J. Phyaiol. (1977), 267, pp. 659-666 With 4 text-ftigure8 Printed in Great Britain
659
ENDOGENOUS PYROGEN-LIKE SUBSTANCE PRODUCED BY REPTILES BY HARRY A. BERNHEIM* AND MATTHEW J. KLUGER From the Department of Physiology, Univereity of Michigan Medical School, Ann Arbor, Michigan 48190, U.S.A.
(Received 20 July 1976) SUMMARY
1. Injection of lizards (Dipso0auruls dorsali8) with rabbit endogenous pyrogen led to a fever. Injections with denatured endogenous pyrogen did not affect body temperature. 2. Injection of lizards with lizard endogenous pyrogen led to a fever of short duration, while injection of denatured lizard endogenous pyrogen produced no change in body temperature. 3. These data support the hypothesis that the febrile mechanism observed in the higher vertebrates has its origins in some primitive vertebrate. INTRODUCTION
Birds (D'Alecy & Kluger, 1975), reptiles (Vaughn, Bernheim & Kluger, 1974; Bernheim & Kluger, 1976), and fishes (Reynolds, Casterlin & Covert, 1976), as well as mammals, develop a fever in response to injection with Gram-negative bacteria. The fever that one observes in mammals is attributed to the synthesis and release of a heat labile protein from the host's leucocytes in response to the action of a bacterial endotoxin. The endogenous (leucocytic) pyrogen seems to act on the hypothalamic neural structures concerned with thermoregulation in such a way as to cause the 'set-point' for body temperature to be raised (i.e. fever) (Bligh, 1973). If the febrile mechanisms of the different classes of vertebrates have a common phylogeny, non-mammalian vertebrates may produce an endogenous pyrogen similar to that of mammals. If this is so then injection of endogenous pyrogen produced by the leucocytes of a representative of one class of vertebrate into another class of vertebrate should cause a fever. The experiments reported here were done to establish whether reptilian leucocytes can synthesize and release a pyrogenic substance, and whether mammalian endogenous pyrogen can cause a fever when injected into a reptilian species. * Present address: Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06519, U.S.A.
660
H. A. BERNHEIM AND M. J. KLUGER METHODS
New Zealand white rabbits (Oryotolagu8 cuniculu8) were used for the production and testing of mammalian endogenous pyrogen. To test the potency of the rabbit endogenous pyrogen, the rabbits were placed in a restrainer and allowed to reach steady state thermal equilibrium (Tre variation of + 0.10 C). The rabbits were then injected (i.v.) with 1 x 108 cell equivalents of endogenous pyrogen. Their rectal temperatures were monitored continuously with a copper-constantan thermocouple until their temperatures returned to base line levels. Desert iguanas (Dip8o8auru8 dorsali) weighing 25-55 g were obtained from a dealer in California. The lizards were housed and maintained as described by Bernheim & Kluger (1976). All lizards were allowed to adjust to the laboratory conditions for at least 2 weeks before their use in these experiments. During experimentation the lizards were allowed free movement in a desert-like environment (Bernheim & Kluger, 1976). Their body temperatures (±+ 0.1 C) were recorded about every 30 sec using copper-constantan thermocouples connected to a Honeywell Electonik 112 multipoint recorder. Each thermocouple was covered with polyethylene tubing, placed about 30 mm into each lizard's cloaca, and taped to their tails. Lizards were then allowed 24 hr to adjust to the experimental chamber. The next day was designated experimental day 1 and was used as the control day. At 09.00 hr on experimental day 2 the lizards received an intracardiac injection of rabbit endogenous pyrogen (1 x 108 cell equivalents) or lizard/endogenous pyrogen (1 x 106, 5 x 106, or 1 x 107 cell equivalents). Body temperatures were recorded for 48 hr after these inoculations. Other lizards were injected with denatured mammalian or reptilian endogenous pyrogen (solutions heated to 90° C for 30 min). Preparation of rabbit endogenous pyrogen was based on methods described by Bornstein, Bredenberg & Wood (1963), with the following exceptions: a 0-2% shellfish glycogen solution was used instead of a 0-1 % solution and the final supernatant solution was not dialysed against a normal salt solution. Lizard endogenous pyrogen was obtained using a modification of the above methods. Either (a) 10 ml. of a sterile 3% thioglycollate-saline solution, or (b) 10 ml. of dead Aeromonas hydrophila-saline solution containing 1 x 1010 bacteria was first injected into the coelomic cavity. (Since lizard endogenous pyrogen resulting from treatments a and b produced similar results, the data were pooled.) The lizards were kept at an ambient (= body) temperature of 370 C for 15-16 hr. They were then killed and the coelomic fluid removed aseptically. The coelom was washed with sterile pyrogen-free saline containing 5 u. heparin/ml. Washes were continued until only clear liquid was recovered. White blood cell and differential cell counts were done (with thioglycollate treatment the yield of polymorphonuclear leucocytes (PMNs) ranged from 7-10 x 10, with bacterial treatment the yield of PMNs ranged from 12- 16 x 10 PMNs). The saline solution containing the leucocytes was then centrifuged at 900 g at 40 C for 20 min and the supernatant discarded. The cell button was then resuspended in 10 ml. saline and incubated at 370 C for 10 min. Then the solution was centrifuged at 900 g for 20 min. The cells were then again resuspended in saline (10 or 20 x 106 cells/ml.) and incubated with gentle shaking at 370 C for 24 hr. After incubation the solution was centrifuged at 900 g for 20 min and the supernatant which contained the endogenous pyrogen-like material was spun at 10,000 g for 30 min, decanted and the supernatant stored at 40 C. The lizard endogenous pyrogen solutions were checked for sterility by incubating samples on blood agar plates at 370 C for 48 hr. Unsterile solutions and any containing haemoglobin were discarded. Data were statistically analysed using the Wilcoxon signed rank test.
REPTILIAN ENDOCENOUS PYROGEN
661
RESULTS
Injection of rabbit endogenous pyrogen (1 x 108 cell equivalents) into three rabbits led to an average increase in temperature of 1.30 C (+ 0.40 C s.E.) with an average latency of 12 min (+ 3 S.E.). Injections of denatured endogenous pyrogen caused no significant rise in body temperature. 43
_ i 2 42 42
t
n=8
41
40
6
E 39 T
37
36 _
Tt
- oControl (day 1) *.- Inject with rabbit EP (day 2) @v-Day 3
10.00
12.00 14.00 Time
16.00
Fig. 1. Effects of an intracardiac injection of rabbit endogenous pyrogen (EP) on the mean hourly body temperature (± s.E.) of eight lizards (Dip8o8auru8 dormali). The injection was made at about 09.00 hr on day 2. The lizards developed a fever 4-5 hr later. This fever continued throughout the second day (day 3).
The injection of rabbit endogenous pyrogen (1 x 108 cell equivalents) into eight lizards produced an elevation in body temperature after a delay of 4-5 hr. The fever continued for the remainder of the day, and into the following day (Fig. 1). The changes in body temperature following injections of denatured endogenous pyrogen did not differ significantly from those during control periods. This showed that the endogenous
662 H. A. BERNHEIM AND M. J. KLUGER pyrogen solution contained no significant amounts of heat-stable endotoxins (Fig. 2). The fever resulting from the injection of lizard endogenous pyrogen (1 x 107 cell equivalents) into 14 desert iguanas had a latency of about 41
40 -
U C)
39
c)0. E
38
co0 37
36 10.00
12.00 14.00 Time
16.00
Fig. 2. Effects of an intracardiac injection of denatured rabbit endogenous pyrogen on the mean hourly body temperature ( ± s.E.) of seven lizards (Dip8o8auru8 dor8ali"). The injection was made at about 09.00 hr on day 2.
3-4 hr and a duration of approximately 5 hr (Fig. 3). (injection of 1 x 106 and 5 x 106 cell equivalents did not produce significant changes in body temperature). Towards the end of the day the lizards' body temperatures returned to base line levels, and remained there during the following day. This fever pattern differs from that caused by the injection of endotoxin into a lizard: the fever then lasted for at least a 48 hr period (Bernheim & Kluger, 1976). Injections of denatured reptilian endogenous pyrogen produced no significant changes in body temperature (Fig. 4). DISCUSSION
Mammalian endogenous pyrogen causes fever when injected into lizards. This indicates that endogenous pyrogen, besides being cross-species reactive (Bornstein & Wood, 1969) is also cross-class reactive. The fever in
REPTILIAN ENDOGENOUS PYROGEN 41
663
P
659
ENDOGENOUS PYROGEN-LIKE SUBSTANCE PRODUCED BY REPTILES BY HARRY A. BERNHEIM* AND MATTHEW J. KLUGER From the Department of Physiology, Univereity of Michigan Medical School, Ann Arbor, Michigan 48190, U.S.A.
