STUDIES ON THE PATHOGENESIS OF FEVER : VII. PRELIMINARY CHEMICAL CHARACTERIZATION OF LEUCOCYTIC PYROGEN

Study of the chemical properties of the pyrogenic component of rabbit polymorphonuclear leucocytes reveals it to contain an essential, non-dialyzable protein which: (a) is precipitated by perchloric acid, (b) is removed by extraction with phenol, (c) is soluble in 50 per cent methanol and 33 per cent saturated ammonium sulfate, and (d) is destroyed by the proteolytic action of both trypsin and pepsin. By combined chemical and chromatographic techniques the leucocytic pyrogen has been purified approximately 50-fold. The partially purified material contains less than 1 per cent carbohydrate, is resistant to periodate oxidation, is unaffected by extraction with butanol and contains at least two immunologically active components when tested by the Ouchterlony gel-diffusion technique. Its chemical properties distinguish it from other known pyrogenic substances which have been implicated in the pathogenesis of fever.     

STUDIES ON THE PATHOGENESIS OF FEVER : XI. QUANTITATIVE FEATURES OF THE FEBRILE RESPONSE TO LEUCOCYTIC PYROGEN

Although the absolute febrile responses of trained individual rabbits injected intravenously with small to moderate doses of leucocytic pyrogen vary over an appreciable range, the relative responses of each rabbit to changes in dosage are satisfactorily reproducible. The quantitative dose-response relationship is characterized by a hyperthermic ceiling at which the intensity of the febrile reaction is relatively constant over a wide dosage range. Only at lower dose levels, where the dose-response curve is reasonably steep, is the magnitude of the fever produced proportional to the amount of pyrogen injected. When sufficiently large doses of LP are injected, the hyperthermic ceiling is exceeded. The fevers thus induced are biphasic in character and, in this way, resemble the usual response to bacterial endotoxin. Similar biphasic fevers result from continuous infusions of relatively low concentrations of LP at a constant rate. Repeated intermittent injections of moderate doses of LP likewise cause prolonged biphasic fevers, but, once the fever has become established, the reaction to each individual injection becomes markedly depressed. When large doses of LP are injected at daily intervals, the characteristic biphasic response occurs only following the first injection. Thereafter a state of tolerance intervenes in which the late secondary rise in temperature fails to occur. This form of tolerance lasts as long as the daily injections are continued but subsides within a few days after the injections are stopped. During the transient tolerance the rabbit's responsiveness to small doses of LP (in the sensitive range of the dose response curve) is depressed. In addition, the amount of endogenous pyrogen mobilized from the tissues by a large dose of LP is not as great as that generated in a normal rabbit. The relations of these findings to biphasic fevers, tolerance, and the accuracy of the conventional method of pyrogen assay are briefly discussed.     

STUDIES ON THE PATHOGENESIS OF FEVER : IX. THE PRODUCTION OF ENDOGENOUS PYROGEN BY POLYMORPHONUCLEAR LEUCOCYTES

Determination of the dose-response curve for rabbit leucocytic pyrogen reveals a hyperthermic "ceiling" at which there is a marked insensitivity to dosage. This finding has important implications in relation to the quantitative assay of leucocytic pyrogen. Polymorphonuclear leucocytes separated from normal rabbit blood possess the capacity to produce less than 5 per cent of the pyrogen generated by the same number of rabbit granulocytes collected from acute peritoneal exudates. Blood granulocytes, separated in the cold from the buffy coat, contain no detectable preformed pyrogen. The amount of preformed pyrogen within exudate granulocytes represents but a small fraction of the pyrogen which the cells are capable of generating when incubated in normal saline at 37°C. It is suggested that the active pyrogen is formed from an inactive precursor within the cells. Under the conditions tested, cell fragments of rabbit granulocytes fail to produce endogenous pyrogen. The fact that the production of pyrogen is blocked at 4°C is in keeping with the hypothesis that it involves metabolic reactions within the cell.     

STUDIES ON THE PATHOGENESIS OF FEVER : XV. THE PRODUCTION OF ENDOGENOUS PYROGEN BY PERITONEAL MACROPHAGES

Macrophages from oil-induced peritoneal exudates in rabbits produce endogenous pyrogen when first activated by incubation in 4 hr exudate fluid and then stimulated by incubation in potassium-free isotonic sodium chloride solution. The failure of earlier investigators to obtain pyrogen from macrophages is explained, and the relevance of macrophage pyrogen to fevers of agranulocytosis and other diseases, in which mononuclear rather than granulocytic exudates predominate, is discussed.     

