Secretion Of Beta-glucuronidase Research Articles

Treatment of chronic proliferative cholangitis with c-myc shRNA

To investigate the feasibility and effectiveness of c-myc shRNA in inhibiting the hyperplastic behavior and lithogenic potentiality of chronic proliferative cholangitis (CPC), in order to prevent stone recurrence and biliary restenosis. An animal model of CPC was established by giving intralumenally 0.5 mL of c-myc shRNA. Then, the effects of c-myc shRNA on hyperplastic behavior and lithogenic potentiality of CPC were evaluated by histological observation, immunohistochemistry, real-time PCR and Western blotting for c-myc, proliferating cell nuclear antigen (PCNA), procollagen III, mucin 5AC, enzymatic histochemistry for beta-glucuronidase, and biochemistry for hydroxyproline in the diseased bile duct. Treatment with c-myc shRNA efficiently suppressed the hyperplasia of biliary epithelium, submucosal gland, and collagen fiber by inhibiting mRNA and protein expression of c-myc. More importantly, it decreased the lithogenic potentiality of CPC by inhibiting the expression of mucin 5AC and the secretion of endogenous beta-glucuronidase. Further investigation indicated that c-myc shRNA-3 had a better inhibitory effect on CPC. Treatment with c-myc shRNA-3 can control CPC and reduce the lithogenic potentiality of CPC.

Membrane receptor‐initiated signaling in 1,25(OH)2D3‐stimulated calcium uptake in intestinal epithelial cells

Demonstrating 1,25(OH)2D3-stimulated calcium uptake in isolated chick intestinal epithelial cells has been complicated by simultaneous enhancement of both uptake and efflux. We now report that in intestinal cells of adult birds, or those of young birds cultured for 72 h, 1,25(OH)2D3-stimulates 45Ca uptake to greater than 140% of corresponding controls within 3 min of addition. Such cells have lost hormone-stimulated protein kinase C (PKC) activity, believed to mediate calcium efflux. To further test this hypothesis, freshly isolated cells were preincubated with calphostin C, and calcium uptake monitored in the presence or absence of steroid. Only cells treated with the PKC inhibitor demonstrated a significant increase in 45Ca uptake in response to 1,25(OH)2D3, relative to corresponding controls. In addition, phorbol ester was shown to stimulate efflux, while forskolin stimulated uptake. To further investigate the mechanisms involved in calcium uptake, we assessed the role of TRPV6 and its activation by beta-glucuronidase. beta-Glucuronidase secretion from isolated intestinal epithelial cells was significantly increased by treatment with 1,25(OH)2D3, PTH, or forskolin, but not by phorbol ester. Treatment of cells with beta-glucuronidase, in turn, stimulated 45Ca uptake. Finally, transfection of cells with siRNA to either beta-glucuronidase or TRPV6 abolished 1,25(OH)2D3-enhanced calcium uptake relative to controls transfected with scrambled siRNA. Confocal microscopy further indicated rapid redistribution of enzyme and calcium channel after steroid. 1,25(OH)2D3 and PTH increase calcium uptake by stimulating the PKA pathway to release beta-glucuronidase, which in turn activates TRPV6. 1,25(OH)2D3-enhanced calcium efflux is mediated by the PKC pathway.

The exocytosis induced in HL-60 cells by 4-hydroxynonenal, a lipid peroxidation product, is not prevented by reduced glutathione

