Inosine binds to A3 adenosine receptors and stimulates mast cell degranulation

We investigated the mechanism by which inosine, a metabolite of adenosine that accumulates to > 1 mM levels in ischemic tissues, triggers mast cell degranulation. Inosine was found to do the following: (a) compete for [125I]N6-aminobenzyladenosine binding to recombinant rat A3 adenosine receptors (A3AR) with an IC50 of 25+/-6 microM; (b) not bind to A1 or A2A ARs; (c) bind to newly identified A3ARs in guinea pig lung (IC50 = 15+/-4 microM); (d) lower cyclic AMP in HEK-293 cells expressing rat A3ARs (ED50 = 12+/-5 microM); (e) stimulate RBL-2H3 rat mast-like cell degranulation (ED50 = 2.3+/-0.9 microM); and (f) cause mast cell-dependent constriction of hamster cheek pouch arterioles that is attenuated by A3AR blockade. Inosine differs from adenosine in not activating A2AARs that dilate vascular smooth muscle and inhibit mast cell degranulation. The A3 selectivity of inosine may explain why it elicits a monophasic arteriolar constrictor response distinct from the multiphasic dilator/constrictor response to adenosine. Nucleoside accumulation and an increase in the ratio of inosine to adenosine may provide a physiologic stimulus for mast cell degranulation in ischemic or inflamed tissues.     

Reagin synthesis in inbred rats. II. Genetic control of reaginic antibody synthesis

Specific reaginic (IgE) and hemagglutinating (IgG) antibodies were quantified after immunization with ovalbumin aluminum hydroxide gel in BN and ACI inbred rats, as well as their F1, F1 X BN backcross, F2, and F3, and F3 progeny. The dissimilarity of these immune responses indicated that reaginic (IgE) antibody synthesis was influenced by polygenic factors, but not sex, and was controlled by loci different from that governing hemagglutinating (IgG) antibody synthesis. In addition, tissue typing of the BN, ACI, F1, and F3 hybrids suggested that reaginic antibody synthesis was not linked to the major rat histocompatibility locus.     

Lack of involvement of mast cell degranulation in the antiarrhythmic effect of preconditioning in rats

It has been proposed that the cardioprotective effects of myocardial ischaemic preconditioning may involve the release of mast cell mediators. The aim of the study was to determine whether mast cells are involved in the antiarrhythmic effect of ischaemic preconditioning in rat hearts. Preconditioning was achieved, both in anaesthetised rats and in rat isolated hearts, by a 3-min temporary occlusion of the left main coronary artery followed by 10 min of reperfusion before a 30-min permanent occlusion. Preconditioning had a marked antiarrhythmic effect, reducing the number of ventricular ectopic beats from 1,176 +/- 69 to 490 +/- 139 and the incidence of ventricular fibrillation from 40% to 0. Administration of the mast cell-stabilising drugs lodoxamide tromethamine and sodium cromoglycate (20 mg/kg/h i.v. 30 min before and throughout experimental protocol) did not modify the antiarrhythmic effect of preconditioning. Sodium cromoglycate, but not lodoxamide tromethamine, itself significantly reduced the number of ectopic beats that occurred over a 30-min period of ischaemia (from 760 +/- 181 to 153 +/- 33 in nonpreconditioned animals). Both drugs abolished the decrease in arterial blood pressure that occurred on coronary artery occlusion. The decrease in arterial blood pressure produced by the mast cell-degranulating compound 48/80 (50 microg/kg; i.v.) was attenuated to a similar degree by both drugs (decreases in pressure of 53 +/- 7, 31 +/- 1, and 25 +/- 3 mm Hg in control, sodium cromoglycate-treated, and lodoxamide tromethamine-treated animals, respectively). In rat isolated hearts, degranulation of mast cells with three consecutive doses of 50 microg of compound 48/80 had no antiarrhythmic effects and did not modify the antiarrhythmic effect of preconditioning. It is concluded that cardiac mast cells do not play a major role in the protection offered by ischaemic preconditioning on arrhythmogenesis in rat hearts.     

Increased growth promoting but not mast cell degranulation potential of a covalent dimer of c-Kit ligand

The native form of soluble c-kit ligand (KL) is a noncovalent dimer. We have isolated a soluble, disulfide-linked dimer of murine KL (KL-CD) by expressing KL in Escherichia coli and refolding the denatured protein under conditions that promote the formation of both noncovalent dimers (KL-NC) and KL-CD. KL-CD exhibits a 10- to 15-fold increase in the ability to stimulate the growth of both the human megakaryocytic cell line MO7e and murine bone marrow-derived mast cells relative to KL-NC. Colony-forming assays of murine bone marrow progenitor cells also reflected this increased potency. However, KL-CD and KL-NC are equally able to prime mast cells for enhanced IgE-dependent degranulation in vitro and activate mast cells in vivo. Improving the growth-promoting activity of KL without changing its mast cell activation potential suggests that KL-CD or a related molecule could be administered in the clinic at doses that stimulate hematopoietic recovery while avoiding significant mast cell activation.     