STUDIES ON THE PATHOGENESIS OF FEVER : XVI. PURIFICATION AND FURTHER CHEMICAL CHARACTERIZATION OF GRANULOCYTIC PYROGEN

Small quantities of highly purified granulocytic pyrogen have been separated from contaminating proteins by disc electrophoresis in polyacrylamide gel. The biologically active material thus isolated was shown to be electrophoretically homogeneous at pH 9 and pH 3.8. Earlier work on the chemical properties of the pyrogen molecule has been extended to include: (a) estimation of its molecular weight by gel filtration; (b) demonstration of free sulfhydryl groups essential for its biological activity; and (c) evidence that it is not inactivated by exhaustive extraction with ethanolether or n-heptane.     

STUDIES ON THE PATHOGENESIS OF FEVER : XIII. THE EFFECT OF PHAGOCYTOSIS ON THE RELEASE OF ENDOGENOUS PYROGEN BY POLYMORPHONUCLEAR LEUCOCYTES

1. Phagocytosis promotes the release of endogenous pyrogen from polymorphonuclear leucocytes. 2. The release of pyrogen, though initiated by the phagocytic event, is not synchronous with it. 3. The postphagocytic release mechanism is not inhibited by sodium fluoride and, therefore, appears not to require continued production of energy by the cell. 4. The release process, on the other hand, is inhibited by arsenite, suggesting the participation of one or more sulfhydryl-dependent enzymes in the over-all reaction. 5. Particle for particle, the ingestion of heat-killed rough pneumococci causes the release of approximately 100 times as much pyrogen as the ingestion of polystyrene beads of the same size. 6. The pyrogen release mechanism of polymorphonuclear leucocytes separated directly from blood, unlike that of granulocytes in acute inflammatory exudates, is not readily activated by incubation of the cells in K-free saline. Despite this difference, both blood and exudate leucocytes following phagocytosis release large amounts of pyrogen, even in the presence of K⁺. The fact that the postphagocytic reaction is uninhibited by the concentrations of K⁺ which are present in plasma and extracellular fluids, suggests that this mechanism of pyrogen release may well operate in vivo. 7. As might be expected from the foregoing observations, the intravenous injection of a sufficiently large number of heat-killed pneumococci causes fever in the intact host. Intravenously injected polystyrene beads, on the other hand, are significantly less pyrogenic. Evidence is presented to support the conclusion that the fever in both instances is caused by pyrogen released from the circulating leucocytes which have phagocyted the injected particles. 8. The possible relationships of these findings to the pathogenesis of fevers caused by acute bacterial infections are discussed.     

STUDIES ON THE PATHOGENESIS OF FEVER : XIV. FURTHER OBSERVATIONS ON THE CHEMISTRY OF LEUKOCYTIC PYROGEN

Leukocytic pyrogen previously reported to contain an essential protein moiety, appears to be a lipid-protein complex having a molecular weight in the range of 10,000 to 20,000. Evidence that it contains essential lipid includes its inactivation by Cu⁺⁺, its lability in alkaline solutions (pH 8.5 and above), and its loss of pyrogenicity when extracted with acid-isooctane. Its solubility in 66% methanol, and the enhancing action of ethanol in freeing it from sonicated cells, suggest the presence of exposed lipid groups at its surface. Once the complex is separated from other proteins, its biological activity is readily destroyed. Although the lipid component is presumed to contain unesterified fatty acid(s), its precise composition is unknown. The finding of lipid in the active complex is in keeping with the hypothesis that the pyrogen is derived from leukocytic membranes.     

STUDIES ON THE PATHOGENESIS OF FEVER : VIII. FURTHER OBSERVATIONS ON THE ROLE OF ENDOGENOUS PYROGEN IN ENDOTOXIN 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.     

STUDIES ON THE PATHOGENESIS OF FEVER : XVII. THE CATIONIC CONTROL OF PYROGEN RELEASE FROM EXUDATE GRANULOCYTES IN VITRO

Evidence has been presented that the release of active endogenous pyrogen from rabbit exudate granulocytes incubated in isotonic NaCl is a relatively prompt energy-dependent process that is preceded by a rise in intracellular pyrogen, and involves a rise in total intracellular cations and an increased permeability of the cell membranes, but does not require the synthesis of new proteins.     

