The role of immunoglobulins in alternative complement pathway activation by zymosan. I. Human IgG with specificity for Zymosan enhances alternative pathway activation by zymosan

Prior absorption of normal human serum (NHS) or C2-deficient human serum (C2D) with zymosan at 0 degrees C results in diminished consumption of C3 and factor B during subsequent incubation of the sera in Mg-EGTA buffer with zymosan at 37 degrees C for 30 min. An acid eluate from the zymosan restores the defect of absorbed NHS and C2D, and also enhances C3 and factor B utilization in hypogammaglobulinemic serum (H gamma S) in a dose-dependent fashion. The activity is specific in that the eluate from zymosan fails to enhance C3 and B depletion in H gamma S or absorbed NHS by lipopolysaccharide or Sepharose. The active component of th zymosan eluate emerges from both Sepharose 4B and Sephacryl S-200 in the region of molecules with m.w. of 150,000. Absorption with protein A-Sepharose removes the activity, demonstrating that it is IgG. Digestion of the IgG with pepsin fails to diminish activity, indicating that the Fc region is not required for activity; reduction to monovalent Fab' fragments, however, abrogates activity. When IgG antibody is bound to Protein A-Sepharose, it fails to enhance C3 depletion in H gamma S by Sepharose, indicating that binding of IgG antibody by the Fab region is necessary for enhancement of alternative pathway activity in human serum.     

Modulation of rat serosal mast cell biochemistry by in vivo dexamethasone administration

To examine steroid-induced biochemical alterations in the mast cell secretory process, rats were injected with intramuscular dexamethasone or saline for 4 days, and serosal mast cells and lung tissue were obtained from each group. Radioligand binding studies utilizing 1-[propyl-1,2-3H]dihydroalprenolol (3H-DHA) demonstrated a 23.1 +/- 0.8% increase in rat lung beta-adrenergic receptors in steroid-treated rats, but the mast cell beta-adrenergic receptors were unaffected. Neither resting mast cell cyclic adenosine 3':5'-monophosphate (cAMP) levels nor the degree of cAMP augmentation induced by isoproterenol were changed by steroid administration. Mast cells from rats treated with dexamethasone released only 48.6 +/- 8.9 and 58.8 +/- 6.0% of the beta-hexosaminidase released from saline-treated rat mast cells when sensitized with anti-dinitrophenyl (DNP) IgE and challenged with DNP-bovine serum albumin antigen or the calcium ionophore A23187, respectively. [3H]serotonin release in cells from steroid-treated rats was 41.8 +/- 7.9 and 87.6 +/- 2.6% of control release stimulated by antigen or A23187, respectively. [14C]arachidonic acid incorporation into mast cell phospholipids followed by antigen or A23187 challenge revealed that cells from dexamethasone-treated rats release 61.3 +/- 15.6% and 62.1 +/- 11.8% of labeled metabolites, respectively, compared to controls. The addition of exogenous arachidonic acid 5 min prior to antigen challenge caused a similar decrease in mediator release in cells from saline- and steroid-treated rats (36.7 +/- 6.1 and 38.4 +/- 0.9%, respectively). When arachidonic acid was added to sensitized cells after specific antigen, no significant changes in beta-hexosaminidase release were noted in either group. Chronic in vivo dexamethasone administration markedly decreases mast cell mediator release without changing resting cAMP levels. The release of arachidonic acid metabolites is reduced in steroid-treated cells, possibly through the inhibition of phospholipases. Exogenous arachidonic acid cannot overcome this inhibition, suggesting that an earlier step in phospholipid metabolism, perhaps involving phospholipase C, may be important.     

