A comparison of the febrile responses of the Brattleboro and Sprague-Dawley strains of rats to endotoxin and endogenous pyrogens

The febrile responses of homozygous (di/di) Brattleboro rats, to both intravenous endogenous pyrogen and to a lipopolysaccharide endotoxin, were compared with those of normal Sprague-Dawley rats. There were no detectable differences between the fever curves of the two strains in response to endogenous pyrogen. Brattleboro rats, which are deficient in the neuropeptide arginine vasopressin (AVP), displayed fevers that were both qualitatively and quantitatively indistinguishable from those of normal Sprague-Dawley rats that do not suffer from congenital diabetes insipidus. It is concluded that the absence of AVP-containing cells in Brattleboro rats is not an important factor in determining the nature of their febrile responses to endogenous pyrogen. More remarkable, however, were the divergent febrile responses of the two strains to intravenously injected endotoxin. Normal rats displayed hypothermic responses, whereas the Brattleboro rats became febrile. By 2 h after the injection of endotoxin, body temperatures in both strains had returned to normal. Three hours after the rats had been exposed to endotoxin, both strains were found to be totally refractory to endogenous pyrogen. However, when both strains of rats were tested to endogenous pyrogen 3 days later, their febrile responses were more than double the magnitude of their initial control responses. These alterations in the febrile responsiveness of rats occurring at different times after the injection of endotoxin appear to be related to the effects that endotoxin has on the cells of the reticuloendothelial system, over the same time course.     

Differences in endogenous pyrogen fevers induced by iv and icv routes in rabbits

We have compared the characteristics of fevers produced by endogenous pyrogen administered by the intravenous (iv) and by the intracerebroventricular (icv) routes in conscious rabbits. Fevers induced by the intracerebroventricular route have a longer latency to onset, a less steep rise in body temperature, and a longer time to peak elevation in body temperature than do fevers induced by the intravenous route. Furthermore, a dose of indomethacin (2 mg/kg) administered intravenously, which is effective in markedly attenuating fevers produced by the intravenous route, was completely without effect on fevers induced by the intracerebroventricular route. On the other hand, when indomethacin (500 micrograms) was infused intracerebroventricularly, it markedly reduced fevers induced by the subsequent injection of endogenous pyrogen into the contralateral cerebral ventricle, but such pretreatment had little effect on fevers elicited by intravenous injections of endogenous pyrogen. It is concluded that the sites of action of endogenous pyrogen in response to intravenous injections of pyrogen are different from those responding to intracerebroventricular injections of pyrogen and that this is manifest in several distinct differences in the characteristics of the two fevers. These results indicate that the intracerebroventricular model of fever production is not appropriate for the study of the normal pathogenesis of fever.     

A study of the variability in the febrile responses of rabbits to endogenous pyrogen

The range of body temperature increases elicited by a standard dose of endogenous pyrogen (0.5 ml/kg iv) was examined in a population of 26 male New Zealand White rabbits. Although the mean maximum increase in rectal temperature was 0.88 +/- 0.06 degree C (SE), individual responses varied from 0.4 degree to 1.5 degree C. Three representative animals that responded to the standard dose of pyrogen with small, intermediate, and large febrile responses were selected and challenged with the same dose of pyrogen on eight separate occasions, and the variability of these responses was examined. There was little variability within the characteristic responses of any particular animal to the repeated challenges. The variability of the febrile responses elicited by both intravenous and intracerebroventricular administration of the same pyrogen was examined and compared using another group of 11 rabbits. The variability in response to the intravenous route was similar to that found in the larger population, whereas the variation in response to the intracerebroventricular route was smaller, and all 11 animals had fevers that were greater than 1 degrees C. It is concluded that the variability of the febrile responses of rabbits to intravenous pyrogen was due to differences between individual sensitivities of animals to the intravenously administered pyrogen. This difference in sensitivity may be due to a difference in the amount of pyrogen that reaches the putative receptor sites, or to a difference in the density or effectiveness of receptor sites in translating the pyrogenic stimulus into a fever response.     

