Release of endogenous pyrogen-activating factor from concanavalin A-stimulated human lymphocytes.

Evidence is presented that the mitogen concanavalin A stimulates human lymphocytes to produce a nonpyrogenic lymphokine (LK) that is capable of activating human monocytes but not granulocytes to produce endogenous pyrogen (EP) in vitro. The potency of this preparation should facilitate further studies to purify and characterize this agent and determine its relation to other know LK. It seems likely that this factor, which we have called EP-activating factor (EPAF), plays a significant role in the development of fever in states of delayed hypersensitivity in man.

Tumor necrosis factor (cachectin) is an endogenous pyrogen and induces production of interleukin 1.

Recombinant human tumor necrosis factor (rTNF alpha) injected intravenously into rabbits produces a rapid-onset, monophasic fever indistinguishable from the fever produced by rIL-1. On a weight basis (1 microgram/kg) rTNF alpha and rIL-1 produce the same amount of fever and induce comparable levels of PGE2 in rabbit hypothalamic cells in vitro; like IL-1, TNF fever is blocked by drugs that inhibit cyclooxygenase. At higher doses (10 micrograms/kg) rTNF alpha produces biphasic fevers. The first fever reaches peak elevation 45-55 min after bolus injection and likely represents a direct action on the thermoregulatory center. During the second fever peak (3 h later), a circulating endogenous pyrogen can be shown present using passive transfer of plasma into fresh rabbits. This likely represents the in vivo induction of IL-1. In vitro, rTNF alpha induces the release of IL-1 activity from human mononuclear cells with maximal production observed at 50-100 ng/ml of rTNF alpha. In addition, rTNF alpha and rIFN-gamma have a synergistic effect on IL-1 production. The biological activity of rTNF alpha could be distinguished from IL-1 in three ways: the monophasic pyrogenic activity of rIL-1 was destroyed at 70 degrees C, whereas rTNF alpha remained active; anti-IL-1 neutralized IL-1 but did recognize rTNF alpha or natural cachectin nor neutralize its cytotoxic effect; and unlike IL-1, rTNF alpha was not active in the mitogen-stimulated T cell proliferation assay. The possibility that endotoxin was responsible for rTNF alpha fever and/or the induction of IL-1 was ruled-out in several studies: rTNF alpha produced fever in the endotoxin-resistant C3H/HeJ mice; the IL-1-inducing property of rTNF alpha was destroyed either by heat (70 degrees C) or trypsinization, and was unaffected by polymyxin B; pyrogenic tolerance to daily injections of rTNF alpha did not occur; levels of endotoxin, as determined in the Limulus amebocyte lysate, were below the minimum rabbit pyrogen dose; and these levels of endotoxin were confirmed by gas chromatography/mass spectrometry analysis for the presence of beta-hydroxymyristic acid. Although rTNF alpha is not active in T cell proliferation assays, it may mimic IL-1 in a T cell assay, since high concentrations of rTNF alpha induced IL-1 from epithelial or macrophagic cells in the thymocyte preparations. These studies show that TNF (cachectin) is another endogenous pyrogen which, like IL-1 and IFN-alpha, directly stimulate hypothalamic PGE2 synthesis. In addition, rTNF alpha is an endogenous inducer of IL-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Fever: effect of drug-induced antipyresis on survival.

To determine whether the prevention of fever affects the survival of an animal infected with pathogenic bacteria, lizards (Dipsosaurus dorsalis) were infected with live Aeromonas hydrophila and received varying doses of sodium salicylate, an antipyretic drug. Twelve lizards received identical injections of bacteria along with a nontoxic dose of sodium salicylate; five animals increased their mean body temperature at least 0.6 degrees C and survived the week, whereas seven did not develop a fever and died within 3 days. These data indicate that in these lizards the prevention of fever by use of an antipyretic drug such as sodium salicylate increases the mortality rate from bacterial infection.

Multiple biological activities of human recombinant interleukin 1.

