 * 0.5 0 -0.5
I:C
POLY
SERUM
I
a)
.1.0 .0.5
NaCI
POLY I OC L
I
I/
0
//9
b)
* 1.0 CH
* 0.5
POLY I:C
SERUM
I
0
_
\.L
L
1J
-0.5
-60
0
60
120
INDUCTION
III.
I
I
L
180 240
BY
L
60
NDV I
4
0
L
60 MINUTES
SERUM
°C
a)
.1,0 *0,5 0
NaCI
NDV
I-
\1
I
/
t g
b) CH
.0.5 0
NDV
SERUM
I I NI."
-
-I1/
,~~~~~
-O.S
IV. NORMAL CONTROLS
c) N Cl
*0.5
I
N
I
Ci
SERUM
0
_-
-60
0
60
120
180
240
300
1132
-
60
0
60 MINUTES
PYROGENS AND FEVER INDUCTION IN RABBITS
VOL. 14, 1976
1133
TABLE 1. Induction of PGE and cyclic AMP in CSF by different exogenous pyrogens in untreated or CHpretreated rabbits (see Fig. 1) Cyclic AMP (pmol/ml, x + PGE (ng/ml, i ± SDb) No. of animals Groupa Induction by:
SD) 56.36 ± 7.61 3.97 + 0.75 12 a 57.57 ± 2.73 4.10 ± 1.02 11 b 50.14 ± 8.34 4.10 ± 1.43 12 a Poly (I:C) 56.02 ± 4.88 4.50 ± 0.58 12 b 60.50 ± 12.70 3.98 + 2.41 12 a NDV 57.30 + 11.20 11 7.55 ± 4.19 b 23.70 ± 9.50 1.87 ± 1.55 12 c Normal concn a Group a, exogenous pyrogens in NaCl-treated animals; b, pretreatment with CH and subsequent injection of exogenous pyrogens; c, untreated animals. a - c, P < 0.001; b - c, P < 0.001. b SD, Standard deviation.
CLP
TABLE 2. Induction ofPGE and cyclic AMP in CSF by differently induced EP in untreated or CH-pretreated rabbits (see Fig. 1) Induction by:
Groupa
No. of animals
PGE (ng/ml,
+± SD)
Cyclic AMPSD) (pmol/ml,
+
52.38 ± 4.67 3.61 ± 1.26 59.34 ± 7.79 4.04 ± 0.65 45.13 ± 13.41 3.22 ± 1.16 Poly(I:C) 49.05 ± 14.30 3.78 ± 1.02 62.31 ± 10.72 4.21 ± 1.94 NDV 65.72 ± 13.95 4.63 ± 2.01 23.70 ± 9.50 1.87 ± 1.55 Normal concn a Group a, EP in NaCl-treated animals; b, pretreatment with CH and subsequent injection of EP; c, untreated animals (see Fig. 1). a - c, P < 0.001; b - c, P < 0.001.
CLP
b
a b a b a b c
6 6 6 6 6 6 12
SD, Standard deviation.
CSF (Tables 1 and 2, group b). Furthermore, CH abolished the effect of EP from various sources, as expected (Fig. 2b). Hyperthermia. As a control, the three fever mediators were also examined during exogenously induced hyperthermia (Fig. 3a). No EP could be demonstrated under these conditions (Fig. 3a), nor were the concentrations ofPGE or cyclic AMP elevated in the CSF (Table 3, group a). In contrast to fever, CH did not prevent hyperthermia (Fig. 3b). PGE and cyclic AMP were also within the normal limits of controls kept at +4°C (Table 3). Since exposition to extreme enviromental temperatures was not found to influence the concentrations of PGE and cyclic AMP, these mediators are apparently not involved in normal temperature regulation. DISCUSSION Three exogenous pyrogens were used to induce fever reactions in rabbits after definite lag periods. During the initial phase of fever, EP
could be demonstrated in the serum. Furthermore, the concentrations of PGE and cyclic AMP in the CSF were elevated twofold in all cases. These observations suggest an analogous fever mechanism. All three pyrogens are known to induce EP (5, 6, 12, 39, 47). The maximal elevation of body temperature observed in the animals that had been injected with EP was proportional to the EP content of the corresponding serum obtained from animals treated with exogenous pyrogens (5, 6). One can assume that EP induced by different agents are identical, although the experimental proof is still lacking
(30). The presence of EP in the blood suggests its participation in the pathogenesis of fever. The results of several experimental approaches argue for its direct action on the thermoregulatory center in the anterior hypothalamus.
