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

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

Suppression of Ag-induced release of EP (IL 1) by spleen cells of specifically desensitized donors: evidence for the role of a suppressor cell

Monocytes or macrophages may be induced to produce IL 1 by activators (e.g., lipopolysaccharide endotoxin) that act directly or by antigens/mitogens (e.g., Con A) that stimulate inducer lymphocytes to release a lymphokine that stimulates macrophages. Using guinea pigs (GP) rendered delayed hypersensitive to ovalbumin (OVA), we investigated the role of spleen cells from normal, sensitized, and specifically desensitized GP in suppressing release of IL 1, measured as endogenous pyrogen (EP), from peritoneal exudates of sensitized GP when incubated with OVA in vitro. Co-cultivation of all three sources of spleen cells with GP peritoneal exudate cells and OVA suppressed EP release as measured in the rabbit fever assay, the effect being most marked with cells from desensitized GP, intermediate with cells from sensitized GP, and least with normal cells. This suppressor activity of spleen cells on in vitro EP release was not explained by nonspecific absorption of EP by the added cells and did not affect EP release by a stimulus that activates macrophages directly (heat-killed staphylococci). It required both lymphocytes and macrophages for its effect, but unlike some other suppressor factors, it was not modified by indomethacin, an inhibitor of prostaglandin release. This appears to be the first reported evidence for cell-mediated suppression of lymphokine-mediated release of IL 1, an important modulator of the immune system through its combined role as a lymphocyte-activating factor and an inducer of fever (EP).     

Studies on the production of endogenous pyrogen by rabbit monocytes: the role of calcium and cyclic nucleotides

Rabbit monocytes stimulated with endotoxin produced endogenous pyrogen, even under conditions of high or low extracellular calcium concentrations. Maximal production occurred when the concentration was in the near-physiological range. Prolonged incubation of cells with a calcium chelator prevented subsequent activation with endotoxin, an effect which was rapidly reversible by re-addition of calcium but not other cations. Addition of small amounts of lanthanum, which acts as a calcium channel blocker, prevented the restoration of pyrogen production, indicating that entry of the added calcium into the monocyte was required. Incorporation of a calcium ionophore into the cell membrane did not stimulate pyrogen production, and no measurable influx or efflux of calcium occurred during stimulation with endotoxin. These observations suggest that a slowly exchangeable calcium pool is necessary for the production of endogenous pyrogen, but that a rise in intracellular calcium is not by itself a necessary or sufficient stimulus. This stands in contrast to other biological systems in which Ca2+ directly couples stimulus and hormone secretion. Incubation of cells with agents shown to increase cyclic 3',5' AMP or cyclic 3',5' GMP levels in monocytes similarly did not stimulate pyrogen production or modulate its production by endotoxin stimulation. Thus, cyclic nucleotides also did not play a detectable role as intracellular messengers in this system. Future work is required to define more clearly the mechanism for the production of endogenous pyrogen, given its marked effects on the immune system through lymphocyte activation and temperature regulation.     

The pyrogenic effect of scarlet fever toxin

Scarlet fever toxin was found to liberate leukocytic pyrogen from granulocytesin vitro. In comparative experiments withSalmonella paratyphi B endotoxin and scarlet fever toxin it was tested whether leukocytes from rabbits tolerant to one of these toxins are able to synthetize and liberate endogenous pyrogen. Leukocytes from rabbits tolerant to endotoxin liberated leukoeytic pyrogen following challenge with endotoxin or with scarlet fever toxin. Leukocytes from animals tolerant to scarlet fever toxin liberated leukocytic pyrogen in the presence of endotoxin, but were insensitive to homologous, i.e. scarlet fever toxin. Similarly, leukocytes from cortisone-treated animals did not liberate leukocytic pyrogen if they were incubated with scarlet fever toxin, but liberation of leukocytic pyrogen did take place under challenge with endotoxin. Leukocytes from normal animals incubated in Hanks solution without toxin did not synthetize endogenous pyrogen.     

Development of a highly sensitive in vitro assay method for biological activity of endotoxin contamination in biological products.