Complementary DNA coding for human monocyte interleukin 1 (IL-1), pI 7 form, was expressed in Escherichia coli. During purification, IL-1 activity on murine T cells was associated with the recombinant protein. Homogeneous human recombinant IL-1 (hrIL-1) was tested in several assays to demonstrate the immunological and inflammatory properties attributed to this molecule. hrIL-1 induced proliferative responses in a cloned murine T cell in the presence of suboptimal concentrations of mitogen, whereas no effect was observed with hrIL-1 alone. At concentrations of 0.05 ng/ml, hrIL-1 doubled the response to mitogen (5 X 10(6) half maximal units/mg). Human peripheral blood T cells depleted of adherent cells underwent a blastogenic response and released interleukin 2 in the presence of hrIL-1 and mitogen. hrIL-1 was a potent inflammatory agent by its ability to induce human dermal fibroblast prostaglandin E2 production in vitro and to produce monophasic (endogenous pyrogen) fever when injected into rabbits or endotoxin-resistant mice. These studies establish that the dominant pI 7 form of recombinant human IL-1 possesses immunological and inflammatory properties and acts on the central nervous system to produce fever.

Mechanisms of fever induced by recombinant human interferon.

Since the early trials using human interferon (hIFN) derived from blood leukocytes or cell lines, fever has been a prominent component of IFN therapy. Human protein impurities might account for the fever to cell-derived hIFN, but recombinant hIFN, free of extraneous human proteins, has produced fever in nearly all recipients during clinical trials. Our present studies were carried out to determine the mechanisms of fever due to recombinant hIFN currently being used in humans. Because recombinant hIFN is produced in Escherichia coli, in these experiments we considered contaminating endotoxin as the cause of fever. Polymyxin B, which blocks endotoxin, had no effect on the pyrogenicity of hIFN in rabbits. In addition, hIFN injected into an endotoxin-resistant strain of mice produced fever. The pyrogenicity of hIFN does not appear to involve production of leukocytic pyrogen (LP), since no circulating LP was detected in rabbits during IFN fever. Furthermore, human mononuclear cells incubated with hIFN in vitro at 10(4)-10(6) U/ml did not release LP. However, hIFN stimulated prostaglandin E2 (PGE2) release from rabbit hypothalamic tissue in vitro. Intracerebroventricular injection of hIFN into the awake cat also produced fever and a rise in PGE2 levels in the cerebrospinal fluid; both effects were reversed by treatment with indomethacin. We conclude that the fever of recombinant hIFN is not due to endotoxin but that hIFN is intrinsically pyrogenic by inducing PGE2 in the hypothalamus.

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.

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.

Is prostaglandin E2 involved in the pathogenesis of fever? Effects of interleukin-1 on the release of prostaglandins.

Interleukin-1 (IL-1) induces the formation of PGE2 from monocytes, fibroblasts, muscle cells, and brain tissue by increasing the intracellular concentrations of CA2+; this cation, in turn, activates a phospholipase which cleaves arachidonic acid from either diacylglycerol or a membrane phospholipid. In addition, IL-1 increases the synthesis of cyclooxygenase, as evidenced by the increased conversion of arachidonic acid into prostaglandins after fibroblasts are pre-incubated with IL-1. Evidence is also presented that fever is caused by interleukin-1-induced prostaglandin E2.

Endogenous pyrogen-like substance produced by reptiles.

1. Injection of lizards (Dipsosaurus dorsalis) 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.

Ability of human leukocytic pyrogen to stimulate brain prostaglandin synthesis in vitro.

Fever is thought to be mediated by leukocytic pyrogen (LP), a polypeptide synthesized by phagocytic leukocytes and which is responsible for the upwards resetting of the hypothalamic thermostat. In an attempt to study the effects of LP directly on brain tissue, purified human LP was incubated with rabbit brain slices in vitro. Because of the well-documented role of prostaglandin (PG) synthesis in both the production of fever and antipyresis, PGE levels were measured on the supernates of brain slices incubated 30 min with LP. Levels of PGE increased 3- to 4-fold in rabbit anterior and posterior hypothalami. In addition, PGE levels were similarly increased in temporal cortex slices when exposed to LP. In another set of experiments, PGE levels increased 4- to 5-fold when brain tissue was incubated with a highly purified preparation of bacterial endotoxin (ET). The ability of ET to increase brain PGE levels was not affected by moderate heating (56 degrees C, 30 min), whereas this temperature destroyed the PGE-inducing properties of LP. The antipyretic ibuprofen markedly reduced the amount of PGE measured in the brain slice supernates after stimulation with LP, suggesting that LP brings about synthesis of PGE and not the release of preformed PG. The results demonstrate that LP is a potent inducer of PGE synthesis in rabbit brain and that receptors for LP are not restricted to the thermoregulatory center, but rather may be distributed throughout the brain.