The effect of reduced glutathione (GSH) was studied on exocytosis triggered by 4-hydroxynonenal in HL-60 cells induced to differentiate towards the granulocytic cell line by dimethylsulfoxide; we measured beta-glucuronidase secretion from cells incubated at 37 degrees C in the presence of 5 mM GSH. GSH addition to the cell suspensions failed to induce any significant change of the exocytosis stimulated by HNE concentrations between 10(-8) and 10(-6) M. In contrast however, 5 mM GSH was able to fully prevent the release of lactate dehydrogenase observed in the presence of 50 microM HNE, a concentration much higher than that able to stimulate the exocytotic secretion. As the activation of phosphoinositide-specific phospholipase C (PLC) has been shown to play a major role in HNE-induced exocytosis, we studied the GSH effect on the breakdown of phosphatidylinositol-4,5-bisphosphate added to plasma membranes isolated from rat neutrophils and incubated in the presence of increasing concentrations of the aldehyde. In neutrophil membranes HNE induced a significant increase of PLC activity when used in the same concentrations as those able to stimulate beta-glucuronidase secretion in DMSO-differentiated HL-60 cells; the presence of 5 mM GSH failed to prevent its action. Our results suggest that these low aldehyde concentrations, which have actually been found in exudates, may increase tissue damage in inflammation through the release of lytic enzymes by neutrophils; it seems unlikely that their effects could be influenced by the levels of -SH groups present in the exudate and by its protein concentration.

A novel nonpeptide ligand for formyl peptide receptor-like 1.

Formyl peptide receptor-like 1 (FPRL1) is a G protein-coupled receptor that binds natural and synthetic peptides as well as lipoxin A(4) and mediates important biological functions. To facilitate its pharmacological characterization, we screened a compound library and identified a substituted quinazolinone (Quin-C1, 4-butoxy-N-[2-(4-methoxy-phenyl)-4-oxo-1,4-dihydro-2H-quinazolin-3-yl]-benzamide) as a ligand for FPRL1. Quin-C1 induces chemotaxis and secretion of beta-glucuronidase in peripheral blood neutrophils with a potency of approximately 1/1000 of that of the peptide agonist WKYMVm. In studies using transfected rat basophilic leukemia (RBL) cell lines expressing either formyl peptide receptor or FPRL1, Quin-C1 induced enzyme release from RBL-FPRL1 but not RBL-FPR cells. Likewise, Quin-C1 selectively stimulates calcium mobilization in RBL-FPRL1 cells, a response that was markedly inhibited by pertussis toxin. Quin-C1 also stimulates phosphorylation of extracellular signal-regulated protein kinases 1 and 2 and induces internalization of an FPRL1 fused to green fluorescent protein. In degranulation assays, both the FPRL1-selective peptide agonist MMK1 and Quin-C1 exhibited lower efficacy and potency than WKYMVm, with EC(50) values of 7.17 x 10(-8) M and 1.88 x 10(-6) M, respectively, compared with the EC(50) value for WKYMVm (2.29 x 10(-8) M). However, Quin-C1 did not induce neutrophil superoxide generation at up to 100 microM. Based on these results, we conclude that Quin-C1 is a novel nonpeptide ligand that binds to FPRL1 and selectively stimulates FPRL1-mediated functions. Quin-C1 is a prototype of substituted quinazolinones based on which further structural modifications may be made to improve its efficacy and potency for FPRL1.

Synthesis and antiinflammatory evaluation of 9-anilinoacridine and 9-phenoxyacridine derivatives.

Mast cells, neutrophils, and macrophages are important inflammatory cells that have been implicated in the pathogenesis of acute and chronic inflammatory diseases. To explore a novel antiinflammatory agent, we have synthesized two types of acridines, 9-anilinoacridine and 9-phenoxyacridine derivatives, for evaluation on the grounds that acridine is a versatile heterocycle possessing a wide variety of biological properties. The title compounds were synthesized by reaction of 9-chloroacridine with appropriate Ar-NH(2) and Ar-OH, and their antiinflammatory activities on inhibitory effects on the activation of mast cells, neutrophils, and macrophages were studied. Three acridine derivatives 4, 10, and 11 were proved to be more potent than the reference inhibitor mepacrine for the inhibition of rat peritoneal mast cell degranulation with similar IC(50) values (16-21 microM). Compound 3 also showed potent inhibitory activity (IC(50) = 8.2 and 4.4 microM, respectively) for the secretion of lysosomal enzyme and beta-glucuronidase from neutrophils. Moreover, compounds 5 and 9 were shown to be efficacious inhibitors of TNF-alpha production in macrophage-like cell lines RAW 264.7. Compounds 2 and 12 were the potent inhibitors of TNF-alpha production in murine microglial cell lines N9. To further explore the cytotoxic properties of these acridine derivatives, (E)-12 was selected for NCI's in vitro disease-oriented tumor cells screen. The results indicated that this compound had no significant cytotoxicity with a mean GI(50) of 58.0 microM. These results indicated that the antiinflammatory effects of acridine derivatives were mediated, at least in part, through the suppression of chemical mediators released from mast cells, neutrophils, and macrophages and that these compounds have the potential to be novel antiinflammatory agents with no significant cytotoxicity.