The pyrogen test in rabbits has been replaced by the bacterial endotoxin test. The endotoxin test, however, showed a considerable discrepancy with pyrogenicity and was, therefore, assumed to have an efficacy limitation in directly predicting harmful biological effects of endotoxin. We developed a sensitive in vitro assay method by making use of tumour necrosis factor alpha (TNF-alpha) induction in RAW264.7 cells, which showed a fine correlation with pyrogenicity in rabbits. RAW264.7 cells maintained by serial subculture under an endotoxin-free condition have gained the similar level of sensitivity as the endotoxin test to allow extensive dilutions of a drug for eliminating adverse effects on the cells. The in vitro TNF-alpha induction assay was shown to be capable to detect quantitatively a synergistic effect of a drug and endotoxin. The synergy is assumed necessary to be taken into consideration to define the limit value for the endotoxin test for guaranteeing the similar level of safety as by the pyrogen test.     

A comparison of the febrile responses of the Brattleboro and Sprague-Dawley strains of rats to endotoxin and endogenous pyrogens

The febrile responses of homozygous (di/di) Brattleboro rats, to both intravenous endogenous pyrogen and to a lipopolysaccharide endotoxin, were compared with those of normal Sprague-Dawley rats. There were no detectable differences between the fever curves of the two strains in response to endogenous pyrogen. Brattleboro rats, which are deficient in the neuropeptide arginine vasopressin (AVP), displayed fevers that were both qualitatively and quantitatively indistinguishable from those of normal Sprague-Dawley rats that do not suffer from congenital diabetes insipidus. It is concluded that the absence of AVP-containing cells in Brattleboro rats is not an important factor in determining the nature of their febrile responses to endogenous pyrogen. More remarkable, however, were the divergent febrile responses of the two strains to intravenously injected endotoxin. Normal rats displayed hypothermic responses, whereas the Brattleboro rats became febrile. By 2 h after the injection of endotoxin, body temperatures in both strains had returned to normal. Three hours after the rats had been exposed to endotoxin, both strains were found to be totally refractory to endogenous pyrogen. However, when both strains of rats were tested to endogenous pyrogen 3 days later, their febrile responses were more than double the magnitude of their initial control responses. These alterations in the febrile responsiveness of rats occurring at different times after the injection of endotoxin appear to be related to the effects that endotoxin has on the cells of the reticuloendothelial system, over the same time course.     

Hog barn dust extract stimulates IL-8 and IL-6 release in human bronchial epithelial cells via PKC activation.

Hog barn workers have an increased incidence of respiratory tract symptoms and demonstrate an increase in lung inflammatory mediators, including interleukin (IL)-8 and IL-6. Utilizing direct kinase assays for protein kinase C (PKC) activation, we demonstrated that dust from hog confinement facilities, or hog dust extract (HDE), augments PKC activity of human airway epithelial cells in vitro. A 5% dilution of HDE typically stimulates an approximately twofold increase in human bronchial epithelial cell (HBEC) PKC activity compared with control medium-treated cells. This increase in PKC is observed with 15 min of HDE treatment, and kinase activity reaches peak activity by 1-2 h of HDE treatment before returning to baseline PKC levels between 6 and 24 h. The classic PKC inhibitor, calphostin C, blocks HDE-stimulated PKC activity and associated IL-8 and IL-6 release. Desensitization to HDE stimulation of PKC activation does not appear to occur because subsequent exposures to HDE after an initial exposure result in further augmentation of PKC. Detoxification of HDE with polymyxin B to remove endotoxin did not change PKC activation or IL-8 release, suggesting that endotoxin is not solely responsible for HDE augmentation of PKC. These data support the hypothesis that HDE exposure augments HBEC IL-8 and IL-6 release via a PKC-dependent pathway.     

A quantitative in vitro assay method for detecting biological activity of endotoxin using prostaglandin E2 induction in rabbit peripheral blood

The pyrogen test and the endotoxin test (the LAL test) have been playing crucial roles in detecting endotoxin in parenteral drugs. The current test methods, however, have disadvantages such as requiring a large number of animals or an inadequacy in evaluation of in vivo endotoxin activity. We attempted to establish a new assay method that can overcome the shortcomings of the current methods. We standardized the in vitro assay method by the use of prostaglandin E2 (PGE2) induction from peripheral blood of rabbits for detecting endotoxin activity. A linear dose-response regression was attained from approximately 0.15 to 5 endotoxin units/ml of Japanese national reference standard endotoxin by the in vitro assay. The assay showed a fine correlation with the pyrogen test but not with the LAL test, when endotoxins from various bacterial sources were tested. The in vitro assay was also shown to have the capability of detecting a synergistic effect of endotoxin and parenteral drugs. The in vitro PGE2 induction test using rabbit blood was, therefore, suggested to be the appropriate test method for guaranteeing the same level of safety of parenteral drugs as the pyrogen test does.     

