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1. The dose-response curve for sustained infusions of leucocyte pyrogen has been demonstrated, and an optimum dose indicated for leucocyte pyrogen clearance experiments. 2. The lungs, liver and small bowel are not significantly involved in removal of leucocyte pyrogen from the circulation in conscious rabbits. 3. A single circulation through one kidney removes up to half of the infused dose of leucocyte pyrogen.
Further studies have been made of a pyrogenic substance which appears in the circulation of rabbits during the course of experimental fever induced by injection of typhoid vaccine. With the use of a passive transfer method and pyrogen-tolerant recipients, the biological properties of this substance have been differentiated from those of the uncleared vaccine in the circulation. The newly identified factor resembles leucocytic pyrogen in the rapidity with which it produces fever and in its failure to exhibit cross-tolerance with bacterial pyrogen. This striking similarity of properties suggests that the circulating factor is of endogenous origin and may arise from cell injury. A close correlation between its presence in the circulation and the existence of fever has been demonstrated. The possible relationship of these findings to the pathogenesis of fever is evident.
The factors present in streptococcal lesion extracts (SLE) which enhanced the lethal and tissue-damaging properties of Gram-negative bacterial endotoxins and streptolysin O were identified with the scarlet fever group of toxins. Toxic manifestations attributed to this group of toxins included lethality, cardiotoxic and other tissue damage, enhancement of toxicity, and pyrogenicity. Of these, the measurement of febrile response in American Dutch rabbits was the most useful parameter of toxicity. In rabbits, repeated daily intravenous injections of 0.125 Lf of a purified erythrogenic toxin immunizes specifically against the pyrogenic activity; this technique was used to type the toxins and to distinguish them from exogenous and endogenous pyrogens; non-specific pyrogens, such as streptococcal endotoxin, were not found in SLE. All types of the Lancefield Group A streptococci tested produced one or or more immunologically distinct toxins in vivo in contrast to Groups B and C which did not produce them; toxins A and B, previously distinguished by neutralization of rash-inducing activity in the skin, were produced in vivo. The A toxin was the most common, as indicated by its presence in extracts prepared with Types 28, 12, 17, and 10 (NY-5); B toxin was found in 10 (NY-5) and 19. A new toxin, designated C, was obtained from a Type 18. In American Dutch rabbits, purified toxin at a concentration of 15 Lf (900,000 STD) neither gave a Dick test nor prepared the skin for the local Shwartzman reaction; by this route, however, in contrast to classical endotoxins, they enhance the lethal and tissue-damaging properties of sublethal doses of these and other toxins. These properties of the immunologic distinct exotoxins as demonstrated in American Dutch rabbits suggest by analogy their importance in the pathogenesis of streptococcal disease in man. Evidence that might implicate them in sequelae, in addition to scarlet fever, is discussed.
The effect of various schedules for inducing tolerance to bacterial endotoxin in donor rabbits upon suitability for demonstration of passive transfer of tolerance to pyrogenicity in normal recipients has been investigated. Long-term treatment of donors, through 5 weeks, is no more effective than a brief series of injections, adding further evidence that tolerance is not attributable to specific antibody to the endotoxin. Qualitative differentiation of the febrile pattern of passively tolerant recipients from that seen in control animals depends upon the magnitude of the test dose of pyrogen. Passively tolerant rabbits respond to endotoxin with an acute leucopenia equivalent to that seen in controls suffering a full biphasic fever. Animals given daily injections of endotoxin continue to show the acute leucopenia, despite the early modification of the course of fever characteristic of endotoxin tolerance. The assumption that the leucopenia reflects damage to the leucocytes, with release of endogenous pyrogen, is not consistent with these findings. Rabbits rendered leucopenic by nitrogen mustard and then given endotoxin exhibit a rapidly developing fever of greater than normal intensity, the exaggeration of the febrile response being proportional to the severity of the induced leucopenia. The implications of these findings for the pathogenesis of endotoxin-induced fever are discussed. The evidence supports the hypothesis that endotoxin produces fever by direct action rather than by release of endogenous leucocytic pyrogen. It is postulated that the lesser fever, in animals having normal numbers of circulating leucocytes, reflects a limitation of available endotoxin by the known rapid sequestration in the white blood cells at the time of the acute leucopenia. It is further suggested that the biphasic febrile response of the normal rabbit results from reinoculation of the blood stream by the temporarily sequestered endotoxin, the RES of the tolerant animal clearing the released endotoxin at a rate sufficient to prevent triggering the second phase of fever.
