Comparison of febrile responsiveness of rats and rabbits to endogenous pyrogen

The fever responses of rats and rabbits were compared in detail using a single common source of semipurified endogenous pyrogen prepared from human monocytes. The characteristics and dynamics of the fever-response curves for each species were examined and their dose-response curves were determined and compared. The fevers displayed by rats were qualitatively similar to those of rabbits, but, typically, they developed and terminated more rapidly than those of rabbits. Rabbits were much more sensitive to the endogenous pyrogen than rats. The threshold dose of pyrogen required to elicit a fever was 5 times lower in the rabbit, and the slope of the rabbit's dose-response curve was 1.5 times steeper than that of the rat. The maximum fevers attainable in rabbits were approximately twice those attainable in rats. It was also shown that the more rapid febrile responses of the rat were not due to the 10-fold smaller mass of the rat; instead, we proposed that this difference was more likely due to a closer diffusional proximity of the pyrogen receptor sites to the circulation in rats. The lower sensitivity of the rat to endogenous pyrogen was attributed to a relative insensitivity of the pyrogen receptor sites in rats in the translation of the endogenous pyrogen stimulus into fever.     

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.     

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.     

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.     

Interleukin 1 (IL 1) as a mediator of crystal arthritis. Stimulation of T cell and synovial fibroblast mitogenesis by urate crystal-induced IL 1

We reported before that monosodium urate (MSU) crystals were potent stimulators of endogenous pyrogen (EP) production from human and rabbit mononuclear phagocytes, and proposed that this property of MSU crystals may be important in the pathogenesis of gout. EP activity is now attributed to interleukin 1 (IL 1) peptides but IL 1 is not the only pyrogenic monocyte-derived cytokine, since both interferon-alpha (alpha-IFN) and tumor necrosis factor (TNF) are also pyrogenic in rabbits. Using a T cell comitogenic assay based on a murine helper T cell clone that does not respond to IFN or TNF, we now report the release of IL 1 activity from human blood monocytes and synovial fluid mononuclear cells (MNC), following stimulation with MSU crystals. MSU-induced supernatants with IL 1 activity were neutralized with rabbit antiserum to human IL 1 and also stimulated the growth ([3H]thymidine incorporation) of long-term fibroblast-like cell lines derived from human synovial rheumatoid exudate. Two other crystals associated with articular inflammation were tested: hydroxyapatite was a much less potent stimulus compared with MSU crystals, and calcium pyrophosphate dihydrate did not stimulate IL 1 release from human monocytes or synovial fluid MNC. As a model for the inflammatory consequences of acute and chronic overproduction of IL 1, gout is the only sterile inflammatory disease where the local and systemic pathology is compatible with such overproduction; raised IL 1 levels have been found at the site of inflammation, and a necessary etiologic agent, crystalline urate, has been shown unequivocally to be a direct activator of mononuclear IL 1 release.     

Release of an endogenous pyrogen from guinea pig leukocytes: the role of T lymphocytes and correlation with suppression (desensitization) of delayed hypersensitivity

The pathogenesis of fever in delayed hypersensitivity (DH) was studied in guinea pigs immunized with either ovalbumin or bovine gamma-globulin in complete Freund's adjuvant. In vitro incubation of sensitized lymphocytes with the specific antigen used for immunization resulted in the elaboration of a lymphokine-like factor that activated either monocytes or neutrophils to release endogenous pyrogen (EP), the protein that causes fever. Specifically sensitized T cells appeared to be responsible for release of this EP-inducing factor. Desensitization of the dermal DH response to antigen was produced by several large injections of antigen and was associated with a reduced capacity of lymphocytes from such animals to activate phagocytic cells to release EP. This may explain the reduced fever (pyrogenic tolerance) that occurs when repeated injections of antigen are given to sensitized animals. Fever and the dermal response to DH seem to be closely linked reactions that have evolved to defend the host against invading pathogens. In both reactions, phagocytic cells appear to be activated by lymphokines derived from T lymphocytes specifically responding to microbial antigens.     

Increased Production of Endogenous Pyrogen and Lysozyme by Blood Monocytes in Sarcoidosis

Blood monocytes from patients with sarcoidosis were incubated in vitro, and secretion of endogenous pyrogen (EP), the protein which mediates fever, and lysozyme (L) were measured. After incubation with endotoxin, monocytes from 5 patients with sarcoidosis released twice as much EP as did monocytes from normal individuals (p < .001). Initial 24-hr secretion of L by monocytes from 6 of 11 additional patients with sarcoidosis exceeded the normal range of values for cells from 11 age- and sex-matched control individuals. Cells with initially augmented secretion rates continued to secrete increased amounts of L for 3 days. A correlation was noted between in vitro secretion of L by monocytes and serum levels of L in the same patient. These studies indicate that circulating mononuclear cells in some patients with sarcoidosis have an increased capacity to secrete EP and/or L prior to tissue localization.     

