Pathogenetic mechanisms in experimental immune fever.

When rabbits sensitized to human serum albumin (HSA) are challenged intravenously with specific antigen, fever develops and two transferable pyrogens can be demonstrated in the circulation. The first appears prior to the development of fever and has properties consistent with soluble antigen-antibody complexes. These have been shown to be pyrogenic when prepared in vitro and to produce a state of febrile tolerance when repeatedly administered. The second pyrogen, demonstrable during fever in donor rabbits, appears to be similar to endogenous pyrogen described in other experimental fevers. It is postulated that the formation of antigen-antibody complexes constitutes an important initial phase of the febrile reaction in this type of immune fever.

Demonstration and characterization of two distinct human leukocytic pyrogens.

Human monocytes and neutrophils were separated from buffy coats of blood obtained from normal donors. Following incubation with heat-killed staphylococci, monocyte preparations contained 20 times more pyrogenic activity in the supernatant media than did supernates from an equal number of neutrophils. During purification of these pyrogens it was discovered that these cell preparations each produced a distinct and different pyrogen. The pyrogen obtained from neutrophils had a mol wt of 15,000 following Sephadex G-75 gel filtration, an isoelectric point of 6.9, and could be precipitated and recovered from 50% ethanol at -10 degrees C. In contrast, the pyrogen derived from monocyte preparations had a mol wt of 38,000, an isoelectric point of 5.1, and was destroyed in cold ethanol. Both molecules were unaffected by viral neuraminidase but biologically destroyed at 80 degrees C for 20 min and with trypsin at pH 8.0. The febrile peak produced by partially purified neutrophil pyrogen occurred at 40 min while that from monocytes was at 60 min. In addition, monocyte pyrogen produced more sustained fevers for the same peak elevation as neutrophil pyrogen. These studies demonstrate for the first time two chemically and biologically distinctive pyrogens derived from circulating human white blood cells and have important implications for our understanding of the pathogenesis of fever in man.

Studies on the origin of human leukocytic pyrogen.

Release of the protein molecule, leukocytic pyrogen, is one of the many reactions exhibited by leukocytes after phagocytosis. After the ingestion of heat-killed S. albus, a 3-4 hr latent period exists, during which human peripheral leukocytes release no pyrogen, yet cellular metabolism is altered in such a way that pyrogen output may subsequently occur in the absence of further phagocytosis. Transcription of messenger RNA and translation of new protein are initial events in the. activation process, since addition of the inhibitors, actinomycin D, and cycloheximide or puromycin, during this period markedly depressed or abolished subsequent pyrogen release. These effects were noted to be dependent upon the time of addition of the inhibitors. None of the inhibitor drugs interfered with cell viability as measured by phagocytosis and hexose monophosphate shunt activity, nor did they alter the pyrogenicity of preformed leukocytic pyrogen. Vincristine did not inhibit pyrogen formation, consistent with its reported failure to alter RNA synthesis in mature human granulocytes. The glycolytic inhibitor, sodium fluoride, blocked pyrogen release both when added prior to particle ingestion or 1 hr after the initiation of phagocytosis. Whereas inhibition of phagocytosis would explain the sodium fluoride effect prior to 1 hr, this was not observed in leukocyte preparations incubated for 1 hr with S. albus before adding sodium fluoride. When sodium fluoride was added to preparations 2 hr after the start of incubation, the LP production was unimpaired. Potassium cyanide had no effect on cell activation or pyrogen release. These findings suggest that the primary energy supply for the activation process is derived from high energy phosphate bonds provided by anaerobic glycolysis. Since the major amount of cell activation appears to occur in the 1st hr after phagocytosis, this energy might be involved in the induction of a genome leading to the transcription of m-RNA and its translation into new protein or is required for polysome integrity during protein synthesis. It is suggested that this new protein may be leukocytic pyrogen itself, or an enzyme responsible for cleaving it from an inactive precursor.

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)

Enhancement of growth of virulent strains of Escherichia coli by interleukin-1.