Fever and antipyresis in the lizard Dipsosaurus dorsalis.

Lizards (Dipsosaurus dorsalis) were placed in a desertlike environment in which the ambient temperature (Ta) at night (1800-0600 h) was 12 degrees C and the day (0600-1800 h) Ta was between 30 and 55 degrees C depending on the location within the chamber. When dead Aeromonas hydrophila (4 X 10(9) organisms) was injected into nine lizards, an elevation in body temperature (Tb) of 2.7 degrees C was observed during the same day. On the day after bacterial injection the lizards' body temperatures averaged 41.6 degrees C, an increase of 4.2 degrees C over their control day Tb. Further investigations on the febrile response of D. dorsalis were conducted at the University of Wisconsin's Biotron, where there exists a simulated desert environment with the light intensity, temperature, and humidity closely parelleling a typical spring day in the southwestern desert of the United States (the natural habitat of Dipsosaurus). In this environment injection of dead bacteria into seven lizards led to an average febrile response of similar magnitude (Tb = 40.5 degrees C) but with a longer latency than that found at the University of Michigan. Injection of 13 lizards with live A. hydrophila (5 X 10(9) organism subcut.) in the simulated desert at Michigan led to a daytime fever averaging 2.3 degrees C (mean Tb = 40.6 degrees C) over a 5-day period. During the 6th and 7th day the lizards' body temperature returned to the normal or afebrile level. Injections of sodium salicylate along with dead A. hydrophila resulted in a dose-dependent attenuation of the febrile response. These results demonstrate that the reptilian febrile response is strikingly similar to avian and mammalian fever and suggest a common origin and perhaps function for the febrile mechanism.

Effect of protein synthesis inhibitors on leukocytic pyrogen-induced in vitro hypothalamic prostaglandin production.

In order to study the antipyretic effect of inhibitors of protein synthesis, hypothalamic tissue was incubated in vitro under controlled conditions and the amount of prostaglandin E2 (PGE2) measured in the supernatant medium. Rabbit anterior hypothalamic tissue was incubated with purified human leukocytic pyrogen (LP) and after 60 minutes the supernatant fluid was assayed for PGE2 by radioimmunoassay. Control tissue incubated with Eagle's medium (MEM) released elevated levels of PGE2; however, the addition of polymyxin B (PmxB), a cationic antibiotic which blocks the activities of bacterial endotoxins, significantly reduced PGE2. In addition, endotoxin added to MEM induced from the brain tissue PGE2 production which could be reduced by the addition of PmxB. Thus, commercial culture media such as MEM may contain sufficient amounts of endotoxin to stimulate brain PGE2 production in vitro. Purified human LP incubated with hypothalamic tissue in the presence of PmxB induced PGE2 production in a dose-dependent fashion. This release could be reduced (p less than 0.001) by the presence of either cycloheximide or puromycin during incubation with LP. The addition of these inhibitors to unstimulated hypothalamic tissue incubations did not reduce background levels of PGE2. It is concluded that the antipyretic effect of protein synthesis inhibitors results in a specific decrease in LP-induced levels of PGE2.

Fever: pathogenesis, pathophysiology, and purpose.

Fever appears to have evolved in vertebrate hosts as an adaptive mechanism for controlling infection. This phenomenon is produced by certain exogenous (largely microbial) stimuli that activated bone-marrow-derived phagocytes to release a fever-inducing hormone (endogenous pyrogen). Endogenous pyrogen, in turn, circulates to the thermoregulatory center of the brain (preoptic area of the anterior hypothalamus) where it causes an elevation in the "set-point" for normal body temperature. Warm blooded animals produced fever by increasing heat production (through shivering) or reducing heat loss (by peripheral vasoconstriction), whereas cold blooded animals do so only by behavioral mechanisms (seeking a warmer environment). This paper discusses current concepts that involve the mechanism of endogenous pyrogen production, the role of central transmittors, and the probable function of fever in combating disease.

Pathogenesis of fever in delayed hypersensitivity: role of monocytes.

