Antigen-stimulated Mast Cells Research Articles

Multiple roles for PI 3-kinase in the regulation of PLCgamma activity and Ca2+ mobilization in antigen-stimulated mast cells.

Cross-linking the IgE-bound FcepsilonRI with polyvalent antigen leads to Ca2+-dependent degranulation from mast cells and basophils, initiating the allergic response. This overview addresses novel roles for PI 3-kinase in the regulation of signaling events that lie downstream of FcepsilonRI-mediated tyrosine kinase activation. The first novel role for PI 3-kinase is in the regulation of PLCgamma activity and is demonstrated by a dramatic inhibition of FcepsilonRI-induced Ins(1,4,5)P3 production after treatment of RBL-2H3 cells with wortmannin, a PI 3-kinase inhibitor. We show that PI 3-kinase lipid products support Ins(1,4,5)P3 production in at least two ways: by promoting translocation and phosphorylation of PLCgamma1 and by direct stimulation of both PLCgamma isoforms. In vitro stimulation of PLCgamma activity by PtdIns(3,4,5)P3 synergizes with activation by in vivo tyrosine phosphorylation for maximal enzymatic activity. A second novel role for PI 3-kinase is in the regulation of antigen-stimulated Ca2+ influx. Compared with control cells, Ca2+ responses are markedly diminished in antigen-stimulated cells after wortmannin pretreatment. Differences include both a longer lag time to the initial elevation in Ca2+ after antigen and an inhibition of the sustained Ca2+ influx phase. However, thapsigargin challenge during the sustained phase demonstrates no difference in the state of the Ca2+ stores in antigen-stimulated cells in the presence or absence of wortmannin. These data suggest that sufficient Ins(1,4,5)P3 is synthesized in wortmannin-treated cells to mobilize intracellular calcium stores and, furthermore, that the affected phase of Ca2+ influx is unlikely to be attributed to capacitative mechanisms. These data are consistent with a model where at least two pathways mediate Ca2+ influx in antigen-stimulated RBL-2H3 cells, one that is dependent on signals from empty stores (capacitative influx) and another that is downstream of PI 3-kinase.

Wortmannin-sensitive phosphorylation, translocation, and activation of PLCgamma1, but not PLCgamma2, in antigen-stimulated RBL-2H3 mast cells.

In RBL-2H3 tumor mast cells, cross-linking the high affinity IgE receptor (FcepsilonRI) with antigen activates cytosolic tyrosine kinases and stimulates Ins(1,4,5)P3 production. Using immune complex phospholipase assays, we show that FcepsilonRI cross-linking activates both PLCgamma1 and PLCgamma2. Activation is accompanied by the increased phosphorylation of both PLCgamma isoforms on serine and tyrosine in antigen-treated cells. We also show that the two PLCgamma isoforms have distinct subcellular localizations. PLCgamma1 is primarily cytosolic in resting RBL-2H3 cells, with low levels of plasma membrane association. After antigen stimulation, PLCgamma1 translocates to the plasma membrane where it associates preferentially with membrane ruffles. In contrast, PLCgamma2 is concentrated in a perinuclear region near the Golgi and adjacent to the plasma membrane in resting cells and does not redistribute appreciably after FcepsilonRI cross-linking. The activation of PLCgamma1, but not of PLCgamma2, is blocked by wortmannin, a PI 3-kinase inhibitor previously shown to block antigen-stimulated ruffling and to inhibit Ins(1,4,5)P3 synthesis. In addition, wortmannin strongly inhibits the antigen-stimulated phosphorylation of both serine and tyrosine residues on PLCgamma1 with little inhibition of PLCgamma2 phosphorylation. Wortmannin also blocks the antigen-stimulated translocation of PLCgamma1 to the plasma membrane. Our results implicate PI 3-kinase in the phosphorylation, translocation, and activation of PLCgamma1. Although less abundant than PLCgamma2, activated PLCgamma1 may be responsible for the bulk of antigen-stimulated Ins(1,4,5)P3 production in RBL-2H3 cells.