Interleukin-1 (IL-1) is a polypeptide cytokine that mediates many physiological responses to infection and inflammation and is a growth factor for certain mammalian cells. Virulent and avirulent clinical isolates of Escherichia coli were grown in culture media in the presence of human IL-1. IL-1 beta, but not tumor necrosis factor or IL-4, enhanced the growth of virulent, but not avirulent, E. coli. This enhancement was blocked by the IL-1 receptor antagonist (IL-1ra). Radiolabeled IL-1 bound to virulent but not avirulent E. coli in a specific and saturable fashion; IL-1ra inhibited this binding. Thus, human IL-1 may recognize a functional IL-1-like receptor structure on virulent E. coli and may be a virulence factor for bacterial pathogenicity.

Cyclic urinary leukopoietic activity in gray collie dogs.

The urinary activities for bone marrow colony formation were measured on consecutive 24-hour urine samples from two gray collie dogs with cyclic neutropenia and from two normal collies. The activity varied cyclically in the gray collies with a peak activity developing during the neutropenic phase, which antecedes the return of blood neutrophils. The activity fell to undetectable levels after the blood neutrophil counts returned to the normal range. The urine of normal dogs showed no activity. Since the dogs with cyclic neutropenia have been shown to have periodic hematopoiesis, these data suggest a regulatory hormonal role for the substance measured by this assay.

Reticuloendothelial system phagocytic function in patients with Hodgkin's disease.

Phagocytic function of the reticuloendothelial system as measured by the clearance of (125)I-labeled aggregated human serum albumin (AA) was studied in patients with Hodgkin's disease. Each more advanced stage of the disease was associated with more rapid clearance of the AA. Production of remission by radiation therapy or by chemotherapy was associated in some patients with slowing of the clearance rate, whereas relapse seemed to be associated with acceleration. Thus, impaired phagocytosis cannot be implicated in the several defects in immunity known to be present in Hodgkin's disease. Furthermore, determination of the clearance rate of AA in patients with Hodgkin's disease may have clinical usefulness as another indicator of extent or activity of disease.

Leukocytic function in hypogammaglobulinemia.

The phagocytic, bactericidal, and metabolic capabilities of circulating blood leukocytes from three adults (two males, one female) with hypogammaglobulinemia and recurrent pneumonia, chronic sinusitis, and intestinal giardiasis were studied. These functions were found to be normal when leukocytes from the patients were incubated in media containing normal human serum. Phagocytosis of Staphylococcus albus and polystyrene balls by both patient and normal leukocytes was diminished when the cells were incubated in hypogammaglobulinemic plasma. A similar defect in opsonization by patient plasma was also noted for pneumococci, Escherichia coli and variably with Staphylococcus aureus. Both patient and normal sera had equivalent levels of heat-labile S. albus opsonins; normal serum, however, contained heat-stable S. albus-specific absorbable opsonins in significantly greater quantities to account for its superior opsonic capacity. The addition of commercial gamma globulin or purified IgG to hypogammaglobulinemic sera restored full S. albus opsonic activity. The relevancy of these observations to the impaired host defenses in these patients will be discussed.

The influence of pyrogen-induced fever on salicylamide metabolism in man.

Salicylamide is metabolized in man by biotransformation to salicylamide glucuronide, salicylamide sulfate, and gentisamide glucuronide. The metabolites are quantitatively and rapidly excreted in urine. Study of the metabolism of this drug in volunteers during episodes of pyrogen-induced fever shows a significant reduction in the half-life (t(1/2)) of the excretion of the drug metabolites. The proportion of the drug transformed to its major metabolite, salicylamide glucuronide, is significantly reduced by fever, with concomitant increase in the proportion of one or both of the other metabolites. Thus, the pattern of urinary metabolites of salicylamide is altered. The shortened t(1/2) of the metabolite excretion is probably due to increased hepatic and renal blood flow known to accompany pyrogen-induced fever. This concept was supported by the observation that when two subjects were placed in a high-temperature environmental chamber, a condition in which hepatic and renal blood flows are known to diminish, the t(1/2) of salicylamide metabolite excretion actually increased. No simple explanation exists to explain the changed metabolite pattern noted during febrile periods. It is most likely to be due to complex interactions between the direct or indirect effects of the pyrogens and the factors affecting the hepatic biotransformation of drugs.

Cyclic hematopoiesis: the mechanism of cyclic neutropenia in grey collie dogs.

