Effect of lysophosphatidylserine on immunological histamine release

The effect of lysophosphatidylserine on immunological histamine release has been studied in rat peritoneal mast cells actively sensitized with horse serum and in human basophils challenged with anti-IgE. In contrast to other lysophospholipids, lysophosphatidylserine enhances the immunological histamine release in rat mast cells. The effect shows the kinetics of a saturable process with an apparent Km for lysophosphatidylserine of 0.26 microM. A similar Km value (0.21 microM) is found when measuring the non-immunological histamine release activated by lysophosphatidylserine plus nerve growth factor. A comparison with phosphatidylserine shows that a half-maximal response to lysophosphatidylserine occurs at a concentration 4-times lower. In addition, the magnitude of the response is higher. At variance with rat mast cells, lysophosphatidylserine does not influence the histamine release elicited by immunological and non-immunological stimuli in human basophils. The histamine secretion in these cells is instead affected by a calcium ionophore or tetradecanoylphorbolacetate, a compound producing activation of protein kinase C.     

Group II phospholipase A2 inhibitors suppressed lysophosphatidylserine-dependent degranulation of rat peritoneal mast cells.

Rat peritoneal mast cells were sensitized with IgE and challenged with the specific antigen in the presence of lysophosphatidylserine (lysoPS), an essential co-factor for rodent connective tissue mast cell degranulation, and the effects of phospholipase A2 inhibitors were examined. Mepacrine, a known inhibitor of phospholipase A2, at concentrations below 10(-5) M and anti-rat 14-kDa group II phospholipase A2 antibody inhibited histamine release, while they did not affect the prostaglandin generation. Like histamine release, prostaglandin generation in IgE- and antigen- challenged rat peritoneal mast cells was dependent on the presence of lysoPS. These results indicate that 14-kDa group II phospholipase A2 may play an essential role in IgE-, antigen-, and lysoPS-dependent degranulation process of rat peritoneal mast cells and that the mechanism whereby it participates may not be due to the production of lysoPS from PS in mast cell membranes.     

Signalling mechanism in the lysophosphatidylserine-induced activation of mouse mast cells.

Lysophosphatidylserine (0.1-1 microM) elicits histamine release in isolated mouse peritoneal mast cells. The effect becomes manifest after a lag of 30 s and reaches completion in 5 min. Maximal activity is observed when serine is in L-configuration. As shown by the activity of a lysophosphatidylserine analogue lacking the OH group in C2 position of glycerol, conversion into phosphatidylserine is not required. When 32PO4-labeled mast cells are challenged 2-5 min with lysophosphatidylserine, the labeling of phosphatidate, phosphatidylinositol and phosphatidylcholine is increased. When [3H]arachidonate-labeled mast cells are used, lysophosphatidylserine increases the appearance of isotopic diacylglycerol and phosphatidate. Like the secretory response, these effects are independent of the presence of extracellular Ca2+. Incubations in the presence of [3H]glycerol show that lysophosphatidylserine does not activate the de novo synthesis of phospholipids. In agreement with a participation of phosphoinositidase C in the action of lysophosphatidylserine, we observe accumulation of inositol phosphates in [3H]inositol labeled mast cells incubated in the presence of Li+. The results suggest that lysophosphatidylserine delivers its stimulus to mast cells, by the activation of phosphoinositide-dependent signalling mechanism.     

Presence of a high-affinity Ca2+- and Mg2+-dependent ATPase in rat peritoneal mast-cell membranes.

Purified perigranular and plasma membranes isolated from rat peritoneal mast cells were examined for Ca2+- and Mg2+-dependent ATPase activity. Isolated perigranular membranes contained only a low-affinity Ca2+- or Mg2+-dependent ATPase (Km greater than 0.5 mM). The plasma membranes contained both a low-affinity Ca2+- or Mg2+-dependent ATPase (Km = 0.4 mM, Vmax. = 20 nmol of Pi/min per mg), as well as a high-affinity Ca2+- and Mg2+-dependent ATPase (Km = 0.2 microM, Vmax. = 6 nmol of Pi/min per mg).     