Is radiation-induced degranulation of mast cells in salivary glands induced by substance P?

Although DNA is the critical target for the lethal effects of irradiation, the precise mechanisms by which irradiation causes damage in tissues and biological systems is not fully understood. In the present study, the number of mast cells and the expression of the neuropeptide substance P (SP) in salivary glands were examined 10 days after a regimen of irradiation. The irradiation was given as a single dose or 5 consecutive days with daily doses of 7 Gy up to a total dose of 35 Gy. In addition, the number of mast cells and the expression of SP were examined 2 and 24 h after a single dose of 7 Gy. Immunohistochemical staining for 5-hydroxytryptamine (5-HT) and staining with avidin peroxidase and toluidine blue were used to detect mast cells. At examination 2 and 24 h after irradiation treatment, no change in the number of mast cells and the pattern of SP expression was observed. Ten days after irradiation there was a remarkable reduction in the number of mast cells in all the three glands, but there was a marked increase in the number of nerve fibers showing SP-like immunoreactivity in the parenchyme. The results show that early time-dependent alterations in the density of mast cells occur in response to irradiation, and that these changes occur concomitantly with changes in the expression of SP. Since the peripheral nervous system is a main regulator of salivary gland function, it is tempting to speculate that the nervous system interacts with mast cells via SP in modulating irradiation provoked tissue responses in salivary glands.     

A neurotensin receptor antagonist inhibits acute immobilization stress-induced cardiac mast cell degranulation, a corticotropin-releasing hormone-dependent process

Stress worsens certain disorders such as migraines or asthma, and has also been implicated in sudden myocardial arrest. It was previously shown that acute psychological stress by immobilization results in dura mast cell degranulation, an effect blocked by pretreatment with antiserum against corticotropin-releasing hormone (CRH). Moreover, CRH was recently shown to induce skin mast cell degranulation. The effect of psychological stress was investigated on rat cardiac mast cells, because their release of coronary constrictive and proinflammatory molecules contributes to myocardial ischemia and possibly arrhythmias. Immobilization of rats for 30 min induced maximal cardiac mast cell degranulation as evidenced by light and electron microscopy. This effect was inhibited by pretreatment with the "antiallergic" drug sodium cromoglycate (cromolyn), which is thought to act primarily through mast cell stabilization. Mast cell degranulation was also blocked by preincubation with antiserum against CRH and was partially inhibited by a CRH type-1 receptor selective antagonist. Sensory neuropeptides did not appear to influence this effect, but a nonpeptide neurotensin receptor antagonist blocked stress-induced cardiac mast cell degranulation. This finding supports the involvement of neuropeptide neurotensin which is present in the heart and is known to trigger mast cell degranulation. These results indicate acute stress could result in local CRH and nonpeptide neurotensin release which could contribute to myocardial pathophysiology through direct or indirect release of cardiac mast cell mediators.     

The src homology 2-containing inositol phosphatase (SHIP) is the gatekeeper of mast cell degranulation

To clarify the role that the src homology 2-containing inositol phosphatase (SHIP) plays in mast cell degranulation, the gene for SHIP was disrupted by homologous recombination in embryonic stem cells. Bone-marrow-derived mast cells from SHIP+/+, +/-, and -/- F2 littermates were compared. SHIP-/- mast cells were found to be far more prone to degranulation, after the crosslinking of IgE preloaded cells, than SHIP+/- or +/+ cells. Intriguingly, IgE alone also stimulated massive degranulation in SHIP-/- but not in +/+ mast cells. This degranulation with IgE alone, which may be due to low levels of IgE aggregates, correlated with a higher and more sustained intracellular calcium level than that observed with SHIP+/+ cells and was dependent upon the entry of extracellular calcium. Immunoprecipitation studies revealed that the addition of IgE alone to normal mast cells stimulates multiple cascades, which are prevented from progressing to degranulation by SHIP. PI 3-kinase inhibitor studies suggested that IgE-induced activation of PI 3-kinase is upstream of the entry of extracellular calcium and that SHIP restricts this entry by hydrolyzing phosphatidylinositol 3,4, 5-trisphosphate. These results show the critical role that SHIP plays in setting the threshold for degranulation and that SHIP directly modulates a "positive-acting" receptor.     