Rat mast cell phospholipase A2: activity toward exogenous phosphatidylserine and inhibiton by N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylserine

The presence of phospholipase A2 in intact rat peritoneal mast cells was investigated by using two synthetic radiolabeled phosphatidylserine (PS) substrates. Incubation of intact cells with 1-oleoyl-2-[3H]oleoyl-PS resulted in the release of a considerable quantity of [3H]oleic acid from the substrate. To establish that [3H]oleic acid release was mediated via direct enzymatic attack at the sn-2 position, we measured release of the [3H]serine moiety from the glycerol backbone of 1,2-dimyristoylphosphatidyl[3H]serine. This activity, which represents the combined actions of phospholipases C and D, was 10-fold lower than [3H]oleic acid release, indicating that neither of these enzymes is required for the release of the preponderance of [3H]oleic acid. These results establish the existence in intact rat mast cells of a phospholipase A2 active toward exogenous PS. Over the concentration range at which exogenous PS activates mast cell secretion, intact mast cells and broken cells possessed nearly equal levels of phospholipase A2 activity, and enzyme activity was 3--4-fold higher toward PS than phosphatidylcholine. Several agents were tested for their ability to inhibit phospholipase A2 in intact mast cells. Of the agents tested, an N-substituted derivative of PS previously identified as an inhibitor of mast cell secretion was shown to be a particularly potent and efficacious inhibitor of mast cell phospholipase A2. The concentration dependence of enzyme inhibition paralleled inhibition of histamine secretion, providing a strong positive correlation between the level of phospholipase A2 in mast cells and the capacity for secretion.     

A major role for phospholipase A2 in antigen-induced arachidonic acid release in rat mast cells.

Cross-linking of IgE receptors by antigen stimulation leads to histamine release and arachidonic acid release in rat peritoneal mast cells. Investigators have reported a diverse distribution of [3H]arachidonate that is dependent on labelling conditions. Mast cells from rat peritoneal cavity were labelled with [3H]arachidonic acid for different periods of time at either 30 or 37 degrees C. Optimum labelling was found to be after 4 h incubation with [3H]arachidonate at 30 degrees C, as judged by cell viability (Trypan Blue uptake), responsiveness (histamine release) and distribution of radioactivity. Alterations in 3H-radioactivity distribution in mast cells labelled to equilibrium were examined on stimulation with antigen (2,4-dinitrophenyl-conjugated Ascaris suum extract). The results indicated that [3H]arachidonic acid was lost mainly from phosphatidylcholine and, to a lesser extent, from phosphatidylinositol. A transient appearance of radiolabelled phosphatidic acid and diacylglycerol indicated phosphatidylinositol hydrolysis by phospholipase C. Pretreatment with a phospholipase A2 inhibitor, mepacrine, substantially prevented the antigen-induced liberation of [3H]arachidonic acid from phosphatidylcholine. It can be thus concluded that, in the release of arachidonic acid by antigen-stimulated mast cells, the phospholipase A2 pathway, in which phosphatidylcholine is hydrolysed, serves as the major one, the phospholipase C/diacylglycerol lipase pathway playing only a minor role.     

Receptors for C3 on rat peritoneal mast cells.

Purified rat peritoneal mast cells adhere to schistosomula of Schistosoma mansoni which have been pre-incubated in fresh normal rat serum. This cytoadherence reaction is dependent on complement and in particular on components of the alternative pathway. Since antibodies to rat C3 but not IgG block the attachment of the cells to the complement-treated larvae, it appears that C3-specific receptors on the mast cell surface are responsible for the adherence phenomenon. These receptors can also be demonstrated by the rosetting of mast cells with rat complement-treated zymosan particles or fluoresceinated bacteria. The key properties of the receptors are their specificity for homologous (rat) complement, their sensitivity to digestion with trypsin, and their functional dependence on Mg++ ions. Thus, the rat mast cell receptors share many of the characteristics of the C3 receptors previously identified on monocytes, macrophages, and polymorphonuclear leukocytes.     

Inhibitory effect of diethylstilbestrol on histamine release by rat mast cells and its relation to the cellular ATP content