Immunoadjuvants enhance the febrile responses of rats to endogenous pyrogen

The febrile responses of male Sprague-Dawley rats to a semipurified endogenous pyrogen produced from human monocytes were characterized by establishing fever dose-response curves. The animals were then injected intravenously with a number of substances that possessed the common properties of stimulating the phagocytic activity of the cells of the reticuloendothelial system and of acting as immunoadjuvants. The substances used were zymosan, lipopolysaccharide endotoxin, and muramyl dipeptide. Three days after any of these immunoadjuvants were injected, the fever sensitivity of the rats was remeasured. In each case, the slope of the fever dose-response curve tripled, and in some instances the response threshold for fever response was reduced by factors of three to eight. Furthermore, the maximum increase in body temperature produced by the endogenous pyrogen was more than doubled after immunoadjuvant treatment. By contrast latex beads, which are also phagocytized by the cells of the reticuloendothelial system but do not subsequently increase their phagocytic index nor do they enhance immune responses, had no effect on the fever sensitivity of rats in response to endogenous pyrogen. In the light of these findings, it is suggested that the febrile responses of rats to endogenous pyrogen are mediated in some manner by cells that possess some of the properties of reticuloendothelial cells. The location of these putative cells must be close to the circulation, because the immunoadjuvants used in this study were, for the most part, large molecular weight molecules that could not cross the blood-brain barrier easily.     

Site of action of calcium channel blockers in inhibiting endogenous pyrogen fever in rats.

We have demonstrated that the Ca2+ channel blocker verapamil, administered intravenously, exerts an antipyretic effect on the febrile responses of rats to intravenously injected endogenous pyrogen (EP). We have also shown that the same intravenous dose of verapamil is ineffective in blocking fevers induced by the microinjection of exogenous prostaglandin E (PGE) into the organum vasculosum laminae terminalis (OVLT) of rats. Experiments were conducted to determine whether the site of this verapamil antipyresis was in the OVLT itself. The febrile responses of six male Sprague-Dawley rats to EP were determined at thermoneutrality. Verapamil (10 micrograms/rat) was microinjected directly into the OVLT, and the febrile responses to the EP dose were redetermined 15-30 min later. In every case the EP fevers were attenuated after verapamil pretreatment. Intra-OVLT injections of verapamil alone were without effect on body temperature. When the same dose of verapamil was injected into the OVLT 15 min before the injection of PGE into the same site, it had no effect on the ensuing PGE-induced fever. In view of the fact that less than 1/250th of the effective systemic dose of verapamil, when injected into the OVLT, was equally effective in blocking the EP fevers, we conclude that verapamil acts within the OVLT to block fever rather than peripherally. Furthermore, because verapamil administered into the OVLT does not block PGE fevers, it is unlikely that PGE produces fever by acting as a Ca2+ ionophore on hypothalamic neurons.     

Enhancement of the febrile responses of rats to endogenous pyrogen occurs within the OVLT region

The febrile responses of male Sprague-Dawley rats to a semi-purified endogenous pyrogen (EP) derived from human monocytes are markedly enhanced 3 days after the animals are intravenously injected with a variety of immunoadjuvants. The present study was designed to investigate the site within the body at which these substances act to produce this febrile-enhancing phenomenon. Stainless steel microinjection cannula guide tubes were implanted within the region of the organum vasculosum lamina terminalis (OVLT) of the rats and control febrile dose-response curves to EP were established. Minute quantities of the immunoadjuvants zymosan, lipopolysaccharide endotoxin, and the synthetic adjuvant peptide, muramyl dipeptide, were microinjected into the OVLT region and 3 days later, the febrile responses of the animals were retested. In each case the febrile response elicited by a standard dose of EP was more than doubled, the slope of the fever dose-response curve was tripled, and the dose threshold was lowered by a factor of four to five. These responses are identical with those produced when much larger amounts of these immunoadjuvants are injected intravenously, and, thus, we conclude that the site of action of these substances in enhancing fever in response to EP resides in or near the OVLT region. It is proposed that EP stimulates a type of reticuloendothelial cell residing within the OVLT to release prostaglandin E, which in turn crosses the blood-brain barrier to effect the changes in the thermoregulatory neurons of the preoptic anterior hypothalamic area that result in fever.     

Fever in rats during normal and dehydrated conditions

The effect of endogenous pyrogen (EP, from rabbit) and endotoxin (Salmonella typhosa) on rectal temperature (Tre) was investigated in normal and dehydrated rats of both sexes. Intraperitoneal injection of either EP or endotoxin did not affect body temperature. In addition, no changes in Tre were observed when endotoxin was injected intravenously in normally hydrated male rats, but significant falls in Tre occurred in normal female rats. However, intravenous injection of EP produced fever in both sexes, but females generally showed smaller responses. A second intravenous injection of endotoxin, given 3 days after the first injection, always produced fever in normally hydrated rats. The pattern of this febrile response was monophasic. In contrast to the response in normal rats, intravenous endotoxin produced significant fevers with a biphasic pattern in dehydrated rats of either sex, but the febrile responses of male rats were greater than those of female rats. On the other hand, there were no significant differences between febrile responses to intravenous EP exhibited by normal and dehydrated animals. These results show that rats of both sexes possess physiological mechanisms capable of producing a fever following intravenous injections of EP.     