Bovine mastitis caused by Listeria monocytogenes: kinetics of antibody responses in serum and milk after experimental infection

The kinetics of antibodies in serum and milk directed against proteins from Listeria monocytogenes were studied using 4 lactating cows after infection was experimentally induced in the udder with four strains of serotypes 4b or 1/2a. Antibodies (IgG and IgA) in samples of composite quarter milk and serum of the cow were measured by indirect ELISA. Microtiter plates were coated with proteins obtained from the culture supernatant of L. monocytogenes 4b. After challenge, an IgG response in serum and milk to listerial infections in the udder occurred for all cows, although the response varied among cows. In sera, the IgG titers reached a peak at 9 to 13 wk after challenge and remained elevated until 21 to 33 wk after challenge. In milk, the IgG titer increased significantly 3 wk after the challenge for all cows. A weak and nonpersistent increase in IgA antibodies also occurred. These results indicate that IMI by L. monocytogenes induced an increase of antibodies in milk, which could be detected with an ELISA test using our antigenic preparation. Therefore, this antigenic preparation could be used for the evaluation of a new method of diagnosis for bovine mastitis caused by L. monocytogenes.     

Regulation of luminol-dependent chemiluminescence and degranulation by bovine neutrophils stimulated with opsonized zymosan

The purpose of this study was to elucidate likely signal transduction pathways in activated bovine neutrophils, by comparing the effects of various inhibitors on the bovine neutrophil respiratory burst and degranulation in vitro. The protein kinase C(PKC) inhibitors staurosporine, and chelerythine, and the beta-adrenergic receptor antagonist DL-propranolol, markedly inhibited opsonized zymosan (OZ) stimulated luminol-dependent chemiluminescence (LDCL). The G-protein inhibitor pertussis toxin (PT), the protein tyrosine inhibitor genistein, and the calcium channel blocker verapamil also reduced LDCL in a dose-dependent manner. In contrast, the lipoxygenase inhibitor zileuton had only a slight effect, and the cyclooxygenase inhibitor indomethacin had no effect on LDCL. The effects of these inhibitors on degranulation was also examined. Staurosporine, propranolol, and pertussis toxin significantly decreased primary granule (beta-glucosaminidase) release in response to OZ. These inhibitors also significantly reduced both phorbol myristate acetate (PMA)-induced primary and secondary granule (lactoferrin) release. Regulation of secondary granule (lactoferrin) release was complex, as it was significantly depressed by propranolol, enhanced by PT and unaffected by staurosporine. These findings suggest that PKC, beta-adrenergic receptors, G-proteins, protein tyrosine kinase(s) and Ca(2+) uptake, may all be involved in some part of the process of bovine neutrophil activation. Moreover, stimulation of LDCL and degranulation may be mediated through distinct signal transduction pathways.     

Regulation of the activity of secreted human lung mast cell tryptase by mast cell proteoglycans

When mast cells from human lungs were stimulated in vitro to degranulate, all of the tryptase secreted was found to be complexed with proteoglycans, three quarters with heparin proteoglycans and one quarter with chondroitin sulphate proteoglycans. Isolation of the tryptase-proteoglycan complexes by fibronectin affinity chromatography and gel filtration on a Sephacryl S-200 column gave the complexes an apparent Mr of 200000, suggesting the presence of heparin and chondroitin sulphate proteoglycans (Mrs=60000) and tryptase (Mr=134000) in a molar ratio of 1:1, equivalent to a mass ratio of about 0.45:1. However, analysis of the total mast cell releasate showed that it contained more proteoglycans (mass ratio of about 2:1) than was needed to complex tryptase. We could demonstrate that the releasate contained two proteoglycan fractions, one complexed (20%) with tryptase and the other not (80%). Incubation of the isolated tryptase-proteoglycan complexes led to rapid monomerisation and inactivation of tryptase, whereas the releasate, containing both complexed and free proteoglycans, retained its tryptase activity for up to at least 18 h. The results indicate that the majority of the proteoglycans secreted by stimulated lung mast cells, although not complexed with the secreted tryptase, are critical for the preservation of its activity.     

Influence of the phosphatidylethanolamine (PE) and bovine serum origins on anti-PE antibody detection by ELISA

The long-term results of external irradiation in locally advanced prostate cancer are poor: relapses are local or distant from infra-clinical disease present at diagnosis. Three clinical randomized trials have shown that hormonotherapy with LH-RH analogue with or without antiandrogen has improved: disease-free survival, local recurrence-free survival and metastasis-free survival (P < 0.001). The EORTC trial 22863 has shown a significant improvement of the overall survival (P = 0.001), with an LH-RH analogue (goserilin acetate, Zoladex) was administered the first day of irradiation and then every 4 weeks for 3 years; for the RTOG trial 85-31 the same LH-RH analogue was administered during the last week of irradiation and given until relapse increases survival of patients with poor differentiated tumours with a Gleason score ranging from 8 to 10 (P = 0.03). Adjuvant hormonal treatment with an LH-RH analogue is recommended, but the optimal duration of the treatment remains unclear.     