Effect of 4‐hydroxynonenal, a lipid peroxidation product, on exocytosis in HL‐60 cells

Our work analysed the effect of 4-hydroxynonenal (HNE), a chemotactic aldehydic end-product of lipid peroxidation, on exocytosis in HL-60 cells. We measured the release of beta-glucuronidase, an enzyme of azurophil granules, from the cells incubated at 37 degrees C for 10 min in the presence of HNE concentrations ranging between 10(-8) and 10(-5) M. The release of lactate dehydrogenase was assayed to test cell viability. HNE (1 microM) was able to induce a significant and strong stimulation of beta-glucuronidase secretion without leading to cytotoxic effects. The finding that HNE could increase the exocytotic secretion from HL-60 cells together with its known chemotactic property supports the hypothesis that this lipid peroxidation product may play an important role as a chemical mediator of inflammation; moreover it is noteworthy that micromolar concentrations of HNE have actually been found in exudates from acute and chronic inflammations.

Androgen regulation of glycosidase secretion in epithelial cell cultures from human epididymis.

The human epididymis and its secretions actively promote sperm fertilizing capacity and provide protection for spermatozoa against harmful influences. Among epididymal secretions, glycosidases have been recently studied and associated with molecular changes on the sperm surface. In the present work, we studied the influence of different concentrations of testosterone, dihydrotestosterone and cyproterone acetate on the secretion of alpha-glucosidase, N-acetyl-glucosaminidase, beta-glucuronidase and alpha-mannosidase by isolated and cultured epithelial cells from human caput, corpus and cauda epididymides. Cell cultures were obtained from aggregates of isolated tubule fragments plated on extracellular matrix-covered multi-well plates. Activities of the glycosidases were measured in conditioned culture media and were higher in the distal regions of the epididymis. Testosterone and dihydrotestosterone significantly increase the enzyme secretion in a concentration-dependent manner. This increase was higher in corpus and/or cauda than in caput epididymis. Cyproterone acetate caused a dose-dependent decrease in glycosidase secretion in cultures from all epididymal regions. It is concluded that the secretion of epididymal glycosidases is regulated by androgen, being stimulated by dihydrotestosterone and testosterone and inhibited by the androgen antagonist cyproterone acetate.

Effect of trans-resveratrol, a natural polyphenolic compound, on human polymorphonuclear leukocyte function.