Effects of metabolic inhibitors on spontaneous and interferon-boosted human natural killer cell activity

Human natural killer (NK) cell activity can be augmented by pretreatment with partially purified preparations of human interferon (IF). Studies have now been performed to determine the metabolic processes required for and involved in spontaneous NK activity and augmentation of cytotoxicity. A 4-hr 51Cr release cellular cytotoxicity assay was used to measure the NK activity, and peripheral blood leukocyte cells (PBL) were treated with: a) x-ray or mitomycin C; b) actinomycin D; or c) emetine, cycloheximide, pactamyhcin, or puromycin to assess the roles of DNA, RNA, and protein synthesis, respectively, in spontaneous NK activity and in boosting by IF. Prolonged incubation (18 hr) of PBL after blockage of synthesis of DNA almost completely abrogated NK activity; however, NK activity could be partially or totally restored to these populations by incubation of the effector cells for 1 hr at 37 degrees C with IF. Blockage of DNA synthesis for 1 hr had no effect on spontaneous NK activity or on boosting by IF. Inhibition of RNA synthesis also had no effect on spontaneous NK activity. Treatment of PBL with actinomycin before exposure to IF prevented boosting, but treatment with the RNA synthesis inhibitor after boosting with IF for 5 to 6 hr no longer had an appreciable effect on cytotoxicity. The effect of protein synthesis inhibitors on spontaneous NK activity was dependent on the inhibitor selected. Emetine and puromycin totally abrogated spontaneous NK activity at concentrations of inhibitor that blocked 3H-leucine incorporation 90% or more. In contrast, cycloheximide and pactamycin had only minimal effects on spontaneous NK activity but totally abrogated the boosting of IF.     

Pyrogen from mouse macrophages causes fever in mice.

Mouse peritoneal macrophages, after phagocytosis, release an endogenous (leucocyte) pyrogen. Intravenous injection of stimulated cell culture supernatant produces a prompt, monophasic fever in mice maintained in a 35 degree environment. The pyrogen is distinct from endotoxin, and resembles cell pyrogens of other species in heat-lability and pronase sensitivity. Human leucocyte pyrogen produces identical responses in mice. Measurement of fever in mice appears to provide a sensitive biological assay for endogenous pyrogens.     

The involvement of protein kinase C in exocytosis in mast cells.

Diacylglycerol generated from inositolphospholipid hydrolysis and tumor-promoting phorbol esters stimulate protein kinase C. The synthetic diacylglycerol 1-oleoyl-2-acetyl-rac-glycerol and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) have been used in pure rat peritoneal mast cells. Both caused histamine release associated with exocytosis. The release by the stimulation of protein kinase C alone in the absence of secretagogues was slow although up to 50% of the histamine content was released by TPA in 120 min. Remarkable potentiation of histamine release was observed when the mast cells were preincubated with TPA before exposure to the calcium ionophore A23187. The potentiation of histamine release corresponded with an intensification of exocytosis. The potentiation is consistent with a participation of protein kinase C in the secretory process. An inhibitory effect due to protein kinase C activity was also demonstrated using TPA and mast cells from sensitized rats. When sensitized mast cells preincubated with 50 nM TPA for 5 min were exposed to the antigen, the histamine release was substantially reduced compared to the sum of the release by the antigen and TPA or by the antigen alone. There was a corresponding decrease in exocytosis. The inhibition of exocytosis and histamine release seems to reflect a regulatory function of protein kinase C for the termination of the response, as demonstrated in other types of cells apparently acting through an inhibition of inositolphospholipid hydrolysis.     

Characterization of Limulus amoebocyte lysate-reactive material from hollow-fiber dialyzers.

Hollow-fiber hemodialyzers containing cellulose-based membranes have been shown to produce positive results with the Limulus amoebocyte lysate test. This study was undertaken to determine whether endotoxin was causing the reaction. Rinses from 45 parallel-plate and hollow-fiber dialyzers from eight different manufacturers were tested before and after treatment with cellulase, using three lysates and four Limulus amoebocyte lysate methods. In addition, four in vitro cellular methods--human leukocytic pyrogen, lymphocytic activating factor, peritoneal macrophage, and arginase release--were used to evaluate endotoxin activity. The substance causing the reaction was identified by chromatographic methods. Results indicate that the Limulus amoebocyte lysate reactive material is cellulose derived and not pyrogenic.     