Common and Distinct Signaling Pathways Mediate the Induction of TNF-α and IL-5 in IgE Plus Antigen-Stimulated Mast Cells

AbstractA small number of signaling cascades represented by mitogen-activated protein kinases, phosphoinositol-3-kinase, protein kinase C, signal transducers and activators of transcription, Ca2+/calcineurin, and a few other molecules are linked to an incomparably large number of surface receptors. Parallel activation of several of these pathways and the existence of isozymes for a number of signal transmitting molecules generate the required complexity and specificity matching the receptor variety. Here we show that the proinflammatory mediator TNF-α and the growth factor IL-5 are activated along common and distinct signaling cascades in allergically stimulated murine mast cells. Both of them are dependent on Ca2+ influx, activation of calcineurin and nuclear factor of activated T cells as well as a member of the atypical PKC family, most likely PKCμ. Additionally, mitogen-activated protein kinases for TNF-α and members of the classical or nonclassical PKCs for IL-5, respectively, were identified as additional required pathways. Inhibition of the classical and nonclassical PKCs, however, does not abrogate IL-5 induction but instead leads to a switch to mitogen-activated protein kinases, which then become essential. The activated branches of this “salvage” signaling cascade are represented by extracellular signal-regulated kinase 1/2 and c-jun NH2 terminal kinase 1 in allergically stimulated mast cells.

Calcium signalling: Sphingosine kinase versus phospholipase C?

A recent study shows that sphingosine kinase and its lipid product have an essential signalling function; they act in the mobilization of calcium ions in antigen-stimulated mast cells. This finding may have relevance to signalling in other cells of the immune system.

Imaging of total intracellular calcium and calcium influx and efflux in individual resting and stimulated tumor mast cells using ion microscopy.

Ion microscopy was employed to investigate intracellular total calcium concentrations and calcium influx, and efflux in resting and antigen-stimulated tumor mast cells (RBL-2H3 cells). The nucleus, a perinuclear region which included the Golgi apparatus (Golgi region), and the remaining cytoplasm were spatially resolved with the Cameca IMS-3f ion microscope in cryogenically prepared cells. In resting cells the nucleus contained about 0.60 mM, the Golgi region about 1.2 mM, and the remaining cytoplasm about 1.0 mM total calcium. Antigen stimulation of rat basophilic leukemia cells resulted in a significant loading of calcium in all three cellular compartments. Antigen stimulation in the absence of extracellular calcium resulted in a significant loss of total calcium from all three intracellular compartments. Influx and efflux of calcium were measured simultaneously in resting and stimulated cells by using stable 44Ca in the extracellular solution, and by imaging mass 40 to determine the native intracellular calcium (40Ca) and mass 44 to localize the 44Ca that entered the cell from extracellular solution. After a 10-min incubation, 0.240 fmol of the total calcium per cell had been replaced with 44Ca, which amounts to about 33% of the total cell calcium. If antigen was present during this incubation there was an additional loss of 0.229 fmol of 40Ca and an added gain of 0.476 fmol of 44Ca per cell, which corresponds to a net increase in total intracellular calcium of 0.247 fmol.

Regulation of AP-1 expression and activity in antigen-stimulated mast cells: the role played by protein kinase C and the possible involvement of Fos interacting protein

We have recently observed that protein kinase C (PKC) was involved in the regulation of the accumulation of mRNAs of the AP-1 components in cultured Abelson-transformed murine fetal-liver-derived mast cells stimulated by exocytotic stimuli. Here we analyzed the probable regulatory effect of PKC on the synthesis and DNA-binding activity of AP-1 complexes in immunologic stimulated mast cells. In this study we used the interleukin-3--dependent murine fetal-liver--derived mast cells that were not transformed by the Abelson oncogene. Study of PKC- depleted cells showed PKC dependency of c-fos mRNA accumulation and protein expression in IgE-Ag stimulated cells. In contrast, the c-jun mRNA accumulation was unaffected by PKC depletion, whereas its protein expression was dependent on this enzymatic activity. This suggests the involvement of PKC in the regulation of translation of c-Jun, a level of c-Jun regulation that was not previously described. The amount of AP- 1 DNA-bound complex was also lowered in PKC-depleted cells. Therefore, PKC plays an important regulatory role in different stages of the signal transduction pathway because of IgE-Ag stimulation. Surprisingly, we have observed that although the amount of total synthesized c-Fos began to increase 15 minutes after immunologic stimulation, the amount of c-Fos associated with Juns did not increase, even after 45 minutes. This association was not affected by PKC. Using a Fos-interacting protein (FIP)-cDNA probe, an expression of 2.9 kb mRNA was detected in these cells. Furthermore, immunologic stimulation caused an increase in the amount of a Fos-containing protein complex that bound to an FIP-binding DNA oligonucleotide. Therefore, we propose that this protein complex that contains most of the immunologically induced c-Fos has an important role in IgE-Ag-stimulated signal transduction.