1. Polymorphonuclear leukocytes (PMN) may contribute to the pathogenesis of acute coronary heart disease (CHD). 2. Epidemiological and laboratory evidence suggests that red wine, by virtue of its polyphenolic constituents, may be more effective than other alcoholic beverages in reducing the risk of CHD mortality. 3 The aim of the present study was to investigate the effects of trans-resveratrol (3,4',5-trihydroxy-trans-stilbene), a polyphenol present in most red wines, on functional and biochemical responses of PMN, upon in vitro activation. 4. trans-Resveratrol exerted a strong inhibitory effect on reactive oxygen species produced by PMN stimulated with 1 microM formyl methionyl leucyl phenylalamine (fMLP) (IC50 1.3+/-0.13 microM, mean+/-s.e.mean), as evaluated by luminol-amplified chemiluminescence. 5. trans-Resveratrol prevented the release of elastase and beta-glucuronidase by PMN stimulated with the receptor agonists fMLP (1 microM, IC50 18.4+/-1.8 and 31+/-1.8 microM), and C5a (0.1 microM, IC50 41.6+/-3.5 and 42+/-8.3 microM), and also inhibited elastase and beta-glucuronidase secretion (IC50 37.7+/-7 and 25.4+/-2.2 microM) and production of 5-lipoxygenase metabolites leukotriene B4 (LTB4), 6-trans-LTB4 and 12-trans-epi-LTB4 (IC50 48+/-7 microM) by PMN stimulated with the calcium ionophore A23187 (5 microM). 6. trans-Resveratrol significantly reduced the expression and activation of the beta2 integrin MAC-1 on PMN surface following stimulation, as revealed by FACS analysis of the binding of an anti-MAC-1 monoclonal antibody (MoAb) and of the CBRM1/5 MoAb, recognizing an activation-dependent epitope on MAC-1. Consistently, PMN homotypic aggregation and formation of mixed cell-conjugates between PMN and thrombin-stimulated fixed platelets in a dynamic system were also prevented by transresveratrol. 7. These results, indicating that trans-resveratrol interferes with the release of inflammatory mediators by activated PMN and down-regulates adhesion-dependent thrombogenic PMN functions, may provide some biological plausibility to the protective effect of red wine consumption against CHD.

Long-term secretion of therapeutic proteins from genetically modified skeletal muscles.

Protein delivery from genetically modified skeletal muscle has been reported previously. However, a stable and prolonged secretion was obtained in immunocompromised or newborn animals only. To evaluate the clinical relevance of this approach, we have transduced myoblasts from an adult beta-glucuronidase-deficient (MPS VII) mouse with retroviral vectors carrying either the human beta-glucuronidase cDNA or the murine erythropoietin (Epo) cDNA. The cells were then grafted into the tibialis anterior muscle of adult immunocompetent MPS VII recipients. Protein expression was controlled either by ubiquitous or muscle-specific transcriptional regulatory elements. Animals were analyzed over an 8-month period. The in situ detection of beta-glucuronidase activity revealed up to 60% of genetically modified myofibers in the recipient muscles. The human desmin promoter and enhancer showed the highest in vivo expression. Secretion of beta-glucuronidase induced a disappearance of lysosomal storage lesions in the liver and spleen of recipient animals. Delivery of Epo led to a permanent increase of hematocrit values over 3 months. These results showed that the transplantation of genetically modified myoblasts allowed a sustained secretion of recombinant proteins at therapeutic levels in immunocompetent adult mice. They suggest that the approach may be considered for human applications.

Additive effects of acidosis and parathyroid hormone on mouse osteoblastic and osteoclastic function.

Patients with end-stage renal disease are acidotic and often develop secondary hyperparathyroidism. Whether acidosis contributes to the bone disease observed in these patients is not clear. To determine whether acidosis and parathyroid hormone (PTH) have additive effects on net calcium efflux (JCa+) from bone and on bone cell function, we measured JCa+, osteoblastic collagen synthesis, and osteoclastic beta-glucuronidase release from neonatal mouse calvariae cultured in control (Ctl, pH approximately 7.4) or acidified (Met, pH approximately 7.1) medium with or without a submaximal concentration of PTH (10(-10) M) for 48 h. Compared with Ctl, from 24 to 48 h JCa+ was increased with Met and with PTH, and the combination of Met + PTH increased JCa+ further. Compared with Ctl, collagen synthesis was decreased with Met and with PTH and decreased further with Met + PTH. There was an inverse correlation between percent collagen synthesis and JCa+. Compared with Ctl, beta-glucuronidase release into the medium was increased with Met and with PTH and increased further with Met + PTH. There was a direct correlation between medium beta-glucuronidase activity and JCa+. Osteoclastic beta-glucuronidase activity correlated inversely with osteoblastic collagen synthesis. During cultures to 96 h, there continued to be greater JCa+ from calvariae incubated with Met + PTH than from those with either treatment alone. Thus acidosis and PTH independently stimulated JCa+ from bone, inhibited osteoblastic collagen synthesis, and stimulated osteoclastic beta-glucuronidase secretion, whereas the combination had a greater effect on each of these parameters than either treatment alone. These findings indicate that acidosis and PTH can have an additive effect on bone cell function and suggest that uremic osteodystrophy may result from a combination of a low pH and an elevated PTH.