Restoration of responsiveness to gonadotropin releasing hormone (GnRH) in calcium-depleted rat pituitary cells.

GnRH-stimulated, but not basal, luteinizing hormone (LH) release from cultured pituitary cells requires extra-cellular calcium. The present studies were designed to show whether cells which had lost responsiveness to GnRH in the absence of extracellular calcium (“Ca²⁺-depleted cells”) could regain responsiveness by readdition of calcium to the media. The addition of calcium-containing medium to cells which were preincubated (75 min) in calcium-free medium resulted in elevated basal LH release. Addition of GnRH to the media in the presence of calcium did not cause additional stimulation of LH release above the elevated basal level. Incubation of Ca²⁺-depleted cells in calcium-containing media for 2 h before measuring responsiveness depressed the basal level to near that seen in control cells and GnRH was able to stimulate LH release, but not to as high a level as in control cells (which were preincubated in 1 mM Ca²⁺-containing media). After incubation of calcium depleted cells in calcium-containing media for 3 h or 5 h, the basal and stimulated levels of LH response were statistically indistinguishable from those seen in control cells.     

Comparison of the rabbit pyrogen test and Limulus amoebocyte lysate (LAL) assay for endotoxin in hepatitis B vaccines and the effect of aluminum hydroxide.

The rabbit pyrogen test and Limulus amoebocyte lysate (LAL) assay have been used to detect endotoxins in vaccines, but interactions between the endotoxins and proteins or aluminum hydroxide can interfere with the results. Currently, the rabbit pyrogen test is used to detect endotoxin in hepatitis B (HB) vaccines even though the HB surface protein, the active ingredient, is over-expressed in and purified from eukaryotic cells which lack endotoxin. Therefore, we examined the possibility of replacing the animal tests with the more efficient LAL test. To this end, we determined whether the aluminum hydroxide in the HB vaccines affects the rabbit pyrogen test and the LAL assay. HB vaccines and HB protein solutions spiked with lipopolysaccharide (LPS) produced almost the same dose-dependent temperature rise in rabbits, indicating that the aluminum hydroxide in the HB vaccine does not interfere with the pyrogenic response in rabbit. In contrast, a spike recovery study showed that aluminum hydroxide interfered with the LAL clot and kinetic assays; however, the LAL clot assay was effective at detecting endotoxin without loss of LAL activity after serial dilution of the samples. Furthermore, there was good correlation in the LAL clot assay between the amount of LPS added and the amount recovered. However, both turbidimetric and chromogenic kinetic assays displayed no correlation between the LPS amount added and recovered. Our results suggest that the LAL clot assay is sensitive and reliable when samples are properly prepared, and can be used to replace the rabbit pyrogen test for the detection of endotoxin in HB vaccines.     

Response of rabbits to pyrogen in a helox environment

1. 1.|The effectiveness of a gas environment consisting of 80% helium and 20% oxygen (Helox) in reducing a rabbit's fever due to an i.v. injection of endotoxin was found to be dependent on the amount of pyrogen injected.

2. 2.|When a relatively large dose of pyrogen was injected, the helox environment used in these experiments reduced the mean maximum temperature reached during the fever from 41.5 to 40.5°C, but the helox did not significantly alter the change in temperature from baseline levels prior to the injection (a 1.4°C increase in air and a 1.1°C increase in helox).

3. 3.|When a relatively low dose of pyrogen was injected, the helox environment increased the change in temperature from baseline at peak fever, but did not produce a significant change in the actual temperatures attained during the fever.

The effects of in vivo administration of endotoxin on the functions and interaction of hepatocytes and Kupffer cells

It was the purpose of this study to determine the effects of the in vivo administration of endotoxin on certain in vitro hepatocyte and Kupffer cell functions. An Alzet osmotic pump that contained endotoxin (LPS, 2.5 mg/100g) was implanted into the peritoneal cavity of 300g guinea pigs and delivered the endotoxin over a period of four days. In vivo administration of LPS did not cause a change in the in vitro release of albumin by isolated hepatocytes. However, when hepatocytes were co-cultured with Kupffer cells there was a significant decrease in albumin release for both control and LPS-treated animals. There was no difference between control and LPS-treated animals in the release of C3 by hepatocytes. However, there was a significant increase over the control group in C3 release by Kupffer cells from LPS-treated animals. When hepatocytes and Kupffer cells were cultured together, their release of C3 was not additive. Kupffer cells from LPS-treated animals released significantly greater amounts of PGE2 than control animals when stimulated in vitro with LPS. Thus, these Kupffer cells appeared to be primed to respond to an in vitro challenge of LPS. Kupffer cells from LPS-treated animals had significantly depressed antibody dependent cellular cytotoxicity (ADCC). This endotoxin model is useful for determining the in vivo effects of endotoxin on cellular function and gives some indirect evidence for the detrimental effects of LPS on the immune system and host defense.     