Suppression of Ag-induced release of EP (IL 1) by spleen cells of specifically desensitized donors: evidence for the role of a suppressor cell

Monocytes or macrophages may be induced to produce IL 1 by activators (e.g., lipopolysaccharide endotoxin) that act directly or by antigens/mitogens (e.g., Con A) that stimulate inducer lymphocytes to release a lymphokine that stimulates macrophages. Using guinea pigs (GP) rendered delayed hypersensitive to ovalbumin (OVA), we investigated the role of spleen cells from normal, sensitized, and specifically desensitized GP in suppressing release of IL 1, measured as endogenous pyrogen (EP), from peritoneal exudates of sensitized GP when incubated with OVA in vitro. Co-cultivation of all three sources of spleen cells with GP peritoneal exudate cells and OVA suppressed EP release as measured in the rabbit fever assay, the effect being most marked with cells from desensitized GP, intermediate with cells from sensitized GP, and least with normal cells. This suppressor activity of spleen cells on in vitro EP release was not explained by nonspecific absorption of EP by the added cells and did not affect EP release by a stimulus that activates macrophages directly (heat-killed staphylococci). It required both lymphocytes and macrophages for its effect, but unlike some other suppressor factors, it was not modified by indomethacin, an inhibitor of prostaglandin release. This appears to be the first reported evidence for cell-mediated suppression of lymphokine-mediated release of IL 1, an important modulator of the immune system through its combined role as a lymphocyte-activating factor and an inducer of fever (EP).     

Antipyrogenic properties of oxytocin

Effects of oxytocin on pyrogenal or endogenous pyrogen-induced fever were studied. Intramuscular injection of oxytocin (0.2 micrograms/kg every half an hour) did not significantly affect the pattern of pyrogenal-induced fever. Constant intravenous injection of oxytocin (0.4 and 4 micrograms/kg/h) 2-4.5 h after pyrogenal decreased the rectal temperature, on an average, by 24% and 31%, respectively. Endogenous pyrogen fever was not attenuated by intravenous oxytocin (4 micrograms/kg/h). The antipyrogenic effect of oxytocin is related to inhibition of endogenous pyrogen synthesis rather than to blockade of its action, which is indicated by a decreased second peak of the temperature curve, inhibition of endogenous pyrogen synthesis in vitro, and persistence of the hyperthermic effect of endogenous pyrogen.     

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.     

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.     

Central and peripheral injections of ACTH (1–24) reduce fever in adrenalectomized rabbits

Central administration of ACTH (1-24) reduces fever in normal rabbits in doses that have no effect on afebrile body temperature. Previous experimental and clinical reports indicate that peripheral administration of both ACTH and corticosteroids reduces fever, and since central injection of corticosteroids can also lower fever it might be that the antipyretic effect of intracerebroventricular (ICV) ACTH (1-24) is due to adrenal stimulation. To learn whether this endogenous central peptide can produce antipyresis independently, ACTH (1-24) was injected ICV in bilaterally adrenalectomized (ADX) rabbits made febrile by IV injections of leukocytic pyrogen (LP). ACTH (250 ng) given ICV reduced fever in these animals and had a slight hypothermic effect when given to the same rabbits when they were afebrile. Doses of 25-75 ng reduced fever without influencing normal body temperature. Intravenous injections of ACTH (2.5 micrograms) also lowered fever caused by IV LP in ADX rabbits. The present findings raise the possibility that release of endogenous central ACTH, and perhaps entry into the brain of circulating ACTH, the release of which is known to increase in fever, limits the magnitude of the febrile response by influencing central temperature controls.     

Pyrogen release in vitro by lymphoid tissues from patients with Hodgkin's disease

The mechanism of fever in patients with Hodgkin''s disease was investigated by examining endogenous pyrogen production by blood, spleen, and lymph node cells incubated in vitro. Blood leucocytes from febrile or afebrile patients with Hodgkin''s disease did not produce pyrogen spontaneously. Spleen cells, however, frequently released pyrogen during initial incubations, unlike spleen cells from patients with non-malignant diseases. Pyrogen production occurred from spleens without observed pathologic infiltrates of Hodgkin''s disease. Lymph nodes involved with Hodgkin''s disease produced pyrogen more frequently than did nodes involved with other diseases. Pyrogen production by tissue cells was prolonged, required protein synthesis, and in some cases was due to mononuclear cells; it did not correlate with fever in the patient. These studies demonstrate spontaneous production of endogenous pyrogen in vitro by lymphoid tissue cells from patients with Hodgkin''s disease.     

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.     