Activation of phosphoinositide hydrolysis by nerve growth factor and lysophosphatidylserine in rat peritoneal mast cells

Histamine secretion in rat peritoneal mast cells stimulated by nerve growth factor requires a synergistic signal delivered by lysophosphatidylserine. To study the signal-transducing system activated by these compounds, phospholipid metabolism has been investigated in these cells. Phospholipid labeling with 32PO4 reveals a 5-9-fold stimulation of phosphatidic acid, phosphatidylinositol and phosphatidylcholine synthesis. Increased synthesis of phosphatidylinositol is also monitored using [3H]inositol incorporation. When [3H]inositol-labeled mast cells are incubated in the presence of Li+, nerve growth factor and lysophosphatidylserine enhance the accumulation of inositol monophosphate, inositol bisphosphate and inositol trisphosphate. Similar to the induced histamine release, accumulation of inositol phosphates (a) does not occur when the two agonists are added separately; (b) is inhibited when lysophosphatidyl-L-serine is replaced by lysophosphatidyl-D-serine; and (c) is enhanced in the presence of extracellular Ca2+. The data suggest that the interactive stimulus of nerve growth factor and lysophosphatidylserine is transmitted through the polyphosphoinositide-phospholipase C system.     

Synergism between lysophosphatidylserine and the phorbol ester tetradecanoylphorbolacetate in rat mast cells

In rat peritoneal mast cells tetradecanoylphorbolacetate (TPA) induced a non cytotoxic histamine release in the absence of extracellular calcium. The addition of calcium prevented the TPA effect but micromolar concentrations of lysophosphatidylserine (lysoPS) converted the calcium-induced inhibition into a stimulation. Other lysophospholipids were inactive. In agreement with a mutual influence between lysoPS and TPA, minimal TPA concentrations enhanced the calcium-dependent histamine release induced by lysoPS in the presence of nerve-growth factor. It is proposed that the calcium-dependent pathway promoted by lysoPS and the activation of protein kinase C by TPA act synergically to induce histamine release from mast cells.     

A defect in mobilization of cholesteryl esters in rabbit macrophages

Macrophages provide an important way for cholesteryl esters to accumulate in tissues in pathologic amounts. We studied cholesteryl ester metabolism in thioglycollate-induced peritoneal macrophages obtained from normocholesterolemic and hypercholesterolemic rabbits. The macrophage preparations from normocholesterolemic rabbit (MN cells) had 26 nmol esterified cholesterol/mg cellular protein, incorporated 1 nmol of labeled oleate into cholesteryloleate/2 h per mg cellular protein and had an acyl-coenzyme A:cholesterol acyltransferase activity of 22 pmol cholesterylpalmitate formed/min per mg protein in isolated membranes. The macrophage preparations from hypercholesterolemic rabbits (MHC cells) contained a 12-fold greater mass of cholesteryl ester, had an 8-times higher rate of formation of cholesteryloleate, and had 3-times more acyl-coenzyme A:cholesterol acyltransferase activity in the isolated membranes. When a cholesterol acceptor (10% fetal bovine serum or 10 mg of lipid-free fetal bovine serum protein) was added to the culture medium of rabbit MHC cells, the MHC cells retained more than 70% of their cholesteryl esters after 48 h of incubation. In contrast, when a cholesterol acceptor (10% fetal bovine serum) was added to the medium of thioglycollate-induced, cholesterol-enriched macrophages from mice, the mice macrophages retained only 19% of their cholesteryl esters after 48 h of incubation. The limited capacity of rabbit macrophages to release unesterified cholesterol from stored cytoplasmic cholesteryl esters to an exogenous acceptor may be related to the propensity of rabbits to develop atherosclerotic lesions.     