STUDIES ON THE PATHOGENESIS OF FEVER : XII. ELECTROLYTIC FACTORS INFLUENCING THE RELEASE OF ENDOGENOUS PYROGEN FROM POLYMORPHONUCLEAR LEUCOCYTES

The metabolic reactions responsible for the release of endogenous pyrogen from rabbit granulocytes incubated in 0.15 M NaCl are specifically inhibited by the presence of K⁺ (and by related alkali metal ions, Rb⁺ and Cs⁺) in the medium. The inhibitory action of K⁺ apparently involves penetration of the cell membrane and is directly antagonized by the cardiac glycoside, ouabain. It is concluded, therefore, that the inhibition of pyrogen release by extracellular K⁺ is due to transport of K⁺ into the cell. Although the precise molecular mechanisms which are responsible for the release of pyrogen from granulocytes incubated in K-free saline have not been elucidated, further study of the process has revealed: (a) that it is preceded by the accumulation of pyrogen within the cell, (b) that it depends upon the catalytic action of one or more sulfhydryl-containing enzymes, (c) that it does not require energy, either from glycolysis or from reactions depending on molecular oxygen, and (d) that its inhibition by K⁺ and by arsenite is qualitatively similar to the depression caused by these same reagents on the release of other leucocytic proteins; i.e., lysozyme and aldolase.     

STUDIES ON THE PATHOGENESIS OF FEVER : VI. THE INTERACTION OF LEUCOCYTES AND ENDOTOXIN IN VITRO

Study in vitro of the interaction of bacterial endotoxin with rabbit polymorphonuclear leucocytes has resulted in the following findings: 1. Incubation of endotoxin and leucocytes in saline results in: (a) the release of leucocytic pyrogen, and (b) the inactivation of endotoxin. 2. Cell-free extracts of leucocytes also inactivate endotoxin. 3. Incubation of leucocytes in "physiological" saline causes rapid discharge of leucocytic pyrogen. In contrast, relatively little pyrogen is released by leucocytes incubated in fresh serum. 4. The release of leucocytic pyrogen in serum is markedly stimulated by the presence of endotoxin. 5. Leucocytes obtained from tolerant rabbits interact with endotoxin in essentially the same manner as leucocytes from normal rabbits. The pertinence of these findings to the pathogenesis of fever and to related information concerning human leucocytes has been discussed.     

STUDIES ON TUBERCULIN FEVER : II. OBSERVATIONS ON THE ROLE OF ENDOGENOUS PYROGEN IN TOLERANCE

Certain characteristics of tolerance which develops to the pyrogenic effects of old tuberculin (OT) in BCG-vaccinated rabbits have been described. Rabbits made tolerant by several injections of OT lost their ability to produce detectable amounts of endogenous pyrogen (EP) in response to the specific agent (OT) but mobilized normal amounts of EP when given a small unrelated stimulus. On the other hand, when this stimulus followed shortly after an initial tuberculin fever of sufficient magnitude, release of additional EP was suppressed, presumably due to an inhibitory effect of the EP previously mobilized by tuberculin. Similarly, a single large dose of endotoxin almost completely suppressed the response of sensitized rabbits to OT given several hours later. Since several spaced injections of the same dosage were ineffective, this phenomenon does not appear to be attributable to the known mechanisms by which endotoxins promote non-specific resistance to toxicity and infection. Tolerance to tuberculin could not be definitely shown following an injection of Newcastle disease virus which also produces a circulating EP, and it has been inferred that endotoxin blocks the pyrogenic action of antigen on host tissues directly rather than through mobilizing EP. On the basis of these observations, the relationship of specific to non-specific tolerance to tuberculin fever has been compared in terms of the ability of such tolerant animals to mobilize EP to heterologous stimuli and it is concluded that the two forms of tolerance are different. Furthermore, the fact that a number of unrelated agents produce tolerance non-specifically supports the concept that there may be a common source of EP released by a number of stimuli, including endotoxins and myxoviruses, as well as antigen in specifically sensitized hosts.     