Regulation of AP-1 Expression and Activity in Antigen-Stimulated Mast Cells: The Role Played by Protein Kinase C and the Possible Involvement of Fos Interacting Protein

AbstractWe have recently observed that protein kinase C (PKC) was involved in the regulation of the accumulation of mRNAs of the AP-1 components in cultured Abelson-transformed murine fetal-liver-derived mast cells stimulated by exocytotic stimuli. Here we analyzed the probable regulatory effect of PKC on the synthesis and DIM A-binding activity of AP-1 complexes in immunologic stimulated mast cells. In this study we used the interleukin-3—dependent murine fetal-liver—derived mast cells that were not transformed by the Abelson oncogene. Study of PKC-depleted cells showed PKC dependency of c-fos mRNA accumulation and protein expression in IgE-Ag stimulated cells. In contrast, the c-jun mRNA accumulation was unaffected by PKC depletion, whereas its protein expression was dependent on this enzymatic activity. This suggests the involvement of PKC in the regulation of translation of c-Jun, a level of c-Jun regulation that was not previously described. The amount of AP-1 DNA-bound complex was also lowered in PKC-depleted cells. Therefore, PKC plays an important regulatory role in different stages of the signal transduction pathway because of IgE-Ag stimulation. Surprisingly, we have observed that although the amount of total synthesized c-Fos began to increase 15 minutes after immunologic stimulation, the amount of c-Fos associated with Juns did not increase, even after 45 minutes. This association was not affected by PKC. Using a Fos-interacting protein (FIP)-cDNA probe, an expression of 2.9 kb mRNA was detected in these cells. Furthermore, immunologic stimulation caused an increase in the amount of a Fos-containing protein complex that bound to an FIP-binding DNA oligonucleotide. Therefore, we propose that this protein complex that contains most of the immunologically induced c-Fos has an important role in IgE-Ag-stimulated signal transduction.

Biosynthesis of platelet-activating factor in cultured mast cells. Involvement of the CoA-independent transacylase demonstrated by analysis of the molecular species of platelet-activating factor.

We have recently demonstrated that arachidonate [20:4(5,8,11,14)] was primarily linked to the hexadecyl (16:0) and octadecenyl (18:1) species of alkylacyl derivatives of glycerolphosphocholine (GroPCho). Consistent with the involvement of arachidonate-specific CoA-independent transacylase in the synthesis of platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-GroPCho), 16:0 and 18:1 PAF species were formed upon antigen stimulation [Joly, F., Breton, M., Wolf, C., Ninio, E. & Colard, O. (1992) Biochim. Biophys. Acta 1125, 305-312]. In the present work, addition of lyso-PAF to mast cells resulted in PAF production. We analyzed the PAF species formed in the presence of a defined lyso-PAF molecular species in order to differentiate between either direct acetylation or involvement of the membrane precursor. The 18:1 lyso-PAF was more effective than the 16:0 in producing PAF which was composed of 95% 18:1 PAF, the balance being 16:0, indicating that part of the acetylated lyso-PAF originated from the cellular pool of alkyl-arachidonyl-GroPCho in resting cells. Consistent with alkyl-arachidonyl-GroPCho species content and acetyltransferase specificity, similar amounts of 16:0 and 18:1 PAF species were formed when mast cells were stimulated with antigen. Supplemented with 16:0 or 18:1 lyso-PAF, antigen-stimulated mast cells responded by 230% and 125% increase in PAF synthesis, respectively. As expected, the amount of the PAF species corresponding to the added lyso-PAF was increased. More interestingly, addition of 16:0 lyso-PAF almost doubled the amount of 18:1 PAF content as compared to antigen alone, thus indicating that the lyso-PAF formed via the CoA-independent transacylase was significantly used for PAF synthesis, despite a large excess of exogenous lyso-PAF. The CoA-independent transacylase, measured using [3H]lyso-PAF as a substrate in sonicates from antigen-stimulated cells, was decreased concurrently with PAF formation. In conclusion, we show that when lyso-PAF is added to mast cells, a direct acetylation may occur. However, PAF is preferentially synthesized through a mechanism involving the CoA-independent transacylase reaction.

Effects of glycyrrhizin and glycyrrhetinic acid on dexamethasone-induced changes in histamine synthesis of mouse mastocytoma P-815 cells and in histamine release from rat peritoneal mast cells