Secretion of plasminogen activator and lysosomal enzymes from mouse skeletal muscle: Effect of denervation

Levels of hydrolytic enzymes increase in skeletal muscle after denervation and their activities in the extracellular matrix appear to be important for interaction between muscle and nerve. Using enzymatic assays for beta-glucuronidase, beta-galactosidase, and plasminogen activator, we show that secretion of these enzymes from mouse skeletal muscle increases after denervation and that drugs interfering with the secretory pathway or the reuptake of enzymes modulate this release. Thus, brefeldin A inhibited secretion of plasminogen activator activity and mannan increased secreted amounts of beta-glucuronidase, but not of beta-galactosidase, in denervated muscle. In innervated muscle, brefeldin A decreased secreted activity of plasminogen activator, but mannan had no effect on secretion of either beta-glucuronidase or beta-galactosidase. Furthermore, secretion of plasminogen activator was temperature dependent. These observations, together with previous studies, suggest that secretion of hydrolytic enzymes from adult skeletal muscle may be of physiological significance in nerve-muscle communication.

Calcium overload toxicity and functional impairment in peritoneal leukocytes elicited by glycogen or interleukin-1 beta.

Although calcium plays an important role in the activation of leukocytes for such processes as eicosanoid biosynthesis, secretion of granular constituents and superoxide generation, sustained high levels of intracellular calcium ions may be toxic. We have previously found that high concentrations of calcium ionophores induce a rapid-onset "calcium overload" toxicity in rat peritoneal leukocytes, in which functional responses such as beta-glucuronidase secretion, superoxide generation and leukotriene B4 synthesis are greatly attenuated, and some leakage of cytoplasmic LDH occurs. We have now compared this phenomenon in peritoneal leukocytes elicited from animals pretreated in three ways: glycogen, interleukin-1 beta (IL-1 beta) alone or glycogen plus IL-1 beta. Peritoneal administration of IL-1 beta caused elicitation of cells which were enriched in eosinophils; however, the functional responses of the cells in all three groups were broadly similar in terms of the ability of the agonists FMLP, PMA and A23187 to initiate superoxide generation, beta-glucuronidase secretion and leukotriene generation. Cells from all three treatment groups showed diminished responsiveness at 10(-5) M A23187, indicative of calcium overload toxicity. This was most evident for the superoxide and beta-glucuronidase responses. Some quantitative differences observed between treatment groups may reflect the different sensitivities of the various cells contained in the mixed leukocyte preparations. We conclude that IL-1 beta induces leukocyte emigration into the peritoneal cavity but that the cell population is different from that induced by glycogen. However, the cells retain susceptibility to calcium overload toxicity.

Platelet dense granule membranes contain both granulophysin and P- selectin (GMP-140)