Acute mast cell leukemia disclosed by vasomotor flushing

INTRODUCTION: Acute mast cell leukemia is a rare and severe disease. We report herein a case associated with a flush syndrome. CASE REPORT: A 44-year-old man, presented with a flush of face and trunk. Bone marrow was infiltrated with immature mast cells. In spite of chemotherapy and bone marrow transplantation the patient deceased. DISCUSSION: Pheochromocytoma, carcinoid tumor, and mastocytosis are associated with a flush syndrome. In our patient the diagnosis was an acute mast cell leukemia. Acute mast cell leukemia can follow systemic mastocytosis or occur de novo. This disease is of poor prognosis. No treatment is available.     

Monoclonal antibody AE-2 modulates carbamate and organophosphate inhibition of fetal bovine serum acetylcholinesterase

The monoclonal antibody AE-2, raised against the human erythrocyte acetylcholinesterase (AChE) dimer (acetylcholine acetylhydrolase, EC 3.1.1.7), binds to other mammalian AChEs, including the tetramer that occurs in fetal bovine serum (FBS). AE-2 partially inhibited the rate of hydrolysis of the charged substrate acetylthiocholine by FBS AChE, whereas it increased the rate of hydrolysis of the neutral substrate indophenyl acetate. Present results show that AE-2 decreases the rate of inhibition of FBS AChE by the positively charged organophosphate amiton-p-toluene sulfonate and the positively charged carbamates pyridostigmine and neostigmine but accelerates inhibition of FBS AChE by the neutral organophosphates paraoxon and diisopropylfluorophosphate. Results suggest that AE-2 may allosterically modulate an anionic site in the catalytic center of FBS AChE.     

Rat interferons. Homologous and heterologus rat serum activity

Serum proteins from normal rat serum and induced with Sindbis virus were separated on Sephadex G-100. Interferon activity was studied in a system of homologous REC and heterologous LG cells. Proteins of normal serum as well as induced serum emerged in two peaks separated by a deep saddle. Four hours after induction, the serum gave two peaks with similar interferon activity in both cell systems. After 8-hour induction, two interferons were also obtained, but activity in the heterologous system was much lower than in the homologous system. Electrophoresis in polyacrylamide gel and on paper showed that interferon activity is connected mainly with the alpha1-globulin fraction.     

Reactivity of native conglutinin in bovine serum with rabbit antibody against recombinant bovine conglutinin with deletion of the N-terminal and collagen-like regions

The reactivity of native bovine conglutinin (Kg) with antibody against recombinant Kg (rKg), with deletion of the N-terminal and collagen-like regions of the native Kg molecule, was studied by sandwich enzyme-linked immunosorbent assay. With anti-recombinant Kg antibody as the coating antibody, rKg reacted with biotinylated homologous anti-rKg and heterologous anti-Kg antibodies as probing antibodies, while native Kg did not. With anti-native Kg antibody as coating antibody, native Kg reacted with biotinylated homologous antibody as probing antibody, while recombinant Kg reacted weakly with both biotinylated homologous and heterologous antibodies. Consequently the N-terminal and collagen-like regions of native Kg molecule are essential to express the complete immunogenicity and/or antigenicity of the native Kg molecule.     

Proteinases of rat mast cells. Peritoneal but not intestinal mucosal mast cells express mast cell proteinase 5 and carboxypeptidase A

Six basic proteins of 26 to 38 kDa with isoelectric points (pI) > or = 8.5 were abundant in proteins separated by two-dimensional SDS-PAGE from adult rat peritoneal mast cells (MC). One was identified previously as rat mast cell proteinase (RMCP) 1, a chymase of 26 to 28 kDa, pI > 9.0. Microsequence analyses showed that two polypeptides of about 29 and 30 kDa had NH2 terminal amino acid sequences homologous to mouse MC proteinase 5 (MCP-5), whereas the amino terminals of the 33, 35, and 36 kDa proteins were homologous to MC carboxypeptidase A (MC-CPA). Rabbit Abs produced against synthetic peptides of the identified NH2 terminal sequences were used in immunoblot studies. At least three proteins reacted with Abs to MC-CPA, whereas Abs to MCP-5 detected three adjacent polypeptides, rather than just the two identified by using microsequence analysis. Removal of oligosaccharide side chains using peptide:N-glycosidase F reduced the heterogeneity of each set of three polypeptides (MCP-5 and MC-CPA) to a band of each protein of a lower M(r). The serine proteinase inhibitor [3H]diisopropylfluorophosphate ([3H]DFP) bound to a proteinase of 30 to 35 kDa, which is probably MC tryptase (pI < or = 6.0). Immunoblot analysis of proteins from intestinal mucosal mast cells showed RMCP-2, but not RMCP-1, MCP-5, or MC-CPA. This is the first report of MCP-5 in the rat and of clearly distinguishable glycosylated forms of MC CPA. These proteinases appear to be restricted in their distribution to selected MC populations, but little is known about their functions.