Limulus amoebocyte lysate assay for detection and quantitation of endotoxin in a small-volume parenteral product.

A Limulus amoebocyte lysate gel-clotting method for the determination of endotoxin in a small-volume parenteral product has been described. Sample dilution with 0.1 M potassium phosphate monobasic buffer (pH 8.0) effectively eliminated assay interference, whereas dilution with water did not. The threshold pyrogenic dose for Escherichia coli EC-2 and O127:B8 endotoxins was determined to be 1.0 ng of endotoxin per kg of body weight. Not more than 1.0 ng of endotoxin (the threshold pyrogenic dose) per the highest recommended human dose or the USP pyrogen test dose per kg of body weight, whichever dose is more stringent, is a logical limit for the quantity of bacterial endotoxin in small-volume parenteral products. Excellent correlation was attained when this criterion was used to compare the Limulus amoebocyte lysate assay with the USP pyrogen test.     

Limulus amoebocyte lysate assay for detection and quantitation of endotoxin in a small-volume parenteral product

A Limulus amoebocyte lysate gel-clotting method for the determination of endotoxin in a small-volume parenteral product has been described. Sample dilution with 0.1 M potassium phosphate monobasic buffer (pH 8.0) effectively eliminated assay interference, whereas dilution with water did not. The threshold pyrogenic dose for Escherichia coli EC-2 and O127:B8 endotoxins was determined to be 1.0 ng of endotoxin per kg of body weight. Not more than 1.0 ng of endotoxin (the threshold pyrogenic dose) per the highest recommended human dose or the USP pyrogen test dose per kg of body weight, whichever dose is more stringent, is a logical limit for the quantity of bacterial endotoxin in small-volume parenteral products. Excellent correlation was attained when this criterion was used to compare the Limulus amoebocyte lysate assay with the USP pyrogen test.     

Applicability of bacterial endotoxins test to various blood products by the use of endotoxin-specific lysates

Endotoxin contamination is a serious threat to the safety of parenteral drugs, and the rabbit pyrogen test has played a crucial role in controlling this contamination. Although the highly sensitive endotoxin test has replaced the pyrogen test for various pharmaceuticals, the pyrogen test is still implemented as the control test for most blood products in Japan. We examined the applicability of the endotoxin test to blood products for reliable detection and quantification of endotoxin. Nineteen types of blood products were tested for interfering factors based on spike/recovery of endotoxin by using 2 types of endotoxin-specific lysate reagents for photometric techniques. Interfering effects on the endotoxin test by the products could be eliminated by diluting from 1/2 to 1/16, with the exception of antithrombin III. However, conventional lysate reagents that also react with non-pyrogenic substances, such as (1–3)-β-d-glucan, produced results that were not relevant to endotoxin content or pyrogenicity. Our results showed that the endotoxin test would be applicable to most blood products if used with appropriate endotoxin-specific lysate reagents.     

Effect of Puromycin on Guinea Pig Lymphotoxin Release and Lectin-Induced Cellular Cytotoxicity

In order to confirm the role of guinea pig lymphotoxin (GLT) in lectin-induced cellular cytotoxicity (LICC), the effect of puromycin, a potent enhancer of GLT activity, on the LICC to target L · P3 cells induced by phytohemagglutinin (PHA) was investigated under serum-free conditions. LICC was completely inhibited by puromycin, when it was added at the initiation of LICC culture, because of the inhibition of the release of GLT from the effector lymph node cells. However, LICC was markedly enhanced when puromycin was added several hours after the initiation of LICC culture. The interpretation of these facts is that GLT release can be inhibited by puromycin, but that the GLT already released exerts an enhanced cytotoxic effect on the target cells in the presence of puromycin. Enhancement of the cytotoxicity by the addition of puromycin several hours after the initiation of LICC culture was observed even after the removal of the GLT present in the supernatant, suggesting that the morphologically intact target cells were already affected by GLT in the early stages of LICC culture.