Rabbits made granulocytopenic with nitrogen mustard have been shown to generate serum endogenous pyrogen when given a fever-producing dose of bacterial endotoxin. This finding is in accord with the hypothesis that endogenous pyrogen plays a central role in the pathogenesis of endotoxin fever. The fact that leucopenic animals produce less serum-endogenous pyrogen than normal animals given the same dose of endotoxin has also been confirmed and suggests that polymorphonuclear leucocytes constitute a major source of the endogenous pyrogen which is demonstrable in the circulation during endotoxin fever.
Fever can be elicited in the rabbit by the intravenous administration of relatively large doses of a synthetic immunoadjuvant, N-acetylmuramyl-L-alanyl-D-isoglutamine, or muramyl dipeptide (MDP). This response could be mediated by endogenous pyrogen because MDP has been shown to induce their production both in vivo and in vitro. The results reported here show that intracisternal injection of minute amounts of MDP could elevate fever without activating the release of endogenous pyrogen in the plasma or in the cerebrospinal fluid. Moreover, indomethacin inhibited hyperthermia produced by intracerebroventricular administration of MDP. Therefore, our findings argue in favor of a direct effect of the glycopeptide on the thermoregulatory centers besides its indirect effect through the production of leukocytic pyrogen. This molecule apparently represents the minimal requirement for the pyrogenicity of bacterial peptidoglycan because administration, even by the intracerebral route, of a mixture of muramic acid and of its dipeptide moiety did not elicit fever.
The role of lymphocytes in the pathogenesis of fever was investigated by stimulating human blood mononuclear cells in a two-way mixed leukocyte reaction (MLR). After 2-7 d of incubation, MLR supernates contained a factor that was not pyrogenic itself when injected into rabbits; however, these supernates, when incubated with human blood monocytes from a third donor, induced the synthesis of LP. The pyrogen-inducing activity was stable at 56 degrees C, destroyed at 70 degrees C, and was neither dialyzable nor removable by adsorption by anti-human leukocytic pyrogen (LP) attached to Sepharose 4B. Production of this factor was not always correlated with increased thymidine incorporation in the MLR. Its production was absent when peripheral lymphocytes were purified over nylon wool. The concentration of mononuclear cells in the MLR varied from 5 X 10(5) to 5 X 10(6)/ml in round-bottomed tubes. Under the latter conditions, some donor cells produced this factor without stimulation in the MLR culture, but when these cells were cultured on flat-bottomed containers at low cell concentration, autologous production was not observed. These experiments demonstrate the production of a human lymphocyte factor (lymphokine) that induces LP synthesis. This pyrogen-inducing lymphokine may be important in the pathogenesis of fever in certain immunologically mediated diseases.
Several groups of streptococci were tested for production of pyrogenic exotoxins (SPE) with Ouchterlony immunodiffusion, a newly developed passive hemagglutination inhibition assay, and an assay for pyrogenicity and capacity to enhance lethal endotoxin shock. With use of these assays, 68 (91%) of 75 group A streptococcal strains were positive for one or more of SPE types A, B, and C; seven were negative for both the known SPE types and antigenically unrelated pyrogenic exotoxins. Group A strains producing both SPE B and C were the most common, and strains producing A alone or AB and AC together were the least common. All of 11 rheumatogenic group A streptococci elaborated SPE C either alone or together with one or both of SPE types A and B. The 10 nephritogenic strains tested were positive for SPE B; five were positive for B alone. In contrast to group A streptococci, non-group A strains (41 tested) did not produce the known SPE types, and 19 of 19 tested were negative for antigenically unrelated pyrogenic exotoxins. Group A strains from Holland, India, and Japan also elaborated SPE. Several group A streptococci used widely in laboratory experiments were tested for SPE types produced.
A novel in-vitro system has been developed for the detection and quantification of pyrogen in pharmaceutical products. The measured variable was evoked secretion of the pyrogenic cytokine interleukin-6 from MONO MAC 6 monocytic cells incubated with the product. The interleukin-6 was detected using a specific and sensitive ELISA developed for this purpose. The test system detected pyrogenic contamination in 3 batches of therapeutic human serum albumin which had caused adverse reactions in recipients. The contamination was not detected in conventional tests: the rabbit pyrogen test and the limulus amoebocyte lysate test.