We recently reported the characterization of a platelet granule membrane protein of molecular weight (mol wt) 40,000 called granulophysin (Gerrard et al: Blood 77:101, 1991), identified by a monoclonal antibody (MoAb D545) raised to purified dense granule membranes. Using immunoelectron-microscopic techniques on frozen thin sections, this protein was localized in resting and thrombin-stimulated platelets. In resting platelets, labeled with antigranulophysin antibodies and immunogold probes, label was localized to the membranes of one or two clear granules per platelet thin section. D545 also labeled dense granules in permeabilized whole platelets and isolated dense granule preparations examined by whole-mount techniques. Expression of granulophysin on the platelet surface paralleled dense granule secretion as measured by 14C-serotonin release under conditions in which lysosomal granule release, as measured by beta-glucuronidase secretion, was less than 5%. After thrombin stimulation, both the surface-connected canalicular system and the plasma membrane were labeled, demonstrating redistribution of granulophysin associated with degranulation. Double labeling experiments with D545 and antibodies to the alpha-granule membrane protein, P-selectin, demonstrated labeling of both P-selectin and granulophysin on dense granule membranes. Distribution of both proteins on the plasma membrane after platelet stimulation was similar. The results demonstrate that granulophysin is localized to the dense granules of platelets and is redistributed to the plasma membrane after platelet activation.

Platelet Dense Granule Membranes Contain Both Granulophysin and P-Selectin (GMP-140)

Abstract We recently reported the characterization of a platelet granule membrane protein of molecular weight (mol wt) 40,000 called granulophysin (Gerrard et al: Blood 77:101, 1991), identified by a monoclonal antibody (MoAb D545) raised to purified dense granule membranes. Using immunoelectron-microscopic techniques on frozen thin sections, this protein was localized in resting and thrombin-stimulated platelets. In resting platelets, labeled with antigranulophysin antibodies and immunogold probes, label was localized to the membranes of one or two clear granules per platelet thin section. D545 also labeled dense granules in permeabilized whole platelets and isolated dense granule preparations examined by whole-mount techniques. Expression of granulophysin on the platelet surface paralleled dense granule secretion as measured by 14C-serotonin release under conditions in which lysosomal granule release, as measured by beta-glucuronidase secretion, was less than 5%. After thrombin stimulation, both the surface-connected canalicular system and the plasma membrane were labeled, demonstrating redistribution of granulophysin associated with degranulation. Double labeling experiments with D545 and antibodies to the alpha-granule membrane protein, P-selectin, demonstrated labeling of both P-selectin and granulophysin on dense granule membranes. Distribution of both proteins on the plasma membrane after platelet stimulation was similar. The results demonstrate that granulophysin is localized to the dense granules of platelets and is redistributed to the plasma membrane after platelet activation.

Differential responsiveness of human neutrophils to the autocrine actions of 1-O-alkyl-homologs and 1-acyl analogs of platelet-activating factor.

The phlogistic actions of six molecular species of platelet-activating factor (PAF) (1-O-alkyl-PAF homologs, 16:0-, 18:0- and 18:1-alkyl-PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC) and their respective 1-acyl-PAF analog counterparts, 16:0-, 18:0- and 18:1-acyl-PAF, 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (AGPC)) were assessed relative to five human neutrophilic polymorphonuclear leukocyte (PMN) functional responses: 1) lysosomal enzyme secretion; 2) specific desensitization to 16:0-AGEPC-induced lysosomal enzyme secretion; 3) O2- production; 4) chemotaxis; and 5) priming for enhanced O2- production. With respect to inducing lysozyme secretion, 18:0-AGEPC was 30- and 75-fold less potent than 16:0-AGEPC and 18:1-AGEPC, respectively, and was 25- and 40-fold less potent for inducing beta-glucuronidase secretion. 18:0-AGEPC was also 10-fold less active than 18:1- and 16:0-AGEPC for inducing O2- production. Thus, the rank order of potency of the alkyl-PAF homologs for inducing both lysosomal enzyme secretion and O2- production was 18:1- greater than or equal to 16:0- much greater than 18:0-AGEPC. In contrast, these three alkyl-PAF homologs had the same potency for desensitizing PMN to subsequent 16:0-AGEPC-induced lysosomal enzyme secretion and for priming PMN for augmented O2- production in response to FMLP or human recombinant C5a. Paradoxically, however, the rank order of potency of the alkyl-PAF homologs for effecting PMN chemotaxis was 18:0- greater than 18:1- much greater than 16:0-AGEPC. At concentrations as high as 1.0 microM, the acyl-PAF analogs did not initiate PMN lysosomal enzyme secretion, O2- production, or chemotaxis. However, the acyl-PAF analogs induced partial PMN desensitization to 16:0-AGEPC. A novel finding of potential (patho)-physiologic significance was the ability of acyl-PAF at nM concentrations to prime PMN for significantly enhanced O2- production after stimulation with FMLP or human recombinant C5a. The priming action of acyl-PAF was due to an increase in the rate as opposed to a prolongation of O2- production. The differing rank orders of potency of the alkyl-PAF homologs and acyl-PAF analogs for stimulating several physiologic responses of the same target cell, the human PMN, support the premise that there may be more than one PAF receptor subtype on the PMN and/or that differences in the biophysical properties of the various molecular species of PAF modulate their interaction with PAF receptor(s) linked to stimulus-response coupling.