STUDIES ON THE PATHOGENESIS OF FEVER : XX. SUPPRESSION AND REGENERATION OF PYROGEN-PRODUCING CAPACITY OF EXUDATE GRANULOCYTES

Suppression of the pyrogen-producing capacity of exudate granulocytes results from incubation of the cells in plasma, serum, or Ringer's solution. When transferred in this state and incubated in isotonic NaCl, the cells release much less pyrogen than untreated exudate cells. The suppressive effect is reversible and appears to involve the cellular uptake of calcium ions. In contrast, regeneration of pyrogen-producing capacity in depleted exudate cells occurs only when the cells are incubated in serum. The process resembles activation and requires the cellular synthesis of protein.     

STUDIES ON THE EFFECT OF CERTAIN MACROMOLECULAR SUBSTANCES ON THE RESPIRATORY ACTIVITY OF THE LEUCOCYTES OF PERIPHERAL BLOOD

The oxygen, uptake of lightly heparinized human blood was found to increase markedly upon the addition of bacterial endotoxins, soluble antigen-antibody complexes, or polystyrene latex particles. The effect apparently reflects a transitory stimulation of respiratory activity of the blood leucocytes. The effect did not occur in the presence of anticoagulant amounts of citrate or ethylenediaminetetraacetate, was inhibited by iodoacetate and fluoride ions, and may be related to the energy-yielding processes involved in histamine release or phagocytosis.     

STUDIES ON THE MECHANISM OF ENDOGENOUS PYROGEN PRODUCTION : III. Human Blood Monocytes

The characteristics of pyrogen production and release by human blood monocytes were investigated. A dose-response assay of monocyte pyrogen in rabbits indicated a linear relationship of temperature elevation to dose of pyrogen at lower doses. Monocytes did not contain pyrogen when first obtained, nor did they release it spontaneously even after 5 days of incubation in vitro. Pyrogen production was apparent 4 h after stimulation by endotoxin or phagocytosis, and continued for 24 h or more. Puromycin, an inhibitor of protein synthesis, prevented both initiation and continuation of pyrogen production and release. Pyrogen-containing supernates retained most pyrogenic activity during overnight incubation even in the presence of activated cells. Lymphocytes appeared to play no role in either initiation or continuation of pyrogen production in these studies.     

STUDIES ON THE PATHOGENESIS OF STAPHYLOCOCCAL INFECTION : III. THE EFFECT OF TISSUE NECROSIS AND ANTITOXIC IMMUNITY

Necrosis of rabbit skin produced by thermal injury was found to result in a striking increase in local infectivity of staphylococci that were coagulase-positive and hemolytic, but no local increase in the infectivity of non-pathogenic staphylococci. Infection produced in necrotic burns extended beyond the area of burn and was characterized by hemorrhage, edema, and necrosis of contiguous normal skin. Such infections, however, never resulted in bacteriemia or metastatic abscesses, and there was no effect of the necrotic burn upon the infectivity of staphylococci injected into normal skin of the burned animal. Recovery of rabbits from severe burn infections was associated with the development of high titers of serum antibody to the alpha hemolysin or dermonecrotoxin of the staphylococcus. Thirty to 100 days after the initial burn infection, it was found that rabbits could no longer be infected in a necrotic burn, although infection induced in normal skin of these resistant animals was no different from that in normal rabbits. Immunity to infection by pathogenic staphylococci in necrotic burns could be induced by vaccination with potent alpha hemolysin toxoid, and this immunity was passively transferable with rabbit antiserum. No strain specificity was detected for this immunity in that immunization with toxoid prepared from bacteriophage type 52/42B/80/81 staphylococci protected animals against infection in a necrotic burn by other typable and non-typable staphylococci. Histopathological study of infected necrotic burns in normal rabbits showed extensive necrosis, hemorrhage, edema, and many masses of bacteria but leucocytic infiltration was observed only at the margin of the infection. In contrast, the infected necrotic burns in animals immunized with alpha hemolysin toxoid showed few bacteria and marked leucocytic infiltration throughout the burn. These experiments have, therefore, demonstrated a significant immunity to infection by pathogenic staphylococci in necrotic tissue but not in normal skin, associated with serum antibody to the alpha hemolysin or dermonecrotoxin of the bacteria. The implications of these findings are discussed.     