1. The intravenous injection into rabbits of 25-50 mug of a preparation of cell-wall mucopeptide (SM) from group A streptococcus produced a rise in rectal temperature and a fall in the number of circulating granulocytes in the blood.2. The pyrogenic response to SM is not dependent on the circulating granulocytes since it occurred also in rabbits made leucopenic by nitrogen mustard (HN 3).3. The pyrogenic response to SM was enhanced after an intravenous injection of 5-hydroxytryptophan (5-HTP).4. Pre-treatment with reserpine caused a much greater attenuation of the pyrogenic response to SM than pre-treatment with alpha-methyl-para-tyrosine (AMPT).5. As the reserpine reduced the 5-hydroxytryptamine (5-HT) and noradrenaline level in the hypothalamus, whereas the AMPT reduced only the noradrenaline level, it is concluded that 5-HT may be involved in the pyrogenic response and the way in which 5-HT may exert this function is discussed.
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.
Streptococcal pyrogenic exotoxin (SPE) isolated from culture filtrates of strain NY-5 (type 10), and separated from other extracellular by differential solubility in ethanol and acetate-buffered saline, has previously been shown to exhibit a wide range of biological activities including erythrogenic activity, pyrogenicity, enhancement of susceptibility to endotoxin shock, blockage of the reticuloendothelial system immmunosuppression, and lymphocyte mitogenicity. Toxin prepared in this way was found to consist of hyaluronic acid and several proteins which could be distinguished by thin-layer polyacrylamide isoelectric focusing (IEF), SPE has been further purified by ion exchange chromatography on QAE-Sephadex columns. One of the fractions isolated from QAE-Sephadex, and shown to be a homogenous protein by thin-layer IEF and Ouchterlony with hyperimmune serum, was highly active erythrogenically, pyrogenically, and in enhancing susceptibility to endotoxin. This fraction was identified as exotoxin A. A second, less active fraction identified as SPE B showed similar activities, but differed from the other fraction antigenically and in net charge and molecular weight. These findings indicate that a single highly purified protein can mediate at least three of the biological activities attributed to SPE and NY-5 produces pyrogenic exotoxins A and B in vitro as well as in vivo.
Group A streptococcal pyrogenic exotoxin (SPE) type C was partially purified by differential solubility in ethanol and acetate-buffered saline. Toxin prepared in this way consisted of protein and hyaluronic acid. After removal of hyaluronic acid, the toxin remained pyrogenic, enhanced susceptibility of rabbits to letahl endotoxin shock, was stable when treated with acid, base, or pepsin, but was inactivated by heat. Toxin further purified by thin-layer isoelectric focusing was pyrogenic and enhanced the susceptibility of rabbits to lethal endotoxin shock. Purified type C toxin appeared homogeneous when tested by Ouchterlony immunodiffusion and migrated as a single protein band in isoelectric focusing polyacrylamide gels (isoelectric point, 6.7) and sodium dodecyl sulfate-polyacrylamide gels (molecular weight, 13,200). The purified toxin was antigenically distinct from A and B SPE, and antisera raised against the purified toxin neutralized pyrogenic activity. The amino acid composition was determined.
Rabbit polymorphonuclear leukocytes were purified from rabbit blood by centrifugation on colloidal silica gradients followed by sedimentation in 4% Ficoll. The purified neutrophils had normal random motility, responded to chemotactic stimuli, phagocytosed zymosan particles, made superoxide, and phagocytosed and killed bacteria. However, they did not secret endogenous pyrogens either spontaneously or in response to stimulation with endotoxin, polyinosine:polycytosine, or muramyl dipeptide. Macrophages isolated on the same gradients secreted some pyrogen spontaneously and secreted considerably more in response to the same three stimuli. This evidence reinforces the idea that macrophages are the only source of endogenous pyrogens, and that pyrogens secreted by cell populations that are rich in neutrophils are to be attributed to the monocytes or macrophages that the cell populations contain.