These results include a fever reaction after administration of EP into the carotid artery and, in particular, after intracisternal injection. In
FIG. 1. Induction offever by exogenous pyrogens in NaCl- or CH-pretreated rabbits and demonstration of EP in serum (see Tables 1 and 2). (a) Fever reaction ofNaCl-pretreated animals; (b) inhibition offever in CHpretreated animals; (c) normal controls. Symbols: J, time of intravenous injection of exogenous pyrogens, CH, NaCl, or serum (EP), respectively; t, heart puncture; I 1, puncture of cisterna magna. Each curve represents the average febrile response of 11 or 12 rabbits, respectively.
1134
INFECT. IMMUN.
SIEGERT ET AL.
eT 10 +
a)
1,0
NaCI
+0,5
BY CLP
EP INDUCED
1.
b) EP
CH
EP
_I
0
-0.5
11.
+ 1,0
NaCI -
EP INDUCED
BY POLY 1I:C
EP
CH
EP
CH
EP
0,5 0
-
0,5
t
1,0
111. EP INDUCED NaCI
BY
NDV
EP
F 0,5
0 -
0.5
-60
I
6
0
60
1 120
2
3
3
180 240 300 360
-
1
6
6
-60
0
60
120
2
30
6
180 240 300 360 MINUTES
FIG. 2. Induction offever by differently stimulated EP in NaCl- or CH-pretreated rabbits (see Tables 1 and 2). (a) Fever reaction ofNaCl-pretreated animals; (b) inhibition of fever by CH pretreatment. Symbols: heart time of intravenous injection of exogenous pyrogens, CH, NaCl, or serum (EP), respectiuely; puncture; a puncture of cisterna magna. Each curve represents the average febrile response of six rabbits. 4,
,
,
the latter case, only 1/500 of the intravenous dose was necessary to induce fever (2, 9, 26). Apparently EP can be transferred from the blood to the CSF and from there exerts its effects on the anterior hypothalamus (3). The original hypothesis suggesting a direct increase in the setting of the thermoregulatory center by EP is no longer supported by the available data. One may assume that EP, as a fever mediator, only initiates mechanisms for stimulation of the neurones regulating body temperature. This is inferred by the activated synthesis of PGE and cyclic AMP after induction of fever by EP-containing serum. This hypothesis is supported by the reaction of rabbits showing complete virus-induced pyrogen tolerance (49). During tolerance, administration of exogenous viral pyrogen fails to induce fever. Furthermore, the concentration of EP in the blood, as well as the concentrations of PGE and cyclic AMP in the CSF, are normal (submitted for publication). After injection of EP, on the other hand, tolerant rabbits exhibit an immediate temperature increase associated with elevated
concentrations of PGE and cyclic AMP in the CSF. It is possible that EP acts via the PGE system; if PGE synthesis from essential precursors is inhibited by antipyretics, there is no fever reaction in spite of the presence of EP (48). Another question that requires consideration is the mechanism by which PGE initiates fever. It has been known for a long time that elevated concentrations of cyclic AMP can be induced in various systems by hormones (45), e.g., epinephrine, as well as by prostaglandins (11). During fever, EP-stimulated prostaglandins may thus exert their effects via a "second messenger," i.e., by altering the concentrations of intracellular cyclic AMP (45). Cyclic AMP and the enzymes for its synthesis and degradation can also be shown to be present in brain tissue, possibly associated with postsynaptic membranes (16, 21). PGE was found to increase cyclic AMP levels in various tissue cultures of the central nervous system and in rat brain homogenates (11, 20). The role of cyclic AMP in fever induction is supported by the finding that cats and rabbits exhibit hyperthermia after in-
PYROGENS AND FEVER INDUCTION IN RABBITS
VOL. 14, 1976 .AT
1135
SERUM
0
C
I
+2,0
+ 1,5
a) 4
I
1,0
+0,5
0
-60
0
60
-60
120
0
+ 1,5
b) + 1,0
+350C
CH + 0,5
0
-0,5
-60
0
60
120
180
240
300
MINUTES
C) +