Two grey collie dogs had regular cyclic fluctuations in the number of all formed elements of the blood. The period lengths for all elements for an individual dog were the same, but the pattern of fluctuation for each element was distinctive. Normal dogs lacked periodic fluctuations.The patterns of day-to-day variation in the normal dogs counts were consistent with a first-order autoregressive process of serial dependence (i.e., each observation of the series depends on the last preceding observation and no others). The grey collie counts showed the same pattern of serial dependence after the component of the over-all variability due to cyclic oscillation was removed. These data suggest that a defect of hematopoietic regulation at the stem cell level leads to periodic interruptions of production of all hematopoietic elements and accounts for the cycles seen in the peripheral blood counts.

The production of antibody against human leukocytic pyrogen.

Human peripheral blood leukocytes were stimulated with killed staphylococci in vitro to release leukocytic pyrogen (LP). Supernates from these stimulated leukocytes were concentrated, emulsified in Freund's complete adjuvant, and injected intradermally into rabbits. After seven monthly booster injections, rabbit antiserum destroyed the pyrogenic activity of human LP, and the titer of this neutralizing ability increased in the subsequent 7 mo. The pyrogen-neutralizing capacity of the rabbit antiserum was recovered in the globulin fraction, the IgG and IgM peaks of Sephadex G-200, and the acid-eluted fraction of a goat anti-rabbit IgG immunoadsorbant. The neutralizing antibody was specific for human LP inasmuch as it had no effect on rabbit, guinea pig, or monkey LP. When coupled to Sepharose, this antibody bound human LP; after acid elution from this immunoadsorbant, LP was recovered without loss of biologic or chemical characteristics. The antiserum was also absorbed with stimulated leukocyte supernates which did not contain LP, and this had no effect on the titer of anti-LP. Crude human LP, eluted from immunoadsorbant columns prepared from absorbed antiserum, contained significantly reduced contaminating protein when evaluated by polyacrylamide gel electrophoresis. These studies have established that specific antibody to human leukocytic pyrogen can be produced. This antibody is useful in the further study and purification of leukocytic pyrogen and its role in the pathogenesis of human fever.

Recurrent fever of unknown etiology: failure to demonstrate association between fever and plasma unconjugated etiocholanolone.

A sensitive method for determination of plasma unconjugated etiocholanolone by double-isotope-derivative dilution has been described. The mean values for normal subjects was 0.038+/-0.003 (SEM) mug/100 ml.40 patients, 20 with familial Mediterranean fever and 20 with other diseases characterized by recurrent fever were studied. The over-all mean concentration of plasma unconjugated etiocholanolone for the patients (febrile or afebrile) was 0.101 +/-0.012 mug/100 ml, significantly above that of normals. Mean plasma values for the patients while they were febrile did not differ from the mean values when they were afebrile. It is suggested that the concentration of plasma unconjugated etiocholanolone is not related to fever in these patients.

Comparison of agents producing a neutrophilic leukocytosis in man. Hydrocortisone, prednisone, endotoxin, and etiocholanolone.

To study the potential application of glucocorticosteroid administration for the measurement of the bone marrow neutrophil reserve response, blood neutrophil count changes were measured in normal subjects after the administration of intravenous hydrocortisone (25, 50, 100, 200, and 400 mg) and oral prednisone (5, 10, 20, 40, and 80 mg). The upper three doses of both steroids increased the blood neutrophil count by approximately 4,000 cells/mm3. The neutrophilia occurring after hydrocortisone (200 mg) and/or prednisone (40 mg) was compared with that observed after endotoxin (0.8 ng/kg) and etiocholanolone (0.1 mg/kg) in 14 normal subjects, 7 patients with Wegener's granulomatosis on cyclophosphamide therapy and 10 patients with chronic idiopathic neutropenia. The normal responses (mean increase of blood neutrophils/mm3 above base line +/- 1 SEM) were: hydrocortisone 4,220 +/- 320, prednisone 4,610 +/- 360, endotoxin 6,060 +/- 880, and etiocholanolone 3,780 +/- 440. In the patient studies, etiocholanolone gave the smallest mean responses, but, in general, the results were similar for all agents. These data indicate that these glucocorticosteroids can be used as equivalent agents to endotoxin and etiocholanolone for measuring the neutrophil reserve response.