Mast Cells Contain Large Quantities of Secretagogue-Sensitive N-Acetylaspartate

Abstract: Mast cells play a central role in both immediate allergic reactions and inflammation. A functional nerve-mast cell interaction has been proposed, given the morphological association between mast cells and neuropeptide-containing peripheral nerves. We now show that purified rat peritoneal mast cells contain large quantities of N -acetylaspartate (NAA; 747.50 nmol/mg of protein). Mast cell levels of NAA were rapidly reduced, by 64.0 and 86.4%, following treatment with compound 48/80 and mastoparan, respectively. These secretagogues strongly decreased mast cell histamine content over the same time period, suggesting also that NAA is stored in secretory granules. The data are the first to show that NAA is present in an immune effector cell type. Because NAA may be involved in myelin synthesis and glutamyl peptide metabolism, NAA released from mast cells following nervous or other stimuli could participate in neuroimmune interactions. Mast cells in multiple sclerosis plaques may contribute to the reported elevations in brain NAA in this disease.     

Isolation of cellular membranes from rat mast cells

Large amounts of membranes enriched either in perigranular membranes or in plasma membranes have been successfully isolated from rat peritoneal mast cells. A cycle consisting of a single sonication pulse to disrupt the mast cells followed by centrifugation to separate the released granules was repeated until 90% of the mast cells were disrupted. This technique resulted in a high yield of intact granules since the released granules were only exposed to the single sonication pulse. The intact granules were separated from plasma membrane fragments by centrifugation through a Percoll gradient. The perigranular membranes were then obtained by osmotic lysis of the purified intact granules. The plasma membrane fraction was enriched 4.5-fold (range, 4.1-6.1) in 5'-nucleotidase activity, a plasma membrane marker enzyme. No suitable marker enzyme activity was found for the perigranular membrane fraction. An important aspect of this procedure is its potential for obtaining both a plasma and perigranular membrane preparation in high yield and purity from the same mast cell preparation.     

Ethanolamine metabolism in cultured bovine aortic endothelial cells

The role of extracellular ethanolamine in phospholipid synthesis was examined in cultured bovine aortic endothelial cells. Serine and ethanolamine were both readily accumulated by these cells and incorporated into phospholipid. Exposing cells to extracellular ethanolamine for 4-6 weeks had no effect on cell growth, yet increased the phosphatidylethanolamine content of these cells by 31% as compared to control cells. The intracellular content of ethanolamine was measured by high performance liquid chromatography, and results showed that the ethanolamine-treated cells contained a significantly greater amount of free ethanolamine compared to control cells (0.62 +/- 0.07 nmol/mg of protein versus 0.27 +/- 0.05 nmol/mg of protein, respectively). Ethanolamine-treated cells also had decreased accumulation and incorporation into lipid of [3H]ethanolamine throughout a 48-h incubation and increased K'm and V'max parameters of ethanolamine transport as compared to control cells. Studies were also done to examine the effect of ethanolamine on the generation of free ethanolamine from phosphatidylserine. In pulse-chase experiments with [3H]serine, a physiological concentration of ethanolamine (25 microM) decreased the amount of 3H-labeled phosphatidylethanolamine produced from 3H-labeled phosphatidylserine by 12 h as compared to the amount of 3H-labeled phosphatidyl-ethanolamine produced in the absence of ethanolamine in the chase incubation. Furthermore, ethanolamine-treated cells accumulated 20% less labeled ethanolamine in the aqueous pool from [3H]serine after 24 h of incubation than did control cells. These results can be explained by isotope dilution with the ethanolamine pool that accumulates in these cells with time when exposed to media supplemented with a physiological concentration of ethanolamine and by an effect of ethanolamine on ethanolamine generation from phosphatidylserine. The results show that an extracellular source of ethanolamine significantly influences the phospholipid metabolism of cultured bovine aortic endothelial cells.     

Proteinase-activated receptor-2-activating peptides induce leukocyte rolling, adhesion, and extravasation in vivo.