(Received 20 July 1976) SUMMARY
1. Injection of lizards (Dipso0auruls dorsali8) with rabbit endogenous pyrogen led to a fever. Injections with denatured endogenous pyrogen did not affect body temperature. 2. Injection of lizards with lizard endogenous pyrogen led to a fever of short duration, while injection of denatured lizard endogenous pyrogen produced no change in body temperature. 3. These data support the hypothesis that the febrile mechanism observed in the higher vertebrates has its origins in some primitive vertebrate. INTRODUCTION
Birds (D'Alecy & Kluger, 1975), reptiles (Vaughn, Bernheim & Kluger, 1974; Bernheim & Kluger, 1976), and fishes (Reynolds, Casterlin & Covert, 1976), as well as mammals, develop a fever in response to injection with Gram-negative bacteria. The fever that one observes in mammals is attributed to the synthesis and release of a heat labile protein from the host's leucocytes in response to the action of a bacterial endotoxin. The endogenous (leucocytic) pyrogen seems to act on the hypothalamic neural structures concerned with thermoregulation in such a way as to cause the 'set-point' for body temperature to be raised (i.e. fever) (Bligh, 1973). If the febrile mechanisms of the different classes of vertebrates have a common phylogeny, non-mammalian vertebrates may produce an endogenous pyrogen similar to that of mammals. If this is so then injection of endogenous pyrogen produced by the leucocytes of a representative of one class of vertebrate into another class of vertebrate should cause a fever. The experiments reported here were done to establish whether reptilian leucocytes can synthesize and release a pyrogenic substance, and whether mammalian endogenous pyrogen can cause a fever when injected into a reptilian species. * Present address: Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06519, U.S.A.
660
H. A. BERNHEIM AND M. J. KLUGER METHODS
New Zealand white rabbits (Oryotolagu8 cuniculu8) were used for the production and testing of mammalian endogenous pyrogen. To test the potency of the rabbit endogenous pyrogen, the rabbits were placed in a restrainer and allowed to reach steady state thermal equilibrium (Tre variation of + 0.10 C). The rabbits were then injected (i.v.) with 1 x 108 cell equivalents of endogenous pyrogen. Their rectal temperatures were monitored continuously with a copper-constantan thermocouple until their temperatures returned to base line levels. Desert iguanas (Dip8o8auru8 dorsali) weighing 25-55 g were obtained from a dealer in California. The lizards were housed and maintained as described by Bernheim & Kluger (1976). All lizards were allowed to adjust to the laboratory conditions for at least 2 weeks before their use in these experiments. During experimentation the lizards were allowed free movement in a desert-like environment (Bernheim & Kluger, 1976). Their body temperatures (±+ 0.1 C) were recorded about every 30 sec using copper-constantan thermocouples connected to a Honeywell Electonik 112 multipoint recorder. Each thermocouple was covered with polyethylene tubing, placed about 30 mm into each lizard's cloaca, and taped to their tails. Lizards were then allowed 24 hr to adjust to the experimental chamber. The next day was designated experimental day 1 and was used as the control day. At 09.00 hr on experimental day 2 the lizards received an intracardiac injection of rabbit endogenous pyrogen (1 x 108 cell equivalents) or lizard/endogenous pyrogen (1 x 106, 5 x 106, or 1 x 107 cell equivalents). Body temperatures were recorded for 48 hr after these inoculations. Other lizards were injected with denatured mammalian or reptilian endogenous pyrogen (solutions heated to 90° C for 30 min). Preparation of rabbit endogenous pyrogen was based on methods described by Bornstein, Bredenberg & Wood (1963), with the following exceptions: a 0-2% shellfish glycogen solution was used instead of a 0-1 % solution and the final supernatant solution was not dialysed against a normal salt solution. Lizard endogenous pyrogen was obtained using a modification of the above methods. Either (a) 10 ml. of a sterile 3% thioglycollate-saline solution, or (b) 10 ml. of dead Aeromonas