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 : 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 : 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 : 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 : 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 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 : 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 : 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 : X. THE EFFECT OF CERTAIN ENZYME INHIBITORS ON THE PRODUCTION AND ACTIVITY OF LEUCOCYTIC PYROGEN

The production of endogenous pyrogen by intact granulocytes obtained from acute peritoneal exudates is blocked by arsenite, iodoacetate, p-chloromercuribenzoate, and N-ethylmaleimide in concentrations of 2 x 10^–4 M. When the concentration of these sulfhydryl-reactive enzyme inhibitors is increased to 2 x 10^–2 M, only the iodoacetate inactivates the pyrogen molecule, whereas the arsenite, the p-chloromercuribenzoate, and the N-ethylmaleimide have no gross effect upon its thermogenic activity. Both diisopropyl fluorophosphate and dinitrofluorobenzene are even more potent inactivators of the pyrogen molecule than iodoacetate, although the action of the DFP cannot be blocked or reversed by known antagonists such as 2-pyridine aldoxime methiodide and hydroxylamine. Proteolytic enzymes, potentially capable of degrading leucocytic pyrogen, are released from polymorphonuclear leucocytes, along with the pyrogen, when the cells are incubated in normal salt solution. These enzymes are readily activated by a sufficient concentration of glutathione (2 x 10^–2 M). They are not present in preparations of partially purified leucocytic pyrogen from which much of the non-pyrogenic protein has been removed. Glutathione by itself, even at concentrations as high as 2 x 10^–1 M, does not affect in the gross the thermogenic activity of the purified pyrogen. The implications of these findings in relation to both the production and the chemical characteristics of leucocytic pyrogen 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 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.     

FURTHER STUDIES ON THE ROLE OF PROTEASES IN THE ALLERGIC REACTION

Protease activity was measured through the hydrolysis of synthetic amino acid esters in body fluids and tissues of guinea pigs, rats, mice, and humans. Significant in vitro activation was observed in serum and lung slices of sensitized guinea pigs on addition of the specific antigen. Increased proteolytic activity was also seen in reverse anaphylaxis. More marked activation occurred when guinea pig serum was treated with peptone and guinea pig or rat serum was treated with agar. Protease activation was demonstrated in specimens of human skin under the influence of a poison ivy extract or croton oil added in vitro. Urinary protease activity of guinea pigs increased significantly during the first hours of anaphylactic shock and very markedly in peptone shock. Peptone shock, elicited in mice pretreated with H. pertussis, was accompanied by a considerable increase in protease activity in the peritoneal fluid as compared with non-pretreated mice which were insensitive to peptone. Proteolytic activity resulting from the activation procedures was due to a number of proteases. The dominant substrate affinity and inhibition patterns suggest that serum and urine proteases are similar to but not identical with plasmin. Anaphylactic activation exhibited patterns different from those resulting from the action of anaphylactoid agents. Tissue enzymes are either of cathepsin- or chymotrypsin-type or mixtures of both. Some of the activated enzymes, although remarkably effective in hydrolyzing amino acid esters, show no activity on protein substrates. This does not justify, however, their designation as "esterases." They probably belong to the class of specific proteases acting only on a single or a small number of functionally significant protein substrates. There is at present sufficient evidence to prove not only that protease activation does occur in anaphylaxis and anaphylactoid conditions but also that it is an important component of the chain of reactions leading to the allergic response.     

TYPHUS FEVER : IV. FURTHER OBSERVATIONS ON THE BEHAVIOR OF RICKETTSIA PROWAZEKI IN TISSUE CULTURES

In tissue cultures grown at 32°C., typhus Rickettsiae increase rapidly within the cytoplasm of infected cells up to about the 14th day. At this time practically every cell is infected and the majority of cells are distended with organisms. This condition remains constant as long as successful cultures of the cells can be maintained (up to 52 days). Loss in virulence does not take place during this period in vitro. The number of Rickettsia-filled cells found in sections and the incubation period of the infection resulting from inoculation of cultures from each age group are definitely correlated. The behavior of typhus Rickettsiae in dividing cells is described and methods of spread of the infection other than by mitosis of cells are discussed. Normal tissues do not become infected in vitroto any considerable extent in spite of prolonged proximity to heavily infected cultures of scrotal sac exudate. Complete anaerobiosis and alterations in pH do not alter the intracellular location of the organism in tissue cultures. The organisms are not seen within nuclei of infected cells. They remain intact and infective for several weeks in cells which are kept alive but not multiplying. They disappear in less than 1 week, however, when the cells undergo degeneration.     

STUDIES ON THE PATHOGENESIS OF RABIES IN INSECTIVOROUS BATS : I. ROLE OF BROWN ADIPOSE TISSUE

Studies on the pathogenesis of rabies in two species of experimentally infected insectivorous Chiroptera, the Mexican free-tailed bat (Tadarida mexicana), a quasi hibernator, and the little brown bat (Myotis lucifugus), a deep hibernator, provided evidence that brown adipose tissue may serve as an extraneural site for storage and multiplication of rabies virus. Although the Mexican free-tailed bat proved to be relatively insusceptible to experimental rabies infection, virus was demonstrated in the brown fat of 22 per cent of those animals shown to be infected by viral assay in white Swiss mice. Rabies infection in this species was most evident 20 to 40 days after intramuscular inoculation of virus. Rabies virus was found to be widely distributed in the little brown myotis 9 to 26 days following inoculation and virus concentrations in some of the tissues approached the level of the stock mouse brain virus suspension used in inoculating these bats. The shorter incubation period and higher virus titers in the tissues assayed reflect the increased susceptibility of Myotis lucifugus as compared with the Mexican free-tailed bat. Virus was demonstrated in the brown fat of 30 per cent of the experimentally infected Myotis. In the experimentally infected Myotis lucifugus and in the Syrian hamster which is highly susceptible to rabies infection, rabies virus was isolated more frequently from the brown fat than from the salivary gland indicating that in a susceptible host brown adipose tissue may be as frequent a site of viral proliferation as salivary gland. Since rabies virus was found to persist for long periods of time in the brown fat of experimentally infected bats and was occasionally demonstrated in this tissue alone, it is suggested that brown adipose tissue provides a mechanism by which these animals may serve as reservoirs for this agent in nature. The possibility that similar mechanisms may be involved in the maintenance of other viral agents during interepidemic periods is discussed.     

STUDIES IN EXPERIMENTAL SYPHILIS : III. FURTHER OBSERVATIONS ON THE POSSIBILITY OF CURE OF SYPHILIS IN THE RABBIT WITH ARSPHENAMINE.

Syphilitic rabbits can be treated with arsphenamine in such a manner as to render the lymph nodes incapable of transmitting the infection to normal rabbits. This can be accomplished if treatment is begun either early or comparatively late in the course of the disease. If treatment is begun early, the animals are almost uniformly susceptible to a second infection, whereas, if it is begun late, they are almost uniformly refractory to a second infection. It is suggested that this refractory state in rabbits may be explained by the existence of an acquired immunity which persists after the abolition of the disease, rather than to the persistence of the first infection. It would appear that it is possible under certain conditions to reinoculate rabbits and produce generalized infection without producing any lesion at the portal of entry.