The effects of glycyrrhizin and its aglycone, glycyrrhetinic acid, on dexamethasone-induced changes in the histamine synthesis of mastocytoma P-815 cells and in the histamine release from antigen-stimulated rat peritoneal mast cells were investigated. Glycyrrhetinic acid but not glycyrrhizin, at concentrations from 20 to 35 μM, almost completely inhibited the dexamethasone-induced increases in both the histamine content and histidine decarboxylase activity of cultured mastocytoma P-815 cells. Glycyrrhetinic acid, however, showed practically no inhibition of [ 3H]dexamethasone binding to the cytoplasmic receptor. On the other hand, glycyrrhetinic acid but not glycyrrhizin markedly inhibited the release of histamine from antigen-stimulated rat mast cells, and intensified the inhibitory activity induced by dexamethasone. Glycyrrhetinic acid inhibited the antigen-induced release and incorporation of [ 3H]arachidonic acid in immunized rat mast cells. The administration of glycyrrhizin into rats, in contrast to the in vitro treatment of the cells with glycyrrhizin, markedly inhibited histamine release from antigen-stimulated rat mast cells. These results suggest that glycyrrhetinic acid inhibited dexamethasone-induced changes in the histamine synthesis of mastocytoma P-815 cells, and in the histamine release from rat mast cells. On the other hand, glycyrrhizin may exert its effect after conversion to glycyrrhetinic acid in vivo.

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.

Biosynthesis of paf-acether. IX. Role for a phosphorylation-dependent activation of acetyltransferase in antigen-stimulated mouse mast cells.

Mouse bone marrow-derived mast cells passively sensitized with monoclonal IgE released paf-acether (platelet-activating factor) and beta-hexosaminidase when challenged with the specific antigen. The formation and the release of paf-acether followed an early increase in the activity of the acetyltransferase, the main enzyme in paf-acether biosynthesis. The antigen-induced activation of the acetyltransferase was dependent on physiologic temperature and on the presence of Ca2+. By using microsomal fractions from unchallenged and challenged mast cells, the Vmax values were 3.5 and 12.0 nmol/min/mg of protein, respectively, whereas in both cases a Km value for acetyl-coenzyme A of 172 microM was measured. The stimulation of acetyltransferase could be mimicked in vitro under experimental conditions which favor phosphorylation, i.e. adding ATP and Mg2+ to lysates from unchallenged mast cells. In contrast, ATP and Mg2+ were uneffective on lysates from challenged cells that exhibited high level of acetyltransferase activity, suggesting that phosphorylation of the enzyme already took place at the time of cell stimulation. Moreover, addition of alkaline phosphatase to microsomal fraction obtained from either antigen-challenged mouse bone marrow-derived mast cells or unchallenged cells, resulted in 52% and 43% loss of acetyltransferase activity, respectively. Phorbol myristate acetate treatment of cells doubled the enzyme activity supporting the phosphorylation hypothesis. Thus, we report on the immunologic activation of a key enzyme for paf-acether synthesis and on the mechanism of this activation in a pure mast cell population. A link between bridging of IgE receptors and the activation of an enzyme critical to the formation of a lipid mediator is thereby evidenced.

Leukotriene C4 production by murine mast cells: evidence of a role for extracellular leukotriene A4.

The glutathione-containing leukotriene C4 (LTC4) is a major mediator of smooth muscle contraction and is released by mast cells when antigen interacts with cell-bound IgE. Antigen-stimulated mast cells undergo phospholipase activation. We report a pathway of LTC4 production by mast cells that does not require phospholipase activation but depends on the interaction of activated neutrophils with unstimulated mast cells, using as an intermediate extracellular leukotriene A4 (LTA4). The epoxide LTA4 is released by neutrophils and, together with leukotriene B4 and 5-hydroxyeicosatetraenoic acid, constitutes the major lipoxygenase metabolites found in supernatants of stimulated neutrophils. Five minutes after activation of neutrophils by calcium ionophore A23187 we measured 136 pmol of extracellular LTA4 per 10(7) neutrophils (range 40-300, n = 7) by trapping the epoxide with alcohols. Therefore, we conclude that LTA4 is not just an intracellular leukotriene precursor but is released as a lipoxygenase metabolite. LTA4 is known to be stabilized by albumin and is efficiently converted by mast cells into LTC4 even at low LTA4 concentrations. The LTA4 complexed to albumin is converted into LTC4 rapidly and completely within 10-15 min. More than 50% of the LTA4 presented to mast cells is metabolized to LTC4 at concentrations of LTA4 between 0.2 and 2 nmol of LTA4 per 10(7) mast cells. This observation establishes a potential physiologic role for extracellular LTA4. Therefore, interactions between various cell types that release or utilize LTA4 may provide an important metabolic pathway for the production of leukotrienes.

Antigen-initiated release of platelet-activating factor (PAF-acether) from mouse bone marrow-derived mast cells sensitized with monoclonal IgE.