Induction of human interleukin-1 by a product of Staphylococcus aureus associated with toxic shock syndrome.

Certain strains of Staphylococcus aureus associated with toxic shock syndrome elaborate material that induces human blood monocytes to secrete interleukin-1 (IL-1). IL-1 was detected both by its ability to cause fever in rabbits using the leukocytic pyrogen (LP) assay and by its mitogenic activity towards thymocytes in the so-called lymphocyte-activating factor (LAF) assay. Anti-human IL-1 prevents the manifestation of both activities. Filtrates of control strains of S. aureus manifest neither activity. Thus, culture filtrates derived from toxic shock syndrome (TSS)-associated strains cause biphasic fever in rabbits when injected intravenously. The fever lasts several hours. Plasma taken at the peak of the fever and injected into a second set of rabbits produces a brief monophasic fever typical of LP. Further, human monocytes release LP when incubated with TSS filtrates in vitro. The monocyte products also stimulate the proliferation of mouse thymocytes in the presence of phytohemagglutinin in a manner characteristic of LAF. A bacterial filtrate is much less effective without an intermediate incubation with monocytes. The stimulation of monocyte IL-1 production is easily quantified, provides a simple method of assaying the TSS toxin, and since it involves human cells, is directly relevant to the human disease. The assay was used to monitor the purification of TSS toxin. Only 0.1 ng/ml of the purified material is required to induce monocyte IL-1 production. It is thus more potent than endotoxin. In contrast to endotoxin, its effect is not blocked by polymyxin B. We conclude that in TSS the sudden fever and probably other components of the acute phase response may be attributed to a massive release of IL-1.

Studies of neutrophil production and turnover in grey collie dogs with cyclic neutropenia.

12 grey collie dogs had cyclic neutropenia with the neutropenia recurring at 11.8+/-0.1-day intervals. The recovery from neutropenia was accompanied by a single wave of myeloid proliferation, an increase in marrow myeloid-labeling indices, and an increase in serum muramidase levels. After recovery from neutropenia during the period when blood neutrophils (PMN) were normal or increased, marrow myeloid precursors became scarce. The decline in marrow precursors and marrow PMN reserves heralded the recurrence of neutropenia. Neither diisopropyl fluorophosphate (DF(32)P) leukokinetic studies nor the rate of development of neutropenia suggested shortened PMN survival as a mechanism for the neutropenia. These studies indicate that the cyclic neutropenia is due to a regularly recurring failure in PMN production.

Periodic hematopoiesis in human cyclic neutropenia.

Human cyclic neutropenia is characterized by severe depression of blood neutrophil levels approximately every 21 days. To investigate the mechanism of cyclic neutropenia four patients were studied with daily complete blood counts, serial bone marrow examinations, marrow reserve testing, serum muramidase determinations, DF(22)P granulocytokinetic studies, and, in one patient, in vivo [(3)H]TdR labeling. Periodogram analysis of the serial blood counts in the latter patient and visual inspection of multiple cycles in the others revealed periodic fluctuations in the levels of blood neutrophils, monocytes, lymphocytes, reticulocytes, and platelets. Rhythmic changes in the morphologic and radioisotopic studies as well as the marrow reserve tests and muramidase measurements were consonant with a mechanism of periodic failure of marrow production rather than peripheral destruction. Human cyclic neutropenia is analogous to cyclic neutropenia in the grey collie dog and may be viewed as the consequence of cyclic hematopoiesis.

Changes in human serum amyloid A and C-reactive protein after etiocholanolone-induced inflammation.