The present studies were designed to investigate the role of monocytes in the pathogenesis of fever in delayed hypersensitivity. Adherent rabbit blood monocytes (from both normal and sensitized donors) were separated on Ficoll-Hypaque gradients and incubated with antigen (Ag; ovalbumin) and sensitized draining-lymph-node lymphocytes (or their supernatants) from rabbits with delayed hypersensitivity, and release of endogenous pyrogen was assayed. Results indicated that monocytes are activated to produce endogenous pyrogen by Ag and suspensions of draining-lymph-node cells or by an agent (lymphokine) in the supernatants of sensitized lymphocytes preincubated with Ag. The release of lymphokine was Ag specific and was correlated with the skin test reactivity of the donor rabbits to the sensitizing Ag. No evidence was found that Ag-antibody complexes or (in the case of sensitized monocytes) cytophilic antibodies play a role in the activity of this lymphokine which appears to act selectively on monocytes rather than on granulocytes.

Prostaglandin E levels in third ventricular cerebrospinal fluid of rabbits during fever and changes in body temperature.

1. A method was devised to sample cerebrospinal fluid (c.s.f.) from the third ventricle of conscious rabbits.2. Levels of PGE were measured in c.s.f. withdrawn from the third ventricle of rabbits exposed to a variety of manipulations of both brain and body core temperatures which mimicked various facets of fever in these animals. These results were compared to the levels of PGE in the c.s.f. of the same rabbits made febrile by I.V. injections of endogenous pyrogen.3. Levels of PGE in ventricular c.s.f. remained unaltered at 2-3 ng/ml. during exposure to cold, hyperthermia due to heat exposure, hypothalamic cooling or hypothalamic heating, whereas during fever produced by endogenous pyrogen, they rose to an average of 11-12 ng/ml. Furthermore, a significant positive correlation was established between the level of PGE measured in the c.s.f. and the subsequent height of the fever produced by the pyrogen.4. Since production of PGE within the brain is not caused by changes in the brain or body temperatures which are comparable to those observed during fever, and yet greater than fivefold increases in the PGE levels in c.s.f. are produced by I.V. injections of endogenous pyrogen, it is concluded that PGE production in the brain is involved in the pathogenesis of fever.

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.

Effects of fever on host defense mechanisms after infection in the lizard Dipsosaurus dorsalis.

Fever has never been proven beneficial in mammals, although it enhances survival in the lizard D. dorsalis infected with Aeromonas hydrophila. We examined the course of the infection and the function of host defence in febrile (41 degrees) and afebrile (35 degrees or 38 degrees) animals using this model. Infected, febrile lizards had sterile blood cultures, and 1-2 logs fewer bacteria in body tissues 6-12 h after infection. Granulocytes appeared early and in large numbers at the site of inoculation in febrile, but not afebrile, animals. We were unable to demonstrate effects of this small range of temperatures on in vitro growth rates of bacteria, on lizard granulocyte chemotactic or phagocytic functions, or upon serum antibody levels. Our results suggest that fever enhances some aspect of the early inflammatory response, leading to increased leucocyte emigration at the local site and containment of the infection.

Dissimilarities between purified human interleukin-1 and recombinant human interleukin-2 in the induction of fever, brain prostaglandin, and acute-phase protein synthesis.

The lymphokine interleukin-2 (IL-2) has been shown to enhance natural cell-mediated cytotoxicity, the generation of cytolytic T lymphocytes, and several other aspects of cellular immune function. The gene coding for human IL-2 has been cloned, and recombinant IL-2 will be available for clinical trials in patients with neoplastic, infectious, and immunodeficiency diseases. The present investigation was undertaken to determine if IL-2 was similar to interleukin-1 (IL-1) in its ability to induce fever and the acute-phase response. These studies were based on recent work with recombinant human interferon (IFN)-alpha, which is intrinsically pyrogenic and capable of producing fever by inducing the synthesis of prostaglandin E2 (PGE2). The prospect that IL-2 might exert similar physiologic effects is of critical concern since elevated temperature, PGE2, and acute-phase reactants may profoundly inhibit natural cell-mediated cytotoxicity. Our studies have shown that recombinant human IL-2 is not intrinsically pyrogenic in rabbits at doses as high as 10,000 units/kg when administered by a single intravenous injection. In contrast to IL-1, IL-2 does not stimulate cultured hypothalamus cells to synthesize PGE2, and, furthermore, IL-2 does not elevate serum C-reactive protein levels. These results predict that the administration of IL-2 to patients in doses that stimulate cellular immune function will not induce fever and other toxic side effects frequently seen in individuals receiving IFN.