Influence of human plasma kallikrein on lysosomal enzyme release from polymorphonuclear leukocytes.

Stimulation of polymorphonuclear leukocytes with kallikrein demonstrated that enzyme acts selectively on the release of lysosomal enzymes of these cells. The release of collagenase, similarly to the release of lysozyme into the incubation medium increased proportionally to kallikrein concentration and the duration of incubation. Kallikrein had a small effect on beta-glucuronidase secretion. No effect on cytoplasm lactate dehydrogenase release was detected. These results suggest that kallikrein, as a soluble stimulus, predominantly induces degranulation of specific granules containing collagenase capable of degrading the connective tissue. Secretion of lysozyme and collagenase requires the presence of active kallikrein. Soybean trypsin inhibitor diminished the enzyme release.

6-2) Release of Liver Microsomal β-Glucuronidase From Hepatocytes in vitro and in vivo by Organophosphates and Hepatotoxic Agents

Liver microsomal beta-glucuronidase is stabilized within microsomal vesicles by complexation with the accessory protein, named egasyn. In this study, we showed that egasyn is identical to one of the carboxylesterase isozymes and organophosphorus and carbamate insecticides, acetanilide which is a specific substrate of egasyn and halothane caused a rapid dissociation of the egasyn-microsomal beta-glucuronidase complex when administered in vivo or when added in vitro to isolated hepatocytes. The dissociation was relatively specific to organophosphates, carbamates, but not pyrethroids. Dissociation of the egasyn-beta-glucuronidase complex in vivo by organophosphates was followed by massive and rapid secretion of microsomal beta-glucuronidase into plasma. From these results, we concluded that release of liver microsomal beta-glucuronidase is the most rapid and sensitive marker to organophosphorus or carbamate insecticide-induced intoxication.

Inhibition of IgG-triggered human eosinophil function by IL-4.

Triggering of eosinophil secretory and cytotoxic functions by stimulation of the IgG and IgE FcR is thought to have major importance in the pathophysiology of tissue eosinophilia. We studied the ability of human rIL-4 to regulate this triggering event in human eosinophils. At doses ranging from 0.1 to 10 pg/ml, IL-4 suppressed eosinophil secretion of beta-glucuronidase and arylsulfatase by up to 65% after stimulation with IgG-coated Sepharose beads. This effect required prolonged preincubation (16 h) of eosinophils with IL-4; no effect was detected after 1 h preincubation. Enzyme secretion stimulated by IgE-coated beads was not affected. Further, IL-4 (after 16 h preincubation), suppressed eosinophil antibody-dependent killing of schistosomula (Schistosoma mansoni) targets by 24 to 39% in four experiments (p less than 0.05). Flow microfluorimetry analysis showed that IL-4 reduced the expression of IgG FcR, but not IgE FcR, suggesting that this mechanism underlies the suppression of IgG-mediated secretion. Taken collectively, these results demonstrate a mechanism for T lymphocyte suppression of IgG-stimulated eosinophil functions via IL-4.