1,0
SERUM
+roC
I
+ 0.5
-60
0
60
120
-60
, 0
60
MINUTES
FIG. 3. Induction of hyperthermia in rabbits at 35°C (see Table 3). (a) Nonpretreated animals; (b) CHpretreated animals; (c) normal controls. Symbols: I time of intravenous injection ofCH or exposition at +35 or 4°C, respectively; heart puncture; a , puncture of cisterna magna. Each curve represents the average febrile response offour rabbits. ,
,
jection of cyclic AMP into the lateral ventrical or the hypothalamus (10, 17, 37). Its concentration in the CSF may represent its biosynthesis in the brain regions bordering the cerebral ventricle system, since the blood brain barrier is not permeable to cyclic AMP (17). The fever reaction after injection of exogenous pyrogens was abolished when animals were pretreated with CH at a concentration that inhibits nucleocytoplasmic protein synthesis by 30 to 50% (41). It has been anticipated
that production of EP would be prevented. Reports dealing with the effect of metabolic inhibitors like CH on EP production in vitro are inconsistent (30, 46). Our experiments do not verify the inhibitory effect that has been described. On the contrary, they favor the concept that there is no de novo synthesis of EP, but rather a liberation from an inactive propyrogen into an active form which is released by the leukocytes. The effect of CH inducing a temporary decrease in body temperature by up to 0.5°C is
1136
SIEGERT ET AL.
TABLE 3. Concentrations of PGE and cyclic AMP in CSF from hyperthermic and CH-pretreated rabbits (see Fig. 3) Cyclic AMP Groupa No. of an- PGE (ng/ml, imals x ± SDI) (pmol/ml, x ± SD) G Pa a b
4 4 4
1.88 ± 3.75 27.20 ± 3.98 1.84 ± 1.61 30.80 ± 10.65 c 1.86 ± 1.62 25.67 ± 3.21 Group a, Hyperthermia at 35°C; b, pretreatment with CH and subsequent hyperthermia at 35°C; c, controls at 4°C. b
SD, Standard deviation.
not believed to be the consequence of an intoxication. This point of view is supported by the observation that the CH effect is reversible (28, 41). Furthermore, the synthesis of the three fever mediators is not interfered with, compared with nontreated febrile animals. Since hyperthermia cannot be prevented by CH, temperature regulation is apparently not affected by this case. Our results favor the view that the induction of fever by various exogenous pyrogens proceeds via similar mechanisms. They initiate the liberation or synthesis of the same mediators (EP, PGE, cyclic AMP) plus an additional CH-sensitive mediator. Such a protein mediator has been postulated earlier in context with the inhibition of pyrexia by CH in rats that had been treated with monoaminooxidase inhibitors (22). In contrast to fever, hyperthermic animals exhibit neither EP in the blood nor elevated concentrations of PGE and cyclic AMP in the CSF. A further difference is the failure of CH to prevent hyperthermia. This observation implies a basic difference in the mechanisms of fever and hyperthermia. ACKNOWLEDGMENT This investigation was supported by a grant from the Deutsche Forschungsgemeinschaft. LITERATURE CITED 1. Absher, M., and W. R. Stinebring. 1969. Endotoxin-like properties of poly I-poly C, an interferon stimulator. Nature (London) 223:715-717. 2. Adler, R. D., and R. J. T. Joy. 1965. Febrile responses to the intracisternal injection of endogenous (leucocytic) pyrogen in the rabbit. Proc. Soc. Exp. Biol. Med. 119:660-663. 3. Atkins, E. 1960. Pathogenesis of fever. Physiol. Rev. 40:580-646. 4. Atkins, E., M. Cronin, and P. Isacson. 1964. Endogenous pyrogen release from rabbit blood cells incubated in vitro with parainfluenza virus. Science 146:1469-1470. 5. Atkins, E., and W. C. Huang. 1958. Studies on the pathogenesis of fever with influenzal viruses. I. The appearance of an endogenous pyrogen in the blood
INFECT. IMMUN.