Proteinase-activated receptor 2 (PAR2) has been suggested to play a role in inflammatory reactions. Because leukocyte-endothelial cell interactions are critical events during inflammatory reactions, and because PAR2 is expressed both on endothelium and leukocytes, we have examined the effects of PAR2-activating peptides (PAR2-APs) on leukocyte rolling and adhesion in mesenteric venules and on leukocyte recruitment into the peritoneal cavity. Using intravital microscopy, leukocyte rolling, flux, and adhesion in rat mesenteric postcapillary venules were quantified. Topical addition of PAR2-APs (10 microM) for 1 min to the superfused venule induced a significant increase in leukocyte rolling and adherence. The increase in leukocyte adherence was not affected by pretreatment with a mast cell stabilizer (sodium cromoglycate) nor by prior degranulation of mast cells with compound 48/80. Nonetheless, both leukocyte rolling and adhesion were completely inhibited by pretreatment with a platelet-activating factor receptor antagonist (WEB 2086). Intraperitoneal injections of a selective PAR2-AP (SLIGRL-NH2) caused a significant increase in leukocyte migration into the peritoneal cavity. The effect of SLIGRL-NH2 on peritoneal leukocyte infiltration was completely inhibited by WEB 2086. These data suggest that PAR2 activation could contribute to several early events in the inflammatory reaction, including leukocyte rolling, adherence, and recruitment, by a mechanism dependent on platelet-activating factor release.     

A novel beta 1 integrin-dependent mechanism of leukocyte adherence to apoptotic cells

Adherence of leukocytes to cells undergoing apoptosis has been reported to be dependent on a variety of recognition pathways. These include alpha V beta 3 (CD51/CD61, vitronectin receptor), CD36 (thrombospondin receptor), macrophage class A scavenger receptor, phosphatidylserine translocated to the outer leaflet of apoptotic cell membranes, and CD14 (LPS-binding protein). We investigated the mechanism by which leukocytes adhere to apoptotic endothelial cells (EC). Peripheral blood mononuclear leukocytes and U937 monocytic cells adhered to human or bovine aortic EC induced to undergo apoptosis by withdrawal of growth factors, treatment with the promiscuous protein kinase inhibitor staurosporine, with the protein synthesis inhibitor and protein kinase activator anisomycin, or with the combination of cycloheximide and TNF-alpha. Expression of endothelial adherence molecules such as CD62E (E-selectin), CD54 (ICAM-1), and CD106 (VCAM-1) was not induced or increased by these treatments. A mAb to alpha V beta 3, exogenous thrombospondin, or blockade of phosphatidylserine by annexin V did not inhibit leukocyte adherence. Further, leukocyte binding to apoptotic EC was completely blocked by treatment of leukocytes but not EC with mAb to beta 1 integrin. These results define a novel pathway for the recognition of apoptotic cells.     

Human mast cells release metalloproteinase-9 on contact with activated T cells: juxtacrine regulation by TNF-alpha

Mast cells, essential effector cells in allergic inflammation, have been found to be activated in T cell-mediated inflammatory processes in accordance with their residence in close physical proximity to T cells. We have recently reported that mast cells release granule-associated mediators and TNF-alpha upon direct contact with activated T cells. This data suggested an unrecognized activation pathway, where mast cells may be activated during T cell-mediated inflammation. Herein, we show that this cell-cell contact results in the release of matrix metalloproteinase (MMP)-9 and the MMP inhibitor tissue inhibitor of metalloproteinase 1 from HMC-1 human mast cells or from mature peripheral blood-derived human mast cells. The expression and release of these mediators, as well as of beta-hexosaminidase and several cytokines, were also induced when mast cells were incubated with cell membranes isolated from activated, but not resting, T cells. Subcellular fractionation revealed that the mature form of MMP-9 cofractionated with histamine and tryptase, indicating its localization within the secretory granules. MMP-9 release was first detected at 6 h and peaked at 22 h of incubation with activated T cell membranes, while TNF-alpha release peaked after only 6 h. Anti-TNF-alpha mAb inhibited the T cell membrane-induced MMP-9 release, indicating a possible autocrine regulation of MMP release by mast cell TNF-alpha. This cascade of events, whereby mast cells are activated by T cells to release cytokines and MMP-9, which are known to be essential for leukocyte extravasation and recruitment to affected sites, points to an important immunoregulatory function of mast cells within the context of T cell-mediated inflammatory processes.     