Calcium channel blockers inhibit endogenous pyrogen fever in rats and rabbits.

We have previously shown that febrile responses in both rats and rabbits are elicited by the intravenous injection of a semipurified endogenous pyrogen (EP) prepared from human monocytes. We are now presenting evidence that these febrile responses are mediated via activation of Ca2+ channels by EP. The febrile responses of male New Zealand White rabbits and Sprague-Dawley rats to a standard dose of EP were determined at their respective thermoneutral ambient temperatures. The animals were then treated with Ca2+ channel blocker verapamil (7.5 mg/kg iv) 30-60 min before the EP challenge. In every case the febrile response to EP was markedly attenuated after verapamil pretreatment, while administration of verapamil by itself had no detectable effect on body temperature. Another Ca2+ channel blocker, nifedipine (5 mg/kg iv), was shown to possess antipyretic activity in rats also. To localize where in the fever pathway these Ca2+ channel blockers were acting, we investigated the effect of verapamil at the same dose on fevers that were produced by microinjection of prostaglandin E (PGE) directly into the brain. These PGE fevers were unaffected by verapamil pretreatment, indicating that the antipyretic action of Ca2+ channel blockers occurs before the formation of PGE in response to EP stimulation. The most likely locus of action is the activation of the enzyme phospholipase A2, which regulates the production of arachidonic acid from cellular phospholipids in the prostanoid cascade.     

Sensitivity of hypothalamic sites to salicylate and prostaglandin.

This study was designed to determine the dose of salicylate necessary to produce substantial antipyresis, and to determine the relationship between the response to salicylate and prostaglandin infused into the same region of the preoptic area of the rabbit. The effect of preoptic infusions of three doses of sodium salicylate, or a control solution, on the fever produced by an intravenous injection of endogenous pyrogen was measured. The pyrogenic response to prostaglandin E1 injected into the same preoptic sites in the same rabbits was also monitored. The results showed that the 50 microgram/microL per hour dose of salicylate did not produce significant antipyresis but that the 100 and 200 microgram/microL per hour doses did. The results also showed a significant correlation between the magnitude of fever produced by prostaglandin E1 and the magnitude of antipyresis produced by sodium salicylate at a particular site. Those sites at which infusion of salicylate produced the most effective antipyresis were also the ones at which prostaglandin E1 produced the largest fevers.     

The pyrogenic effect of scarlet fever toxin

Scarlet fever toxin was found to liberate leukocytic pyrogen from granulocytesin vitro. In comparative experiments withSalmonella paratyphi B endotoxin and scarlet fever toxin it was tested whether leukocytes from rabbits tolerant to one of these toxins are able to synthetize and liberate endogenous pyrogen. Leukocytes from rabbits tolerant to endotoxin liberated leukoeytic pyrogen following challenge with endotoxin or with scarlet fever toxin. Leukocytes from animals tolerant to scarlet fever toxin liberated leukocytic pyrogen in the presence of endotoxin, but were insensitive to homologous, i.e. scarlet fever toxin. Similarly, leukocytes from cortisone-treated animals did not liberate leukocytic pyrogen if they were incubated with scarlet fever toxin, but liberation of leukocytic pyrogen did take place under challenge with endotoxin. Leukocytes from normal animals incubated in Hanks solution without toxin did not synthetize endogenous pyrogen.     

Neuropeptide alpha-MSH antagonizes IL-6- and TNF-induced fever.

The pro-opiomelanocortin-derived peptide melanocyte stimulating hormone (alpha-MSH) antagonizes the fever induced by several stimuli including endotoxin, endogenous pyrogen, and certain cytokines. To determine if alpha-MSH can antagonize the pyrogenic action of recombinant IL-6 and TNF directly within the central nervous system, the cytokines were injected with and without alpha-MSH (200 ng) into a lateral cerebral ventricle of rabbits and rectal temperature was monitored continuously. Central administration of both cytokines caused fever. However, when alpha-MSH was injected after cytokine administration, the fevers were markedly reduced. The results are consistent with previous observations on the antipyretic effect of alpha-MSH and they show that the peptide can act within the brain to antagonize pyrogenic actions of specific cytokines believed to be important in CNS mediation of fever.     