following intravenous injection of virus. J. Exp. Med. 107:383-401.
6.
Atkins, E., and W. B. Wood. 1955. Studies on the pathogenesis of fever. I. The presence of transferable
pyrogen in the blood stream following the injection of
typhoid vaccine. J. Exp. Med. 101:519-528. 7. Beeson, P. B. 1948. Temperature elevating effect of substances obtained from polymorphonuclear leuco-
cytes. J. Clin. Invest. 27:524. 8. Bennett, I. L., R. R. Wagner, and V. S. Le Quire. 1949. Pyrogenicity of influenza virus in rabbits. Proc. Soc. Exp. Med. Biol. 71:132-133. 9. Bornstein, D. L., C. Bredenberg, and W. B. Wood. 1963. Studies on the pathogenesis of fever. XI. Quantitative features of the febrile response to leucocytic pyrogen. J. Exp. Med. 117:349-364. 10. Clark, W. G., H. R. Cumby, and H. R. Davies. 1974. IV. The hyperthermic effect of intraventricular cholera enterotoxin in the unanaesthetized cat. J. Physiol. 240:493-504.
Collier, H. O., and A. C. Roy. 1974. Morphine-like drugs inhibit the stimulation by E prostaglandins of cyclic AMP formation by rat brain homogenate. Nature (London) 248:24-27. 12. Cox, C. G., and G. W. Rafter. 1971. Pyrogen and enzyme release from rabbit blood leucocytes promoted by endotoxin and polyinosinic polycytidylic acid. Bio11.
chem. Med. 5:227-236. 13. Feldberg, W., and K. P. Gupta. 1973. Pyrogen fever and prostaglandin-like activity in cerebrospinal fluid. J. Physiol. 228:41-53. 14. Feldberg, W., K. P. Gutpa, A. S. Milton, and S. Wendlandt. 1972. Effect of bacterial pyrogen and antipyretics on prostaglandin activity in cerebrospinal fluid of unanaesthetized cats. Br. J. Pharmacol. 46:550551. 15. Fleetwood, M. K., G. W. Gander, and F. Goodale. 1975. Effect of metabolic inhibitors on pyrogen production by rabbit leucocytes. Proc. Soc. Exp. Biol. Med.
149:336-339.