STUDIES ON THE PATHOGENESIS OF STAPHYLOCOCCAL INFECTION : IV. THE EFFECT OF BACTERIAL ENDOTOXIN

Intracutaneous and intravenous injection of pyrogenic, non-lethal doses of bacterial endotoxin were found to increase the infectivity of pathogenic but not non-pathogenic staphylococci in rabbit skin. The increased infectivity of the microorganism was characterized by accelerated multiplication at the site of inoculation and by the production of necrosis and hemorrhage locally. Histologically, the infection of skin in endotoxin-prepared animals was characterized by necrosis, masses of bacteria, but absence of leukocytic infiltration into the area of bacterial growth. The infectivity of staphylococci in skin of endotoxin-prepared rabbits could be controlled by antibody to the alpha hemolysin of the microorganism. The effect of endotoxin upon staphylococcal infection was demonstrable only within 4 hours after injection of the endotoxin. It could not be prevented with chlorpromazine or dibenamine and was closely related to the effect of endotoxin upon leukocytes. It was suggested that the effect of endotoxin upon leukocytes was probably responsible for its influence upon staphylococcal infection. The implications of these findings in the pathogenesis of staphylococcal infection are discussed.     

STUDIES ON THE PATHOGENESIS OF STAPHYLOCOCCAL INFECTION : II. THE EFFECT OF NON-SPECIFIC INFLAMMATION

Studies have been described in which the effect of early and late or established inflammation, upon staphylococcus infection of rabbit skin has been evaluated. Inflammation was produced in skin by thermal, chemical, bacterial and immunological injury, and it was found that the area of inflammation was more susceptible to staphylococcus infection than was normal skin if the bacteria were injected within 2 to 3 days after the injury. When staphylococci were injected into an area of inflammation of over 3 days' duration, there appeared to be an increase in local resistance to infection. The way in which inflammation was produced seemed to have a little influence upon the effects observed. This influence of non-specific inflammation upon staphylococcus infection was compared with the influence of specific bacterial hypersensitivity, which also is associated with an increase in infectivity of the microorganism in sensitized animals. It was concluded that specific bacterial hypersensitivity probably increases susceptibility to infection with the staphylococcus in the same way as non-specific inflammation. The general significance of non-specific inflammation upon infection is also discussed.     

STUDIES ON TUBERCULIN FEVER : III. MECHANISMS INVOLVED IN THE RELEASE OF ENDOGENOUS PYROGEN IN VITRO

In a search for the source of the circulating endogenous pyrogen (EP) that mediates tuberculin-induced fever, tuberculin was incubated in vitro with various tissues of rabbits sensitized by intravenous infection with BCG. Evidence was obtained that tuberculin specifically stimulates cells in the blood of sensitized rabbits to generate pyrogen in vitro, whereas both lymph node and spleen cells from the same donors were inactive. Since normal blood cells, incubated in plasma of sensitized donors, were similarly activated, it is postulated that circulating antibodies play a role in sensitizing cells (presumably granulocytes) to release pyrogen on contact with tuberculin) both in vitro and in vivo. Release of endogenous pyrogen in vitro may be a sensitive means of detecting immunologic reactions between antigen and specifically sensitized blood cells-in other allergic states accompanied by fever.     

STUDIES ON THE PATHOGENESIS OF STAPHYLOCOCCAL INFECTION : I. THE EFFECT OF REPEATED SKIN INFECTIONS

The influence of repeated staphylococcal infection of rabbit skin upon the characteristics of the experimentally induced lesion was studied. It was found that the repeated infection was associated with the development of delayed hypersensitivity unaccompanied by the appearance of demonstrable serum antibody. The delayed hypersensitivity to the staphylococcus resulted in an increased infectivity of the organism in skin of the sensitized animal, characterized by intensification of the lesions seen with large bacterial inocula and the induction of abscesses with inocula incapable of producing any lesion in normal rabbit skin. Similarly, the severity of experimentally induced pyoarthrosis was greater in sensitized than in normal rabbits. Induction of delayed hypersensitivity by vaccination of rabbits with washed heat-killed staphylococci resulted in the same increased severity of the infection and an increase in infectivity of the microorganism. In contrast to the observations of cutaneous and joint infection, the sensitized animals appeared to be less susceptible to severe infection of the anterior chamber of the eye. The role of immunity and hypersensitivity in staphylococcal infection is discussed and the possibility that non-specific inflammation may influence staphylococcal infection in the same way as specific hypersensitivity is indicated. Studies to further elucidate this are presented in the following pages.