The effect of diethylstilbestrol, a synthetic estrogen, on mast cell secretion was investigated. The results showed that 50 microM diethylstilbestrol inhibited histamine release from rat peritoneal mast cells in the presence and absence of glucose, but did not affect 45Ca uptake stimulated by concanavalin A. Diethylstilbestrol also inhibited histamine release induced by compound 48/80, exogenous ATP, or ionophore A23187. Since estradiol benzoate, hexestrol and daidzein were not inhibitory, the inhibitory action of diethylstilbestrol must be independent of its estrogenic activity. The ATP content of mast cells decreased to less than 0.1 nmol/10(6) cells on treatment with 50 microM diethylstilbestrol at 37 degrees C for 15 min. This effect of diethylstilbestrol in decreasing the ATP content of mast cells correlated well with its inhibitory effect on histamine release. Diethylstilbestrol at 50 microM depleted the cells of ATP at 37 degrees C, but not at 0 degrees C, whereas [3H]diethylstilbestrol ( [monoethyl-3H]diethylstilbestrol) binding to rat mast cells was the same at 0 and 37 degrees C. It is concluded that diethylstilbestrol reduced the ATP content of rat mast cells by inhibiting metabolism of the cells, and consequently inhibited degranulation.     

Glucose inhibits insulin release when not promoting the entry of calcium into the beta-cells

The effect of antigen on the metabolism of polyphosphoinositides was investigated in sensitized rat peritoneal mast cells. Addition of antigen to rat peritoneal mast cells prelabelled with [3H]arachidonic acid resulted in a very rapid decrease in the level of phosphatidylinositol 4-phosphate (DPI) within 5 sec, which appeared to precede the breakdown of phosphatidylinositol (PI), while there was no significant decline of PI 4,5-bisphosphate (TPI). The reduced levels of these phosphoinositides returned almost to control or even slightly higher values by 300 sec in parallel with the antigen-stimulated [32P]phosphate incorporation into these lipids. This early and transient disappearance in DPI prior to that in PI was also observed in [3H]glycerol-prelabelled cells. These data suggest that DPI degradation upon stimulation by antigen in mast cells may be an initial step in the histamine release process.     

Phagocytosis-induced release of arachidonic acid from human neutrophils

The phospholipids of human neutrophils were labeled with [³H] arachidonic acid and [¹⁴C] palmitic acid. Phagocytosis of opsonized zymosan resulted in rapid release of free arachidonic acid but not of palmitic acid. Arachidonic acid was not released when the cells were exposed to unopsonized zymosan, zymosan-activated serum, or phorbol myristate acetate. These observations suggest that phagocytosis of opsonized zymosan results in the activation of a phospholipase A₂.     

Does intracellular histamine mediate mast cell histamine release?

Previously, we demonstrated that through binding a novel intracellular receptor of microM affinity (HIC), histamine mediates, and the HIC antagonist N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine. HCl (DPPE) inhibits, platelet aggregation and serotonin granule secretion; the latter response is dependent upon the same processes that mediate histamine release from mast cell granules. We now show that, as for platelet serotonin release, DPPE blocks concanavalin A-stimulated mast cell histamine release with a potency (IC50 = 30 microM) greater than the H1-antagonist, pyrilamine (IC50 = 150 microM) or the H2-antagonist cimetidine (IC50 = 5 mM), correlating with rank order of potency to inhibit 3H-histamine binding in rat brain membranes and liver microsomes. We postulate that histamine release from mast cells is mediated at HIC by second messenger intracellular histamine. However, unlike platelets, mast cells do not appear to rely on newly synthesized histamine. Rather, as for calcium, histamine may be mobilized from bound stores to mediate histamine secretion.     

Evidence for de novo synthesis of phosphatidylinositol coupled with histamine release in activated rat mast cells

Phospholipid metabolisms in rat mast cells activated by ionophore A23187 and compound 48/80 were examined with reference to 'phosphatidylinositol (PI) cycle'. The addition of A23187 to [3H]glycerol-prelabeled mast cells induced a marked accumulation of the radioactivity in 1,2-diacylglycerol(DG) and phosphatidic acid(PA) within 10 to 30 sec. A great enhancement of [3H]glycerol incorporation into PA and PI was also detected during histamine release. On the other hand, 48/80 was far less effective than A23187 both in producing 1,2- DG and PA and in accerelating [3H]glycerol incorporation into PA and PI, despite the comparable ability of histamine release. The activity of Ca2+ uptake into mast cells, as measured by pulse-labeling with 45Ca2+, was increased when exposed to both of two agents. These data provide circumstantial evidence that phospholipid metabolisms, mainly de novo PI synthesis, may be a part of the triggering events for Ca2+ mobilization and secretory process. The PI metabolism induced by two different stimulants appears to behave in a different manner.     