16. Florenco, N. T., R. J. Barrnett, and P. Greengard. 1971. Cyclic 3',5'-nucleotide phosphodiesterase: cytochemical localization in cerebral cortex. Science
173:745-747. 17. Gessa, G. L., G. Krishna, J. Forn, A. Tagliamonte, and B. B. Brodie. 1970. Behavioral and vegetative effects produced by dibutyryl cyclic AMP injected into different areas of the brain, p. 371-381. In P. Greengard and E. Costa (ed.), Role of cyclic AMP in cell function. Raven Press, New York. 18. Gillman, S. M., D. L. Bornstein, and W. B. Wood. 1961. Studies on the pathogenesis of fever. VIII. Further observations on the role of endogenous pyrogen in endotoxin fever. J. Exp. Med. 114:729-739. 19. Gilman, A. G. 1970. A protein binding assay for adenosine 3',5'-cyclic monophosphate. Proc. Natl. Acad. Sci. U.S.A. 67:305-312. 20. Gilman, A. G. 1972. Regulation of cyclic AMP metabolism in cultured cells of the nervous system, p. 389410. In P. Greengard, R. Paoletti, and G. A. Robison (ed.), Advances in cyclic nucleotide research. Raven Press, New York. 21. Gilman, A. G., and M. Nirenberg. 1971. Regulation of adenosine 3',5'-cyclic monophosphate metabolism in cultured neuroblastoma cells. Nature (London) 234:356-357. 22. Graham-Smith, D. G. 1972. The prevention by inhibitors of brain protein synthesis of the hyperactivity and hyperpyrexia produced in rats by monoamine oxidase inhibition and the administration of L-tryptophan or 5-methoxy-N,N-dimethyl-tryptophan. J. Neurochem. 19:2409-2422. 23. Hahn, H. H., D. C. Char, W. B. Postel, and W. B.
VOL. 14, 1976
PYROGENS AND FEVER INDUCTION IN RABBITS
Wood. 1967. Studies on the pathogenesis of fever. XV. The production of endogenous pyrogen by peritoneal macrophages. J. Exp. Med. 126:385-394. 24. Jaffe, B. M., H. R. Behrmann, and C. W. Parker. 1973. Radioimmunoassay measurement of prostaglandins E, A, and F in human plasma. J. Clin. Invest. 52:398405. 25. Kanoh, S., and H. Kawasaki. 1966. Studies on myxovirus pyrogen (I). Interaction of myxovirus and rabbit polymorphonuclear leucocytes. Biken J. 9:177-184. 26. King, M. K., and W. B. Wood. 1958. Studies of the pathogenesis of fever. IV. The site of action of leucocytic and circulating endogenous pyrogen. J. Exp.
Med. 107:291-303. 27. Kozak, M. S., H. H. Hahn, W. J. Lennarz, and W. B. Wood. 1968. Studies on the pathogenesis of fever. XVI. Purification and further chemical characterization of granulocytic pyrogen. J. Exp. Med. 127:341357. 28. Lau, R. Y., D. Van Alstyne, R. Berckmans, and A. F. Graham. 1975. Synthesis of reovirus-specific polypeptides in cells pretreated with cycloheximide. J. Virol. 16:470-478. 29. Lindsay, H. L., P. W. Trown, J. Brandt, and M. Forbes. 1969. Pyrogenicity of poly I * poly C in rabbits. Nature (London) 223:717-718. 30. Moore, D. M., P. A. Murphy, P. J. Chesney, and W. B. Wood. 1973. Synthesis of endogenous pyrogen by rabbit leucocytes. J. Exp. Med. 137:1263-1274. 31. Orczyk, G. P., and H. R. Behrmann. 1972. Ovulation blockade by aspirin or indomethacin-in vivo evidence for a role of prostaglandin in gonadotropin secretion. Prostaglandins 1:3-20. 32. Philipp-Dormston, W. K. 1975. Prostaglandine in Zentralnervensystem des Kaninchens. Hoppe-Seyler's Z. Physiol. Chem. 356:263-263. 33. Philipp-Dormston, W. K., and R. Siegert. 1974. Identification of prostaglandin E by radioimmunoassay in cerebrospinal fluid during endotoxin fever. Naturwissenschaften 61:134-135. 