Nerve growth factor modifies the expression of inflammatory cytokines by mast cells via a prostanoid-dependent mechanism

Nerve growth factor (NGF) is well recognized to have a number of potent effects on mast cells, including increasing mast cell numbers in vivo and inducing mast cell degranulation in vitro. More recently, NGF has been demonstrated to induce PGD2 production by mast cells through the induction of mast cell cyclooxygenase expression. We have observed that NGF at doses as low as 10 ng/ml will induce IL-6 production and inhibit TNF-alpha release from rat peritoneal mast cells in the presence of lysophosphatidylserine as a cofactor. NGF synergizes with LPS treatment of peritoneal mast cells (PMC) for the induction of IL-6. Examination of the mechanism of this phenomenon has revealed that NGF can induce both rat PMC and mouse bone marrow-derived cultured mast cells to produce substantial levels of PGE2. This response is maximal at later time points 18-24 h after NGF activation. The ability of NGF to induce PGE2 is not dependent on mast cell degranulation. Other stimuli capable of inducing IL-6, such as LPS, do not induce production of this prostanoid. Inhibition of cyclooxygenase activity by PMC using either flurbiprofen or indomethacin inhibited both the NGF-induced PGE2 synthesis and the NGF-induced alterations in TNF-alpha and IL-6 production. These results suggest a role for mast cell-derived prostanoids in the regulation of local inflammatory responses and neuronal degeneration after tissue injury involving induction of NGF production.     

Nerve growth factor induces mast cell degranulation without changing intracellular calcium levels

Nerve growth factor (NGF) induces degranulation of rat peritoneal mast cells (RPMC) in a dose-dependent manner, providing direct evidence for its action on non-neuronal tissues. Activation of RPMC by NGF depends on lysophosphatidylserine and extracellular calcium. NGF-mediated RPMC degranulation is not coupled to a transient increase in intracellular free calcium ([Ca2+]i). It is suggested that NGF has a unique mode of action independent of [Ca2+]i and presumably also without involving protein kinase C activation as indicated by the effects of phorbol esters and NGF on antigen-evoked [Ca2+]i rise.     

Mast cells contain spleen-type prostaglandin D synthetase

Prostaglandin D synthetase activity in the cytosol (100,000 x g, 1-h supernatant) fraction of peritoneal mast cells of adult rats (105.0 nmol/min/mg protein) was the highest among such activities in various rat tissues and cells. As judged by the absolute requirement for glutathione for the reaction (Km = 300 microM), the Km value for prostaglandin H2 (200 microM), and insensitivity of the activity to 1 mM 1-chloro-2,4-dinitrobenzene, the enzyme in mast cells was similar to rat spleen prostaglandin D synthetase and differed from rat brain prostaglandin D synthetase or glutathione S-transferase, all of which catalyze the isomerase reaction from prostaglandin H2 to prostaglandin D2. In immunotitration analyses, the activity in mast cells showed a titration curve exactly identical with that of the purified spleen-type enzyme and almost completely absorbed by an excess amount of antibody against this enzyme, but it remained unchanged after incubation with antibodies against the brain-type enzyme and glutathione S-transferase isozymes thus far purified. In Western blot after two-dimensional electrophoresis of crude extracts of mast cells, a single immunoreactive spot was observed with antibody against the spleen-type enzyme at the same position as that of the purified enzyme (Mr = 26,000, pI = 5.2). Furthermore, the immunoreactive protein obtained from mast cells showed the same peptide fingerprints as those of the purified spleen-type enzyme, after partial digestion with Staphylococcus aureus V8 protease or trypsin. In immunoperoxidase staining, the immunoreactivity of the spleen-type enzyme was found in the cytosol of tissue mast cells in various organs such as thymus, intestine, stomach, and skin of adult rats. These findings indicate that prostaglandin D2 is produced by the spleen-type synthetase in mast cells of various tissues.     