Induction of circulating tumor necrosis factor (TNF alpha) as the mechanism for the febrile response to interleukin-2 (IL-2) in cancer patients.

Fever is frequently observed in cancer patients treated with high-dose recombinant human interleukin-2 (rIL-2). The preincubation of rIL-2 with polymyxin B, an antibiotic that inhibits the biologic effects of endotoxins, did not diminish the pyrogenicity of IL-2 in New Zealand rabbits, indicating that IL-2-induced fever is not due to contaminating endotoxins. In contrast to interleukin-1 (IL-1), tumor necrosis factor (TNF), and interferon alpha, which cause fever through their effects on arachidonic acid metabolism in the hypothalamus, IL-2 was unable to induce prostaglandin E2 synthesis in hypothalamic cells or fibroblasts in vitro, suggesting that IL-2 is not intrinsically pyrogenic. To determine if IL-2-induced fever is mediated indirectly through the generation of pyrogenic cytokines, culture supernatants from IL-2-stimulated human peripheral blood mononuclear cells were screened for the presence of pyrogens by direct injection into rabbits and by measuring the amounts of IL-1 alpha, IL-1 beta, and TNF alpha by specific radioimmunoassays (RIA). All three cytokines were readily detected by RIA in these supernatants, which in turn caused fever when injected into rabbits. Furthermore, in six of six cancer patients treated with rIL-2, elevated levels of TNF alpha were detected in the plasma by RIA 2 hr after IL-2 administration. Plasma TNF levels increased from pretreatment values of 14 +/- 7 to 765 +/- 150 pg/ml 2 hr after an IL-2 injection. These results strongly implicate IL-2-induced pyrogenic cytokines, in particular TNF alpha, as a major cause of the fever and possibly other aspects of the acute-phase response associated with IL-2 therapy.

Studies on the molecular nature of human interleukin 1.

Adherent human blood monocytes were stimulated with heat-killed Staphylococcus albus or Escherichia coli lipopolysaccharide in the presence of 35S-methionine-, [3H]leucine-, or 14C-labeled amino acids. After incubation, interleukin 1 (IL 1) activity in the supernatant medium was purified over an anti-human IL 1 immunoadsorbent followed by gel filtration and chromatofocusing. The purity of the IL 1 was assessed by fluorography of one- and two-dimensional SDS-polyacrylamide gel electrophoresis. Isoelectric and chromatofocusing of low m.w. proteins (less than 20,000 m.w.) revealed three charged 18,000 m.w. species of IL 1 with approximate pI's of 7, 6, and 5, with the most abundant form at pI 7. During the purification procedures, lymphocyte co-mitogenic activity, fever in rabbits, and prostaglandin E2 release from dermal fibroblasts co-eluted in the same fractions. In addition, these fractions were active when injected into endotoxin-resistant C3H/HeJ mice for the production of fever, the induction of serum amyloid A protein, a decrease in serum iron concentration, and an increase in the number of circulating neutrophils. Fluorography revealed homogeneous bands with an m.w. of about 18,000 which correlated with these biological activities. The specific activity of the pI 6 or 5 IL 1, as judged by the ratio of T cell co-mitogenic activity to incorporated radiolabeled amino acid, was at least 10-fold greater than that observed for the pI 7 form. This result suggests that the amino acid compositions of the two 18,000 m.w. acidic forms are unrelated to the pI 7 species. These results also demonstrate that the pI 7 human monocyte IL 1 is the predominant 18,000 m.w. form synthesized and, furthermore, that homogeneous pI 7 IL 1 exhibits multiple biological properties on various tissues by modulating immunologic, inflammatory, metabolic, and neurologic functions. Data are also presented for the existence of a high m.w. (32,000) human pro-IL 1 molecule as the predominant monocytic intracellular form. This pro-IL 1 is degraded artifactually during isolation to lower m.w. forms in the presence of an extracellular serine protease activity. These data are consistent with a model for IL 1 secretion in which pro-IL 1 is first synthesized within the cell and is processed during or after extracellular transport.