34. Philipp-Dormston, W. K., and R. Siegert. 1974. Prostaglandins of the E and F series in rabbit cerebrospinal fluid during fever induced by Newcastle disease virus, E. coli-endotoxin, or endogenous pyrogen. Med. Microbiol. Immunol. 159:279-284. 35. Philipp-Dormston, W. K., and R. Siegert. 1974. Plasma prostaglandins of the E and F series in rabbits during fever induced by Newcastle disease virus, E. coliendotoxin, or endogenous pyrogen. Z. Naturforsch. Teil C 29:773-776. 36. Philipp-Dormston, W. K., and R. Siegert. 1975. Adenosine 3',5'-cyclic monophosphate in rabbit cerebrospinal fluid during fever induced by E. coli-endotoxin. Med. Microbiol. Immunol. 161:11-13. 37. Philipp-Dormston, W. K., and R. Siegert. 1975. Fever produced in rabbits by N6,O2'-dibutyryl adenosine
1137
3',5'-cyclic monophosphate. Experientia 31:471-472. 38. Rafter, G. W., S. F. Cheuk, D. W. Krause, and W. B. Wood. 1966. Studies on the pathogenesis of fever. XIV. Further observations on the chemistry of leukocytic pyrogen. J. Exp. Med. 123:433-444. 39. Siegert, R. 1957. Natur und Wirkungsmechanismus der Virus-pyrogene. Klin. Wochenschr. 35:652-652. 40. Siegert, R., and P. Braune. 1964. The pyrogens of myxoviruses. I. Induction of hyperthermia and its tolerance. Virology 24:209-217. 41. Siegert, R., W. K. Philipp-Dormston, K. Radsak, and H. Menzel. 1975. Inhibition of Newcastle disease virus-induced fever in rabbits by cycloheximide. Arch. Virol. 48:367-373. 42. Siegert, R., W. Pollmann, and H. L. Shu. 1967. Zur chemischen Natur des endogenen, durch Myxoviren induzierten Pyrogens. Z. Naturforsch. Teil B 22:320323. 43. Siegert, R., H. L. Shu, W. Pollmann, and H. Menzel. 1967. Untersuchungen uber Viruspyrogene. I. Mitteilung: Versuche zur Abtrennung des pyrogenen Prinzips von Myxoviren. Z. Naturforsch. Teil B 22:155158. 44. Steinberg, D., M. Vaughan, P. J. Nestel, 0. Strand, and S. Bergstrom. 1964. Effects of prostaglandins on hormone-induced mobilization of free fatty acids. J. Clin. Invest. 43:1533-1540. 45. Sutherland, E. W., I. 0ye, and R. W. Butcher. 1965. The action of epinephrine and the role of the adenyl cyclase system in hormone action. Recent Prog. Horm. Res. 21:623-646. 46. Taborsky, I. 1972. Effect of cycloheximide on in vitro formation of rabbit granulocyte pyrogen induced with influenza A/PR& virus or endotoxin. Acta Virol. 16:376-381. 47. Taborsky, I., J. Doskocil, and V. Zajicova. 1974. Pyrogenic activity of double-stranded RNA from Escherichia coli f2 phage. Acta Virol. 18:106-112. 48. Vane, J. R. 1971. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature (London) New Biol. 231:232-235. 49. Wagner, R. R., I. L. Bennett, and V. S. Lequire. 1949. The production of fever by influenzal viruses. I. Factors influencing the febrile response to single injections of virus. J. Exp. Med. 90:321-334. 50. Weiss, B., and E. Costa. 1968. Regional and subcellular distribution of adenyl cyclase and 3',5'-cyclic nucleotide phosphodiesterase in brain and pineal gland. Biochem. Pharmacol. 17:2107-2116. 51. Westphal, O., A. Nowotny, 0. Loderitz, H. Hurni, E. Eichenberger, and G. Schonholzer. 1958. Die Bedeutung der Lipid-Komponente (Lipoid A) fur die biologischen Wirkungen bakterieller Endotoxine (Lipopolysaccharide). Pharm. Acta Helv. 33:401-411. 52. Wood, W. B. 1955. The pathogenesis of fever. Am. J. Med. 18:351-353.