Eicosanoid-mediated increase in glucose and lactate output as well as decrease and redistribution of flow by complement-activated rat serum in perfused rat liver

Rat serum, in which the complement system had been activated by incubation with zymosan, increased the glucose and lactate output, and reduced and redistributed the flow in isolated perfused rat liver clearly more than the control serum. Heat inactivation of the rat serum prior to zymosan incubation abolished this difference. Metabolic and hemodynamic alterations caused by the activated serum were dose dependent. They were almost completely inhibited by the cyclooxygenase inhibitor indomethacin and by the thromboxane antagonist 4-[2-(4-chlorobenzesulfonamide)-ethyl]-benzene-acetic acid (BM 13505), but clearly less efficiently by the 5'-lipoxygenase inhibitor nordihydroguaiaretic acid and the leukotriene antagonist N-(3-[3-(4-acetyl-3-hydroxy-2-propyl-phenoxy)-propoxy]-4-chlorine-6-meth yl- phenyl)-1H-tetrazole-5-carboxamide sodium salt (CGP 35949 B). Control serum and to a much larger extent complement-activated serum, caused an overflow of thromboxane B2 and prostaglandin F2 alpha into the hepatic vein. It is concluded that the activated complement system of rat serum can influence liver metabolism and hemodynamics via release from nonparenchymal liver cells of thromboxane and prostaglandins, the latter of which can in turn act on the parenchymal cells.     

Serotonin storage pools in basophil leukemia and mast cells: characterization of two types of serotonin binding protein and radioautographic analysis of the intracellular distribution of [3H]serotonin

We studied binding of serotonin to protein(s) derived from rat basophil leukemia (RBL) cells and mast cells. We found two types of serotonin binding protein in RBL cells. These proteins differed from one another in molecular weight and eluted in separate peaks from sephadex G-200 columns. Peak I protein (KD = 1.9 X 10(-6) M) was a glycoprotein that bound to concanavalin A (Con A); Peak II protein (KD1 = 4.5 X 10(-8) M; KD2 = 3.9 X 10(-6) M) did not bind to Con A. Moreover, binding of [3H]serotonin to protein of peak I was sensitive to inhibition by reserpine, while binding of [3H]serotonin to protein of peak II resisted inhibition by that drug. Other differences between the two types of binding protein were found, the most significant of which was the far more vigorous conditions of homogenization required to extract peak I than peak II protein. Neither peak I nor peak II protein resembled the serotonin binding protein (SBP) that is found in serotonergic neurons of the brain and gut. Electron microscope radioautographic analysis of the intracellular distribution of [3H]serotonin taken up in vitro by RBL cells or in vivo by murine mast cells indicated that essentially all of the labeled amine was located in cytoplasmic granules. No evidence for a pool in the cytosol was found and all granules were capable of becoming labeled. The presence of two types of intracellular serotonin binding proteins in these cells may indicate that there are two intracellular storage compartments for the amine. Both may be intragranular, but peak I protein may be associated with the granular membrane while peak II protein may be more free within the granular core. Different storage proteins may help to explain the differential release of amines from mast cell granules.     

Stability of DNA/anti-DNA complexes.

Human monocytes in culture release small amounts of prostaglandin E (PGE) into the medium. Addition of Fc fragments of IgG to human monocyte monolayer cultures results in a marked increase in PGE release; Fab fragments, monomeric IgG, and human serum albumin have no effect. An IgG1 myeloma has no effect on PGE levels but addition of the heat aggreagted protein results in a marked increase of PGE secretion. Exposure of the cells to Con A, which binds to a specific monocyte plasma membrane receptor, also results in a large increase in PGE release. The magnitude of the increase in PGE secretion produced by exposure of the monocytes to these ligands greatly exceeds the stimulation observed after the addition of antigen-activated mononuclear cell supernatants, zymosan, Sephadex beads, or endotoxin, to monocyte cultures. Prostaglandin E2 (PGE2) accounts for approximately 70% of the total prostaglandins released by stimulated cells. After addition of Indomethacin to monocyte cultures, the stimulatory effects of the ligands on PGE release are inhibited. Addition of Con A to monocyte cultures results in an increased incorporation of [3H]-arachidonic acid into PGE2. These results suggest that this ligand stimulates synthesis as well as release of this prostaglandin.     