The search for an endogenous activator

Certain febrile diseases are unaccompanied by infection or apparent hypersensitivity. In myocardial infarction or pulmonary embolism, for example, fever has been attributed to inflammation and/or tissue necrosis. Exogenous (microbial) pyrogens stimulate both human and animal monocytes/macrophages to produce endogenous pyrogen (EP) in vitro. To determine if plasma and cellular endogeneous mediators (EMs) of inflammation induced EP production, human mononuclear cells (M/L) were incubated for 18 hours with varying amounts of EM and the supernates assayed for EP in rabbits. Neutrophils (PMNs), which do not generate EP and yet are a feature of acute inflammation, were tested. Neither viable, phorbol myristic acetate-stimulated PMNs nor sonicated PMNs, red blood cells, or M/L stimulated human monocytes to produce EP. Human C3b and C5a, which mediate phagocytosis and chemotaxis, respectively, were also inactive. Despite its chemoattractant properties, the synthetic peptide FMLP failed to induce EP release. Since Poly I:Poly C (PIC: a synthetic, double-stranded RNA) is a potent pyrogen in rabbits, we investigated PIC, as well as a native, single-stranded RNA (from E. coli) and DNA (from calf thymus). None was active in vitro, and only PIC caused fever when given to rabbits intravenously. In summary, we have been unable to find an endogenous activator of EP from human monocytes to explain fevers associated with inflammation alone.     

Effects of nitric oxide synthase inhibitors on the febrile response to muramyl dipeptide and lipopolysaccharide in rats

We have administered aminoguanidine, a relatively specific inhibitor of inducible nitric oxide synthase, and N-nitro-L-arginine methyl ester (L-NAME), an unspecific nitric oxide synthase inhibitor, to rats made febrile with the gram-positive pyrogen, muramyl dipeptide and gram-negative pyrogen, lipopolysaccharide. Sprague-Dawley rats, housed individually at approximately 25 degrees C with a 12:12 h light:dark cycle (lights on 0700 hours), were injected (at 0900 hours) intraperitoneally with 50 mg/kg aminoguanidine, 25 mg/kg or 50 mg/kg L-NAME, and intramuscularly with 500 microg/kg muramyl dipeptide or 100 microg/kg lipopolysaccharide. Pyrogen injections were spaced at least 14 days apart. Body temperature was measured throughout the study in unrestrained animals using radio-telemetry. Neither muramyl dipeptide nor lipopolysaccharide-induced fevers were affected by aminoguanidine. However, L-NAME administration inhibited muramyl dipeptide and lipopolysaccharide-induced fevers, but only for the 1st 2-4 h of the fevers (two-way ANOVA, P<0.05). After the initial inhibition, lipopolysaccharide fevers developed normally. Therefore, constitutively expressed nitric oxide synthase appears to be involved in the initial phases of fever genesis of gram-negative and gram-positive fevers in rats. On the other hand, inducible nitric oxide synthase appears not to play a role in these fevers.     

Central arginine vasopressin and endogenous antipyresis.

Arginine vasopressin (AVP) is a centrally synthesized nonapeptide that exerts classical endocrine effects as well as a host of centrally mediated actions. A strong case can be argued in support of a neurotransmitter-neuromodulator role for AVP. Acting within the central nervous system (CNS), AVP has been demonstrated to be involved in the modulation of febrile body temperature. Because AVP acts to reduce pyrogen-induced fevers, but not normal body temperature, its actions are deemed to be antipyretic. However, to demonstrate an endogenous antipyretic function, AVP must be shown to be active during conditions where fever is naturally suppressed. This review will focus on five such conditions where the absence of pyrogen-induced fever can be linked to the endogenous activity of AVP within the brain. In the neonatal rat pup, the use of specific antagonists to the AVP receptor has revealed a role for CNS AVP in the absence of fever following peripheral injections of bacterial endotoxin. These results may help to explain a similar lack of fever in other newborn species. In parturient animals a reduced or absent febrile response has been linked to the increased presence of AVP within the septal area of the brain. The combined use of AVP receptor antagonism as well as immunohistochemistry has shown enhanced AVP activity within the ventral septal area of the rat and guinea pig brain during tolerance to intravenous pyrogens. These results suggest that the mechanism of fever suppression following repeated systemic injections of bacterial pyrogen includes centrally acting AVP.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of low ambient temperature on the febrile responses of rats to semi-purified human endogenous pyrogen