The effect of α-MSH on fever caused by Staphylococcus aureus cell walls in rabbits

The role of endogenous pyrogens induced by gram-positive bacterial pyrogens is not known. Intravenous alpha-MSH (2.5 micrograms) significantly reduced only the first phase of the biphasic thermal response to IV S. aureus cell walls (5 x 10(7)). Intracerebroventricular alpha-MSH (200 ng) had no effect on the fever response. The fall in serum iron concentration was significantly attenuated by the IV alpha-MSH but was not affected by the ICV alpha-MSH. Intravenous alpha-MSH had no effect on fever or the serum iron response caused by muramyl dipeptide (MDP). We conclude that the first phase of the thermal response to S. aureus cell walls is mediated by an endogenous pyrogen (EP) and the second phase of the response by a mechanism not involving EP, but possibly a muramyl peptide.     

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.     

The neutrophil-activating protein of Helicobacter pylori crosses endothelia to promote neutrophil adhesion in vivo

Helicobacter pylori induces an acute inflammatory response followed by a chronic infection of the human gastric mucosa characterized by infiltration of neutrophils/polymorphonuclear cells (PMNs) and mononuclear cells. The H. pylori neutrophil-activating protein (HP-NAP) activates PMNs, monocytes, and mast cells, and promotes PMN adherence to the endothelium in vitro. By using intravital microscopy analysis of rat mesenteric venules exposed to HP-NAP, we demonstrated, for the first time in vivo, that HP-NAP efficiently crosses the endothelium and promotes a rapid PMN adhesion. This HP-NAP-induced adhesion depends on the acquisition of a high affinity state of beta(2) integrin on the plasma membrane of PMNs, and this conformational change requires a functional p38 MAPK. We also show that HP-NAP stimulates human PMNs to synthesize and release a number of chemokines, including CXCL8, CCL3, and CCL4. Collectively, these data strongly support a central role for HP-NAP in the inflammation process in vivo: indeed, HP-NAP not only recruits leukocytes from the vascular lumen, but also stimulates them to produce messengers that may contribute to the maintenance of the flogosis associated with the H. pylori infection.     

Blunted febrile response to intravenous endotoxin in starved rats

The effects of fasting on the febrile responses to intravenous injection of bacterial lipopolysaccharide (LPS; endotoxin) of Escherichia coli were investigated in rats. Ad libitum-fed rats (C) produced a biphasic fever with an increase in the temperature difference between brown adipose tissue and colon and shivering activity (SA). Measurement by a direct calorimeter showed no particular changes in heat loss. Rats starved for 4 days (F4) responded to intravenous LPS with a monophasic fever accompanied by an increase in SA only. However the maximal rise in colonic temperature (Tco) did not differ from C rats. Subsequent 2-day fasting reduced SA and the maximal fever height. Endogenous pyrogen (EP) injected intravenously produced a prompt rise in Tco followed by prolonged hyperthermia in C rats. In the F4 rats, there was no such sustained rise in Tco as a result of intravenous EP. The response in Tco to intravenous prostaglandin E2 (PGE2) was the same in fed and starved rats. The administration of LPS, EP, and PGE2 into the lateral ventricle evoked a similar extent of hyperthermia in C and F4 rats. Because the second phase of fever has been shown to occur after pyrogens are translated into a febrile stimulus within the blood-brain barrier, it is assumed that the functional changes of the blood-brain barrier such as in the permeability of pyrogens or in the sensitivity of pyrogen receptors resulted in the absence of the second phase of fever in starved rats.     

Passage of immunomodulators across the blood-brain barrier

The question is considered of how and where cytokines, such as interleukin 1 (IL-1), that are released into the circulation during the host defense response, reach and interact with the central nervous system to produce fever or act as neuroimmunomodulators. Evidence is presented suggesting a role for a brain circumventricular organ (CVO) in this respect. Several interactions between a specific CVO, the organum vasculosum laminae terminalis (OVLT) and endogenous pyrogen (EP) in the production of fever are reviewed. A more general hypothesis is developed on a role for the brain CVOs in monitoring the blood concentrations of several proteins and complex polypeptides such as the circulating endocrines that are regulated via the autonomic nervous system. A proposed connection between the release of prostaglandin E (PGE) at the blood-brain interface in response to infection and the ability of the brain to maintain an immunoprivileged status in the face of exposure of its CVOs to foreign antigens is discussed.     

Macrophage hyperreactivity to endotoxin induced by streptococcal pyrogenic exotoxin in rabbits

Pretreatment of rabbits with streptococcal pyrogenic exotoxin (SPE) resulted in an enhancement of their febrile response to subsequent endotoxin challenge. This suggested that SPE may enhance the macrophage capacity to respond to endotoxin in vivo to produce an endogenous pyrogen. It was also demonstrated that peritoneal macrophages derived from SPE-treated rabbits exhibited hyperreactivity to endotoxin in vitro as assessed by endotoxin-induced increase in glucose consumption. These data indicate that SPE has the ability to enhance macrophage reactivity to endotoxin.