Effects of immune complexes, zymosan preparations and ionophore A 23187 on leukotriene formation in human leukocytes

Incubation of human leukocytes with opsonized zymosan or IgG immune complexes led to a time dependent release of leukotrienes (LT) B4 and C4. After 3-4 min, the levels of LTB4 were 93 and 35 pmol/3*10(7) cells, respectively [corrected]. These amounts were 2-4 times lower than those released by leukocytes stimulated with the calcium ionophore A 23187. The levels of LTC4 were 8 and 20 times lower than those of LTB4 after incubation with opsonized zymosan or immune complexes, respectively. Heat-inactivation of the serum prior to zymosan coating decreased the effect of opsonized zymosan. Uncoated zymosan was an even weaker stimulus of leukotriene formation. These results suggest that both complement factors and immunoglobulins play a pivotal role in activating leukotriene synthesis in a mixed suspension of human leukocytes.     

Differential activation of membrane phospholipid turnover by compound 48/80 and ionophore A23187 in rat mast cells

Membrane phospholipid turnover was investigated during histamine release from rat mast cells. Addition of calcium ionophore A23187 (0.5 microgram/ml) to mast cells prelabeled with [3H]glycerol induced the rapid and progressive increase in phosphatidic acid (PA) and 1,2-diacylglycerol (DG), which was concomitant with the small rise in phosphatidylinositol (PI). Loss of the level in triacylglycerol (TG) was very marked. Polyamine compound 48/80 (5 micrograms/ml) was shown to cause rises in PA, 1,2-DG, and PI without any significant changes in TG. Both stimuli increased incorporation of exogenous [3H]glycerol into phospholipids, indicating the involvement of de novo synthesis in phospholipid metabolism. Studies with [3H]arachidonic acid-labeled mast cells showed an enhanced liberation of radioactive arachidonate and metabolites upon histamine release. There were associated decreases of radioactivity in phosphatidylcholine (PC) and TG when exposed to A23187, while phosphatidylethanolamine (PE) was degraded as a result of 48/80 activation. The transient increases of [3H]arachidonoyl-1,2-DG and PA were caused by 48/80, while A23187 showed a gradual rise in the radioactivity in these two lipid fractions. These findings reflect activation of phospholipase C. When mast cells were activated by low concentrations of A23187 (0.1 microgram/ml) and 48/80 (0.5 microgram/ml), different behaviors of PI metabolism were observed. An early degradation of PI and a subsequent formation of 1,2-DG and PA suggest that the lower concentrations of these agents stimulate the PI cycle initiated by PI breakdown rather than de novo synthesis. These results demonstrate that marked and selective changes in membrane phospholipid metabolism occur during histamine release from mast cells, and that these reactions seem to be controlled by the coordination of degradation and biosynthesis, depending on the type and the concentration of stimulants. A23187 stimulates arachidonate release perhaps via the cleavages of PC and TG, whereas 48/80 liberates arachidonate from PE.     

Inhibition of IgE-mediated histamine release from rat basophilic leukemia cells and rat mast cells by inhibitors of transmethylation