hydrophila-saline solution containing 1 x 1010 bacteria was first injected into the coelomic cavity. (Since lizard endogenous pyrogen resulting from treatments a and b produced similar results, the data were pooled.) The lizards were kept at an ambient (= body) temperature of 370 C for 15-16 hr. They were then killed and the coelomic fluid removed aseptically. The coelom was washed with sterile pyrogen-free saline containing 5 u. heparin/ml. Washes were continued until only clear liquid was recovered. White blood cell and differential cell counts were done (with thioglycollate treatment the yield of polymorphonuclear leucocytes (PMNs) ranged from 7-10 x 10, with bacterial treatment the yield of PMNs ranged from 12- 16 x 10 PMNs). The saline solution containing the leucocytes was then centrifuged at 900 g at 40 C for 20 min and the supernatant discarded. The cell button was then resuspended in 10 ml. saline and incubated at 370 C for 10 min. Then the solution was centrifuged at 900 g for 20 min. The cells were then again resuspended in saline (10 or 20 x 106 cells/ml.) and incubated with gentle shaking at 370 C for 24 hr. After incubation the solution was centrifuged at 900 g for 20 min and the supernatant which contained the endogenous pyrogen-like material was spun at 10,000 g for 30 min, decanted and the supernatant stored at 40 C. The lizard endogenous pyrogen solutions were checked for sterility by incubating samples on blood agar plates at 370 C for 48 hr. Unsterile solutions and any containing haemoglobin were discarded. Data were statistically analysed using the Wilcoxon signed rank test.
REPTILIAN ENDOCENOUS PYROGEN
661
RESULTS
Injection of rabbit endogenous pyrogen (1 x 108 cell equivalents) into three rabbits led to an average increase in temperature of 1.30 C (+ 0.40 C s.E.) with an average latency of 12 min (+ 3 S.E.). Injections of denatured endogenous pyrogen caused no significant rise in body temperature. 43
_ i 2 42 42
t
n=8
41
40
6
E 39 T
37
36 _
Tt
- oControl (day 1) *.- Inject with rabbit EP (day 2) @v-Day 3
10.00
12.00 14.00 Time
16.00
Fig. 1. Effects of an intracardiac injection of rabbit endogenous pyrogen (EP) on the mean hourly body temperature (± s.E.) of eight lizards (Dip8o8auru8 dormali). The injection was made at about 09.00 hr on day 2. The lizards developed a fever 4-5 hr later. This fever continued throughout the second day (day 3).
The injection of rabbit endogenous pyrogen (1 x 108 cell equivalents) into eight lizards produced an elevation in body temperature after a delay of 4-5 hr. The fever continued for the remainder of the day, and into the following day (Fig. 1). The changes in body temperature following injections of denatured endogenous pyrogen did not differ significantly from those during control periods. This showed that the endogenous
662 H. A. BERNHEIM AND M. J. KLUGER pyrogen solution contained no significant amounts of heat-stable endotoxins (Fig. 2). The fever resulting from the injection of lizard endogenous pyrogen (1 x 107 cell equivalents) into 14 desert iguanas had a latency of about 41
40 -
U C)
39
c)0. E
38
co0 37
36 10.00
12.00 14.00 Time
16.00
Fig. 2. Effects of an intracardiac injection of denatured rabbit endogenous pyrogen on the mean hourly body temperature ( ± s.E.) of seven lizards (Dip8o8auru8 dor8ali"). The injection was made at about 09.00 hr on day 2.
3-4 hr and a duration of approximately 5 hr (Fig. 3). (injection of 1 x 106 and 5 x 106 cell equivalents did not produce significant changes in body temperature). Towards the end of the day the lizards' body temperatures returned to base line levels, and remained there during the following day. This fever pattern differs from that caused by the injection of endotoxin into a lizard: the fever then lasted for at least a 48 hr period (Bernheim & Kluger, 1976). Injections of denatured reptilian endogenous pyrogen produced no significant changes in body temperature (Fig. 4). DISCUSSION
Mammalian endogenous pyrogen causes fever when injected into lizards. This indicates that endogenous pyrogen, besides being cross-species reactive (Bornstein & Wood, 1969) is also cross-class reactive. The fever in
REPTILIAN ENDOGENOUS PYROGEN 41
663
P