Secondary amyloidosis is a complication of diseases characterized by recurrent acute inflammation. In this study, a standardized stimulus which induced fever and inflammation was given to six normal subjects (19-24 yr old) to follow the fluctuation in concentration of serum amyloid A (SAA), the precursor of the secondary amyloid fibril protein. After a single intramuscular injection of etiocholanolone (0.3 mg/kg), blood samples were drawn twice a day for 12 days for determination of SAA by solid phase radioimmunoassay. From a base line of <100 mug/ml, the SAA concentration began rising within 12 h to a maximum value at about 48 h of 1,350-1,800 mug/ml in three males and 380-900 mug/ml in three females and returned to base line by 4-5 days. The SAA response showed a similar time response to C-reactive protein (CRP), a well-documented acute phase protein which was assayed semiquantitatively by capillary tube precipitin reaction. CRP, but not SAA, showed a quantitative correlation with the amount of fever induced by etiocholanolone. One subject exhibited a second rise in SAA and CRP concentrations after acute over-indulgence with alcohol, suggesting that acute liver damage may have caused an acute phase reaction. Thus, a controlled episode of fever and inflammation produced a prompt and prolonged elevation of SAA and CRP concentrations. Unlike SAA, CRP has not been implicated in the pathogenesis of amyloidosis, although its relationship to the P component of amyloid has recently been established.

Effects of etiocholanolone-induced fever on plasma antipyrine half-lives and metabolic clearance.

The plasma half-life and metabolic clearance rate of antipyrine, a drug metabolized by hepatic microsomal enzymes, were determined in 33 normal volunteers during a basal state and during fever induced with a single intramuscular injection of etiocholanolone. Of the 14 normal volunteers who achieved significant fever (fever index greater than 50), in 11 plasma antipyrine half-life was prolonged after a single oral dose of 10 mg/kg and antipyrine metabolic clearance rate was decreased. There was no significant change of these mean values in 19 normal volunteers who failed to develop significant fever (fever index smaller than 50). Therefore, under the conditions of this study plasma antipyrine half-life was prolonged, probably due to impaired hepatic metabolism, during etiocholanolone-induced fever, although no correlation was observed between the magnitude of fever and the extent to which plasma antipyrine half-life was prolonged. Failure to obtain such a correlation may be attributable to the very small range of temperature elevation, extending from 37.9 degrees C to 39.2 degrees C, in the group of 14 subjects achieving significant etiocholanolone-induced fever (fever index greater than 50). A higher dose of antipyrine (18 mg/kg) suppressed induction of fever by etiocholanolone; antipyrine is the only orally administered drug thus far shown to be effective in repressing etiocholanolone-induced fever.

The hypereosinophilic syndrome: analysis of fourteen cases with review of the literature.

Several closely related disease entities make up the idiopathic hypereosinophilic syndrome (HES). The syndrome is manifest by persistent and prolonged eosinophilia with organ damage. A group of 14 patients had hematologic, cardiac, and neurologic abnormalities attributable to this disease. Patient survival and response to chemotherapy was significantly better in this group than in previously reported patients. The etiology of HES remains unknown, as does the mechanism of tissue damage.

Human cyclic neutropenia: clinical review and long-term follow-up of patients.

Human cyclic neutropenia is a distinctive disorder of unknown cause characterized by regularly recurrent episodes of profound neutropenia, which have a periodicity of about 3 weeks. This periodicity remains constant and is remarkably consistent among patients. Although blood elements other than neutrophils are nt depleted, essentially all patients experience a cycling of monocyte counts with monocyte cycles of the same length as but reciprocal to neutrophil cycles. Cycling of platelet and reticulocyte numbers also may occur. Patients experience a clinical syndrome of recurrent illness characterized by malaise, fever, aphthous stomatitis, and cervical adenopathy. Incidental infections may occur with neutropenia but respond readily to antibiotics. The clinical course is benign compared with others conditions in which similar degrees of neutropenia occur. The only life-threatening complication encountered during long-term follow-up of patients was the occurrence of spontaneous peritonitis, segmental bowel necrosis, and septicemia which required surgical intervention. Most patients develop the disease in childhood, but a significant number of patients develop the disease in adulthood as an apparently acquired condition. The disease occurs equally in both sexes and is familial in some. Studies of marrow morphology, myelopoiesis, and neotrophil kinetics have shown that cyclic neutropenia is primarily a disease of abnormally regulated neutrophil production. The judicious use of antibiotics, careful oral and dental care, and patient education are the mainstays of management. Alternate-day corticosteroids have been used successfully to abate the recurrent signs and symptoms, and in one patient the disease was gradually corrected by alternate day prednisolone. Human cyclic neutropenia is of special investigative interest because clarification of this disease may contribute greatly to an understanding of the normal control of myelopoiesis.