Mouse bone marrow mast cells sensitized with monoclonal IgE and activated with specific antigen released 2.8 +/- 0.5 ng of platelet-activating factor (1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) (PAF-acether)/ 10(6) cells. The PAF-acether was identified by its ability to aggregate fully aspirin-treated washed rabbit platelets in the presence of an adenosine diphosphate (ADP)-scavenger complex, by its co-chromatography with [3H]-labeled semi-synthetic PAF-acether and synthetic 1-0-octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, and by its inactivation by phospholipases A2, C, and D and not by lipase A1. The antigen-initiated release of PAF-acether, leukotriene C4 (LTC4), and leukotriene B4 (LTB4), and the secretion of the granule marker beta-hexosaminidase were not diminished by washing the cells before challenge, indicating that they were due to the interaction of antigen with the IgE fixed on the cell membrane and not to phagocytosis of immune complexes formed in the fluid phase. The parallel antigen-induced dose-response relationship, along with the superimposable time-course of the extracellular appearance, of beta-hexosaminidase, PAF-acether, and both leukotrienes indicated that the origin of these diverse mediators was from a common cell type with IgE-Fc receptors. Ethanol extraction of antigen-stimulated bone marrow-derived mast cells revealed the early transient appearance of a cell-associated platelet-aggregating activity, the action of which on platelets, like PAF-acether, was independent of ADP and arachidonic acid metabolism. The cell-associated activity contained a novel product that eluted at 13 min during high performance liquid chromatography (HPLC) (solvent hexane:n-propanol:water, 46:46:8), permitting resolution from PAF-acether and lyso-PAF-acether (1-O-alkyl-sn-glyceryl-3-phosphorylcholine), which eluted at 29 min and 30 min, respectively. The cell-associated material, which differs from lyso-PAF-acether, the putative precursor of PAF-acether, in being active in the bioassay on platelets may represent a newly recognized intermediate in the generation of PAF-acether. As the transiently present cell-associated intermediate has not been previously recognized, its detection may depend upon the relatively unique properties of the bone marrow-derived mast cell system in which IgE-dependent activation leading to product generation is complete within 5 min.(ABSTRACT TRUNCATED AT 400 WORDS)

Selective phosphorylation of the IgE receptor in antigen-stimulated rat mast cells.

Purified rat serosal mast cells were sensitized with mouse immunoglobulin E (IgE) anti-2,4-dinitrophenyl antibody, partially depleted of phosphate, labeled with [32P]orthophosphate, and stimulated with dinitrophenylated bovine serum albumin or control protein. After 15-120 seconds at 37 degrees C, the cells were extracted with nonionic detergent. IgE receptors were purified by repetitive affinity chromatography and were analyzed by NaDodSO4/polyacrylamide gel electrophoresis and radioautography. Antigenic stimulation of intact rat mast cells produced a rapid and marked increase in the phosphorylation of the surface-exposed alpha component of the IgE receptor. However, phosphorylation of the 33,000 Mr beta component of the IgE receptor was not altered significantly by antigen stimulation. This suggests that the selective increase in phosphorylation of the IgE receptor alpha component may be part of the physiologic mediator secretion process triggered by antigen.

Movement of strontium ions into mast cells and its relationship to the secretory response.

1. Non-stimulated mast cells take up (89)Sr and (45)Ca. There is a rapid phase of uptake of both labels which is complete in 1 min and the (89)Sr uptake is similar in magnitude to the (45)Ca uptake. A further slower phase of uptake occurs in the period from 1 to 30 min of incubation. The magnitude of this slower phase is about 6 times greater for (89)Sr than for (45)Ca.2. Non-stimulated and antigen-stimulated mast cells accumulate Sr which can be measured by atomic absorption spectroscopy. There is a direct relationship between Sr accumulation and histamine secretion which is independent of whether or not the cells are stimulated. 10% histamine secretion is associated with Sr accumulation of 0.25 f-mole/cell.3. The time course of (89)Sr uptake is similar to the time course of histamine secretion in non-stimulated cells.4. The uptake of (89)Sr is linearly related to the external Sr concentration in the range 0.5-16 m-mole/l. for both stimulated and non-stimulated cells.5. Ca, 10-1000 mumole/l. inhibits (89)Sr uptake in non-stimulated cells.6. Inhibition of glycolysis and oxidative phosphorylation prevents spontaneous histamine secretion in the presence of Sr without blocking the accumulation of Sr by the cells.7. Dibutyryl cyclic AMP, 10 m-mole/l. produces only 18% inhibition of (89)Sr uptake whereas histamine secretion is inhibited by 45% by the same concentration. The antigen-stimulated Sr uptake on the other hand can be completely inhibited by dibutyryl cyclic AMP, 10 m-mole/l.8. The uptakes of (89)Sr and Sr by unstimulated mast cells after 60 min incubation are dependent on extracellular H ion concentration. Both uptakes increase with increasing pH over the range pH 7-8.5.