We evaluated cross-reactivity of antibodies against staphylococcal and streptococcal pyrogenic toxins. Monoclonal antibodies against staphylococcal enterotoxin (ET) C1 and streptococcal pyrogenic exotoxin (SPE) A were tested for reactivity with homologous and heterologous pyrogenic toxins in vitro. Ten immunoglobulin G1 anti-ET C1 monoclonal antibodies showed little or no cross-reactivity in an enzyme-linked immunosorbent assay, but many of these could neutralize the mitogenic effect of ET B, SPE A, or both. Two immunoglobulin M anti-ET C1 monoclonal antibodies and eight immunoglobulin M anti-SPE A monoclonal antibodies showed extensive cross-reactivity in the enzyme-linked immunosorbent assay and the mitogenicity neutralization assay. No cross-reactivity was observed with SPE C or toxic shock syndrome toxin 1. Rabbits immunized against ET B, ET C1, or SPE A were resistant to challenge with the immunizing toxin. In addition, reciprocal immunity was stimulated by the two ETs, and immunity to SPE A provided protection against ET B but not ET C1. These results show that staphylococcal and streptococcal pyrogenic toxins which share sequence homology have common antigenic determinants which may not be detected in Ouchterlony immunodiffusion assays.
The pyrogenic response to supernatant fluids obtained from human peripheral blood mononuclear cells (PBMC) stimulated with staphylococcal enterotoxin A (SEA) was characteristic of a response to an endogenous pyrogen in that it was brief and monophasic and was destroyed by heating supernatant fluids at 70 degrees C for 30 min. The febrile responses were in parallel with the levels of interleukin-1 (IL-1), tumor necrosis factor (TNF), interferon-gamma (IFN-gamma), IL-2, and IL-6 in supernatant fluids obtained from PBMC treated with SEA. Both the pyrogenicity and the levels of IL-1, TNF, IFN-gamma, IL-2, and IL-6 in supernatant fluids started to rise at 6 to 18 h and reached their peak levels at 24 to 96 h after SEA incubation. Both the fever and the increased levels of IL-1, TNF, IFN-gamma, IL-2, and IL-6 in supernatant fluids obtained from the SEA-stimulated PBMC were decreased by incubating SEA-PBMC with anisomycin (a protein synthesis inhibitor), aminoguanidine (an inhibitor of inducible nitric oxide synthase [NOS]), or dexamethasone (an inhibitor of NOS). The febrile response to supernatant fluids obtained from the SEA-stimulated PBMC was attenuated by adding either anti-IL-1beta, anti-TNF-alpha, or anti-IFN-gamma monoclonal antibody (MAb) to supernatant fluids. The antipyretic effects exerted by anti-IL-1beta MAb were greater than those exerted by anti-TNF-alpha or anti-IFN-gamma MAb. The data suggest that SEA acts through the NOS mechanisms in PBMC to stimulate synthesis of pyrogenic cytokines (in particular, the IL-1beta).
N-acetylmuramyl-L-alanyl-D-isoglutamine, or muramyl dipeptide (MDP), is a synthetic immunoadjuvant analogue of a bacterial peptidoglycan subunit that has a definite pyrogenic effect in the rabbit. Some adjuvant-active derivatives such as murabutide [MDP(Gln)-OnBu] or murametide [MDP(Gln)-OMe] are not pyrogenic. Murabutide did not stimulate human or rabbit cells to release endogenous pyrogen (EP), but murametide induced EP production at the same dosage levels as MDP. Moreover, plasma from rabbits treated with murametide transferred into untreated recipients elicited a febrile response typical of EP fever and comparable with that induced by plasma from MDP-treated animals. Murametide not only inhibited the central effect of EP that is generated but also the effect of an extra dose of EP administered later by the intravenous route. Moreover, pretreatment of rabbits with murametide decreased fever responses induced by certain high-molecular-weight exogenous pyrogens as mediated through the production of EP.
It has been proposed that pyrogens may produce their febrile response by the release of prostaglandins in the hypothalamus. To test this theory, prostaglandin E1 (PGE1) was injected into a lateral ventricle in dosages of 2-200 ug into conscious newborn lambs, ages 4-168 h. Fiifteen of 40 injections were followed by rises in rectal temperature but the remainder were followed either by no change or by falls. Temperature responses did not appear to be related to age and a variation in responses to the same dosage of PGE was often observed. Some lambs were able to develop fevers in response to intravenous bacterial pyrogen yet did not develop fever after intraventricular PGE 1. Intraventricular bacterial pyrogen (3 ng) produced no change in body temperature, whereas three of four injections of 300 ng pyrogen caused fever. The results suggest that the newborn lamb may be able to develop a fever independently of the central involvement of PGE1. Alternatively, the intraventricular approach may not be useful for the study of the central control of body temperature in the newborn lamb.