Identification of P-glycoprotein at the membrane of mast cell secretory granules. An immunofluorescence and protein A-gold electron microscopical investigation

The presence of P-glycoprotein has been investigated in rat peritoneal mast cells by means of immunofluorescence and immunogold electron microscopy, using the specific monoclonal antibody JSB-1. Immunofluorescence studi es showed that the glycoprotein is primarily concentrated in mast cell granules, and little is localized at the plasma membrane. Electron microscope observations revealed a marked accumulation of colloidal gold particles at the granule-coating membranes, whereas decoration of the plasma membrane is much less intense. When mast cells are stimulated to exocytate with compound 48/80, both immunofluorescence and electron microscopy showed concentration of P-glycoprotein reactivity at the plasma membrane level. Indeed, fusion of the granule with the plasma membrane allowed transfer of immunoreactive P-glycoprotein material from the granule-coating membrane to the cell surface membrane. These findings confirmed the presence of P-glycoprotein in mast cells; it is predominantly localized in the granules and is exposed on the cell surface only after exocytosis, suggesting, therefore, a possible physiological role for P-glycoprotein in the secretion of certain mediators. This revised version was published online in November 2006 with corrections to the Cover Date.     

Metabolism of ochratoxin A by primary cultures of rat hepatocytes.

Association of ochratoxin A with cultured rat hepatocytes occurs at 4 degrees C, and the saturation level in the medium is 0.3 mM ochratoxin A, with maximal binding after 60 min. At 37 degrees C the level of cell-associated ochratoxin A increased up to 6 h and remained at 2 nmol of toxin per mg of cell protein for 30 h. With increasing concentrations of ochratoxin A, increasing amounts of the toxin accumulated in the cells; saturation occurred at a concentration of 0.3 mM. Ochratoxin A was metabolized by hepatocytes at 37 degrees. (4R)-4-Hydroxyochratoxin A appeared in the medium at a maximal level (about 30 nmol/mg of cell protein) at an ochratoxin A concentration of 0.25 mM after 48 h of incubation. Small amounts of (4S)-4-hydroxyochratoxin A were detected only after incubation for 22 h or longer.     

Identification of P-glycoprotein at the membrane of mast cell secretory granules. An immunofluorescence and protein A-gold electron microscopical investigation

The presence of P-glycoprotein has been investigated in rat peritoneal mast cells by means of immunofluorescence and immunogold electron microscopy, using the specific monoclonal antibody JSB-1. Immunofluorescence studi es showed that the glycoprotein is primarily concentrated in mast cell granules, and little is localized at the plasma membrane. Electron microscope observations revealed a marked accumulation of colloidal gold particles at the granule-coating membranes, whereas decoration of the plasma membrane is much less intense. When mast cells are stimulated to exocytate with compound 48/80, both immunofluorescence and electron microscopy showed concentration of P-glycoprotein reactivity at the plasma membrane level. Indeed, fusion of the granule with the plasma membrane allowed transfer of immunoreactive P-glycoprotein material from the granule-coating membrane to the cell surface membrane. These findings confirmed the presence of P-glycoprotein in mast cells; it is predominantly localized in the granules and is exposed on the cell surface only after exocytosis, suggesting, therefore, a possible physiological role for P-glycoprotein in the secretion of certain mediators. This revised version was published online in November 2006 with corrections to the Cover Date.     

Development of mast cells in vitro. II. Biologic function of cultured mast cells.

Mast cells were obtained by long term culture of rat thymus cells on rat embryonic fibroblast monolayers. Pure mast cell preparations obtained culture were incubated with 125I-labeled rat E myeloma protein to study receptors for IgE on their surface. When the cells were obtained after 35 to 45 days culture, the average number of receptors per mast cell was 100,000 to 400,000. An equilibrium constant of the binding reaction between their receptor and rat IgE was in the order of 108 M-1. The histamine content of the cultured mast cells was 0.2 to 5 mug/106 cells. The measurement of histamine content in mast cells recovered after different periods of culture suggested that the histamine content increased with maturation. Even after 45 to 50 days culture, the histamine content of cultured mast cells was significantly lower than that in rat peritoneal mast cells. The cultured mast cells were passively sensitized in vitro with rat IgE antibody against Nippostrongylus brasiliensis. The sensitized cells released histamine upon incubation with the antigen. It was also found that cultured mast cells released histamine upon exposure to compound 48/80. These results indicated that cultured mast cells have physiologic functions similar to those of normal rat mast cells, but they have not reached full maturation.