The receptors for ATP and fMetLeuPhe are independently coupled to phospholipases C and A2 via G-protein(s). Relationship between phospholipase C and A2 activation and exocytosis in HL60 cells and human neutrophils

The relationship between phospholipase A2 and C activation and secretion was investigated in intact human neutrophils and differentiated HL60 cells. Activation by either ATP or fMetLeuPhe leads to [3H]arachidonic acid release into the external medium from prelabelled cells. This response was inhibited when the cells were pretreated with pertussis toxin. When the [3H]arachidonic acid-labelled cells were stimulated with fMetLeuPhe, ATP or Ca2+ ionophore A23187, and the lipids analysed by t.l.c., the increase in free fatty acid was accompanied by decreases in label from phosphatidylinositol and phosphatidylcholine. Moreover, incorporation of label into triacylglycerol and to a lesser extent phosphatidylethanolamine was evident. Activation of secretion was evident with ATP and fMetLeuPhe but not with A23187. The pharmacological specificity of the ATP receptor in HL60 cells was investigated by measuring secretion of beta-glucuronidase, formation of inositol phosphatases and release of [3H]arachidonic acid. External addition of ATP, UTP, ITP, adenosine 5'-[gamma-thio]triphosphate (ATP[S]), adenosine 5'-[beta gamma-imido]triphosphate (App[NH]p), XTP, CTP, GTP, 8-bromo-ATP and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) to intact HL60 cells stimulated inositol phosphate production, but only the first five nucleotides were effective at stimulating secretion or [3H]arachidonic acid release. In human neutrophils, addition of ATP, ITP, UTP and ATP[S] also stimulated secretion from specific and azurophilic granules, and this was accompanied by increases in cytosolic Ca2+ and in [3H]arachidonic acid release. The addition of phorbol 12-myristate 13-acetate (PMA; 1 nM) prior to the addition of either fMetLeuPhe or ATP led to inhibition of phospholipase C activity. In contrast, this had no effect on phospholipase A2 activation, whilst secretion was potentiated. Phospholipase A2 activation by either agonist was dependent on an intact cell metabolism, as was secretion. It is concluded that (1) activation of phospholipase C does not always lead to activation of phospholipase A2, (2) phospholipase A2 is coupled to the receptor independently of phospholipase C via a pertussis-toxin-sensitive G-protein and (3) for secretion to take place, the receptor has to activate both phospholipases C and A2.

Differential Effects of Interleukin-1 Alpha and Interleukin-1 Beta on Human Peripheral Blood Eosinophils

Interleukin-1 (IL-1) plays a major role in the response to infection, inflammation, and immunological challenge. Eosinophils participate in the host response to parasitic infection and allergic and hypersensitivity diseases. The role of IL-1 in these disease states has not been extensively explored. We have reported that purified human monocyte derived IL-1 (mIL-1), a mixture of the two IL-1 forms but predominantly consisting of IL-1 beta, modulates eosinophil oxidative metabolism and enzyme secretion. Although the two major species of IL-1 (IL-1 alpha and IL-1 beta) have identical specific activities on T cells, we now report the selective effects of human recombinant IL-1 (hrIL-1) alpha and hrIL-1 beta on eosinophil function. Whereas hrIL-1 beta caused a significant increase in arylsulfatase secretion (235.4 +/- 29% of resting secretion, P less than or equal to .01) and beta-glucuronidase secretion (135.8 +/- 9.6% of resting secretion, P less than or equal to .02) similar to our experience with mIL-1, hrIL-1 alpha had no effect on enzyme secretion. However, a mixture of hrIL-1 alpha and hrIL-1 beta reproduced the ability of mIL-1 to inhibit the oxidative response to suboptimal doses of phorbol myristate acetate (PMA). When eosinophils were separated into subpopulations by density gradients, we found that eosinophil responses to IL-1 differed among the populations. These results suggest that eosinophil subpopulations respond selectively to each form of IL-1.