The febrile responses of Sprague-Dawley rats to semi-purified human endogenous pyrogen were studied at a thermoneutral ambient temperature (26 degrees C) and in the cold (3 degrees C). It was found that while rats developed typical monophasic febrile responses at thermoneutrality, febrile responses were absent in the cold-exposed rats. Experiments were conducted to determine whether this lack of febrile responses in cold-exposed rats was due to an inability of these animals to generate or retain heat in the cold. Thermogenesis and vasoconstriction were stimulated in cold-exposed rats by selectively cooling the hypothalamus, using chronically implanted thermodes. It was shown that, using this stimulus, metabolic rate could be increased by more than 50 percent and body temperature could be driven up at a rate of 5 degrees C/hour in rats exposed to the cold. Therefore, it was concluded that the lack of febrile responses of cold-exposed rats to pyrogen is in no way due to a physical or physiological inability to retain heat. Instead, it appears that in some manner cold exposure suppresses the sensitivity or responsiveness of the rat to pyrogenic stimuli.     

Transiently enhanced LPS-induced fever following hyperthermic stress in rabbits

Hyperthermia has been shown to induce an enhanced febrile response to the bacterial-derived endotoxin lipopolysaccharide (LPS). The aim of the present study was to test the hypothesis that the enhanced LPS-induced fever seen in heat stressed (HS) animals is caused by leakage of intestinal bacterial LPS into the circulation. Male rabbits were rendered transiently hyperthermic (a maximum rectal temperature of 43°C) and divided into three groups. They were then allowed to recover in a room at 24°C for 1, 2 or 3 days post-HS. One day after injection with LPS, the post-HS rabbits exhibited significantly higher fevers than the controls, though this was not seen in rabbits at either 2 or 3 days post-HS. The plasma levels of endogenous LPS were significantly increased during the HS as compared to those seen in normothermic rabbits prior to HS. LPS fevers were not induced in these animals. One day post-HS, rabbits that had been pretreated with oral antibiotics exhibited significantly attenuated LPS levels. When challenged with human recombinant interleukin-1 instead of LPS, the 1-day post-HS rabbits did not respond with enhanced fevers. The plasma levels of TNF increased similarly during LPS-induced fevers in both the control and 1-day post-HS rabbits, while the plasma levels of corticosterone and the osmolality of the 1-day post-HS rabbits showed no significant differences to those seen prior to the HS. These results suggest that the enhanced fever in the 1-day post-HS rabbits is LPS specific, and may be caused by increased leakage of intestinal endotoxin into blood circulation.     

Clinical fever: its history, manifestations and pathogenesis.

A short summary of some aspects of the history of clinical fever is presented with special reference to its association with inflammation. The role of bacterial endotoxins and endogenous pyrogen (released from inflammatory cells) in the genesis of human fevers is reviewed. Clinical diseases are tabulated within various broad categories and discussed in relation to the frequency with which they are associated with fever and the probable pathogenetic mechanisms involved. Certain unresolved discrepancies are emphasized in the light of our present knowledge.     

Is the endogenous antipyretic neuropeptide alpha-MSH responsible for reduced fever in aged rabbits?

The reduced febrile response in aged man has been noted since the beginning of clinical thermometry. Our previous research on aged rabbits and squirrel monkeys disclosed a similar reduced fever, presumably due to a decrease in central receptors for endogenous pyrogen. However, because central alpha-melanocyte stimulating hormone (MSH) appears to have a potent role in physiological control of fever, it may be that increased release of the peptide is responsible for the reduced febrile response in aged animals. To test this idea, antiserum specific to MSH was administered intracerebroventricularly to rabbits of known age. The antiserum given according to three schedules of treatment augmented fever caused by IV injections of interleukin-1 (IL-1) in young (less than 2 years) male and female rabbits. Aged female rabbits (3-5+ years) and females aged 2-3 years showed significant augmentation of fever only after pretreatment plus acute injection of antiserum. A single ICV injection of MSH (200 ng) reduced fever in all groups with the greatest antipyretic effect in the aged females. The results indicate that while aged rabbits have an increased antipyretic response to central MSH, binding of the endogenous peptide does not result in marked increases in fever in these animals. Thus, whereas a change in central MSH sensitivity may contribute to reduced fever in aged homeotherms, a reduction in central pyrogen receptors appears to be the most parsimonious explanation.     

Differetial sensitivity of spontaneously hypertensive rats to the hypothermic effect of dopaminergic drugs

The dopaminergic agonist apomorphine produces dose-related hypothermia in naive rats as does L-DOPA in carbidopa-pretreated rats. The hypothermic responses to these two dopaminergic drugs were significantly more pronounced and prolonged in the spontaneously hypertensive rat than in normotensive Wistar control rats. The greater sensitivity of the SHR to these drugs was reflected as a leftward shift of the dose-response curves for apomorphine- and L-DOPA-induced hypothermias.