This study evaluated the effect of inhibitors of transmethylation on histamine release from rat mast cells and rat basophilic leukemia cells. IgE-mediated histamine release from rat basophilic leukemia cells (RBL-2H3 cells) was inhibited by 3-deazaadenosine (DZA) in the presence of L-homocysteine thiolactone (Hcy) or the combination of adenosine, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), and Hcy in a dose-dependent fashion. There were no significant changes in the cellular cAMP levels by these inhibitors. Histamine release induced by anti-IgE or dextran from normal rat mast cells was also blocked by DZA plus Hcy in a dose-dependent manner. DZA at 10(-3) M in the presence of 10(-4) M Hcy or the combination of 10(-3) M adenosine, 10(-4) M EHNA, and 10(-3) M Hcy inhibited lipid (perhaps phospholipid) methylation into RBL-2H3 cells without affecting choline incorporation. In the presence of 10(-3) M DZA plus 10(-4) M Hcy there was a 170-fold increase in [35S]AdoHcy with the concomitant appearance of 3-deaza-AdoHcy when the cells were incubated with [35S]methionine, thus indicating that these drugs inhibited methylation reaction(s) through the intracellular accumulation of AdoHcy and 3-deaza-AdoHcy. In contrast, histamine release from rat mast cells induced by the calcium ionophore A23187, compound 48/80, polymyxin B, or ATP was not inhibited by these compounds. These results suggest that IgE- or dextran-mediated histamine release involves methylation reactions(s), whereas the other secretagogues bypass this early step.     

Somatostatin enhances binding of [3H]estradiol to a cytosolic protein in rat pancreas. Possible role of oligopeptide coligands in secretion

The cytosol fraction of rat pancrease can bind [3H] estradiol specifically and extensively. In contrast to the rat uterus, the binding protein in pancreas requires an accessory factor as a coligand in the steroid-binding reaction. Removal of this accessory factor by passage of the cytosol through CM Affi-Gel blue columns renders eluate fractions virtually incompetent with respect to binding of [3H]estradiol (10 nM). Certain synthetic oligopeptides such as N-benzoyl-L-argininyl-p-nitroanilide, as well as an endogenous accessory factor, can reactivate binding of [3H]estradiol. Thus, localization of the protein that binds [3H]estradiol following chromatography with CM Affi-Gel blue columns can be determined readily by assaying eluate fractions in the absence and presence of either accessory factor or N-benzoyl-L-argininyl-p-nitroanilide. Addition of somatostatin (tetradecapeptide referred to as SRIF14; somatotropin release inhibiting factor) to the activatable, but incompetent, eluate fractions, also enhanced binding of [3H]estradiol. The effect of SRIF14 was biphasic. The threshold concentration required for activation of [3H]estradiol binding was about 1 microM, and maximal stimulation occurred at 25 microM. At higher concentrations of SRIF14, binding declined and reached basal levels at about 75 microM. The concentrations of somatostatin required for activation of binding of [3H]estradiol in vivo may be lower than those indicated above since 1) preparations containing [3H]estradiol-binding protein also contained an SRIF14 peptidase. Following incubation of [125I-Tyr1]SRIF14 with these preparations there was loss of binding of radiolabeled peptide with SRIF14 antiserum. 2) The biphasic nature of SRIF14 activation may reflect feedback inhibition of [3H]estradiol binding by a degradation product of SRIF14. Since SRIF14 has been identified in the delta- (or D-) islet cells of the pancreas, and in concentrations that may be in the microM range, the possibility is raised that these cells serve a paracrine function with respect to acinar cell secretion.     

An antibody that blocks insulin-like growth factor (IGF) binding to the type II IGF receptor is neither an agonist nor an inhibitor of IGF-stimulated biologic responses in L6 myoblasts

To better define the biologic function of the type II insulin-like growth factor (IGF) receptor, we raised a blocking antiserum in a rabbit by immunizing with highly purified rat type II IGF receptor. On immunoblots of crude type II receptor preparations, only bands corresponding to the type II IGF receptor were seen with IgG 3637, indicating that the antiserum was specific for the type II receptor. Competitive binding and chemical cross-linking experiments showed that IgG 3637 blocked binding of 125I-IGF-II to the rat type II IGF receptor, but did not block binding of 125I-IGF-I to the type I IGF receptor, nor did IgG 3637 block binding of 125I-insulin to the insulin receptor. In addition, IgG 3637 did not inhibit the binding of 125I-IGF-II to partially purified 150- and 40-kDa IGF carrier proteins from adult and fetal rat serum. L6 myoblasts have both type I and type II IGF receptors. IGF-I was more potent than IGF-II in stimulating N-methyl-alpha-[14C]aminoisobutyric acid uptake, 2-[3H]deoxyglucose uptake, and [3H]leucine incorporation into cellular proteins. IgG 3637 did not stimulate either 2-[3H]deoxyglucose uptake, N-methyl-alpha-[14C]aminoisobutyric acid uptake, or [3H]leucine incorporation into protein when tested alone. Furthermore, IgG 3637 at concentrations sufficient to block type II receptors under conditions of the uptake and incorporation experiments did not cause a shift to the right of the dose-response curve for stimulation of these biologic functions by IGF-II. We conclude that the type II IGF receptor does not mediate IGF stimulation of N-methyl-alpha-[14C]aminoisobutyric acid and 2-[3H]deoxyglucose uptake and protein synthesis in L6 myoblasts; presumably, the type I receptor mediates these biologic responses. The anti-type II receptor antibody inhibited IGF-II degradation in the media by greater than 90%, suggesting that the major degradative pathway for IGF-II in L6 myoblasts utilizes the type II IGF receptor.     

Inhibitory effects of GMCHA-OPhBut on phospholipid methylation and histamine release in mast cells activated by concanavalin A, anti-IgE, and antigen

[3H]Methyl group incorporation and histamine secretion in rat mast cells induced by anti-IgE and con A were strongly inhibited by trans-4-guanidinomethylcyclohexanecarboxylic acid 4-tert-butylphenyl ester (GMCHA-OPhBut), a strong and specific inhibitor for pH 7 tryptase (Muramatsu et al. (1988) Biol. Chem. Hoppe-Seyler 369, 617-625) which is present in rat mast cells. The IC50s for these events were of the order of 10(-6) M. Addition of GMCHA-OPhBut after the maximal increase in [3H]methyl group incorporation in rat mast cells activated by con A and anti-IgE induced rapid reduction of the methylated phospholipid, and the later histamine release was strongly suppressed. Mast cells were prepared with Mg2+-free Tyrode-HEPES solution, and challenged with anti-IgE with or without Mg2+. With Mg2+, [3H]methyl group incorporation was enhanced, and histamine was secreted time-dependently. Without Mg2+, [3H]methyl group incorporation fell to one-third, whereas histamine secretion was not affected. These results were incompatible with the above results. From these results it was strongly suggested that a trypsin-like protease, probably pH 7 tryptase, is involved not only in the early events, such as activation of phosphatidylethanolamine methyltransferase I and/or II, but also in the late events such as histamine release, and phospholipid methylation is not associated with histamine secretion.     

Metabolism of lysophosphatidylserine, a potentiator of histamine release in rat mast cells

Lysophosphatidylserine (lysoPS) strongly enhances degranulation of rat mast cells induced by concanavalin A (Con A). In the present paper, the metabolism of exogenous lysoPS in intact mast cells was investigated. Incubation of mast cells with 1-stearoyl-sn-glycero-3-phospho-[3-3H]serine resulted in the rapid binding of lysoPS to mast cells and the time-dependent formation of a considerable amount of [3H]phosphatidylserine. No other radiolabeled lipid metabolites were detected. These results suggest that phosphatidylserine (PS) is synthesized through acylation of lysoPS incorporated into mast cells. Most of the lysoPS associated with mast cells was removed by washing with bovine serum albumin, whereas PS newly formed from lysoPS was not. The cells washed with albumin showed no appreciable histamine release upon subsequent addition of Con A. A different set of experiments was performed using lysoPS analogs which were modified at the hydroxyl group at position 2 of glycerol to avoid acylation. 1-Stearoyl-2-O-methyl-glycero-3-phosphoserine showed almost the same potentiating activity as 1-stearoyl-lysoPS, although the former does not have the free hydroxyl moiety at position 2 of the glycerol residue. The enhancing activity of another lysoPS analog, 1-stearyl-propanediol-3-phosphoserine, which lacks the hydroxyl group altogether, was quite similar to that of 1-stearyl-lysoPS. From these results we conclude that the acylation of lysoPS bears no relation to its potentiating activity and that lysoPS acts toward mast cells as lysoPS itself without any conversion to PS. The effect of replacement of an ester bond at position 1 of glycerol in lysoPS with an ether bond, and the phospholipid composition of rat mast cells are also discussed.