Mycophenolic acid inhibits the degranulation of rat peritoneal mast cells.

The effect of mycophenolic acid (MPA), a potent inhibitor of inosine monophosphate dehydrogenase, on the degranulation of rat peritoneal mast cells (RMC) was studied. RMC were pretreated for 48 hr with 0.1-10 microM MPA before the cells were sensitized with IgE and triggered with specific Ag. The net amount of [3H]5-HT released from granules was decreased by 44 and 32% with 1 and 10 microM MPA treatment, respectively. MPA inhibition of degranulation was completely reversed by the addition of 30 microM guanosine to the incubation medium. There was no difference in the apparent number or affinity of IgE binding sites between control and MPA-treated RMC. MPA pretreatment also had no effect on the IgE receptor-mediated production of PGD2 in RMC. These results suggest that depletion of intracellular GTP pools by MPA can disrupt the signaling between the IgE receptor and the secretory granules and that, under these same conditions, the release and metabolism of arachidonic acid are unaffected.     

Viability and recovery from degranulation of isolated rat peritoneal mast cells

Using a culture system that allows prolonged maintenance of purified populations of peritoneal mast cells, we have examined them following stimulation by non-immunologic or immunologic agents. Employing phase-contrast microscopy of living cells and various pharmacological manipulations, we have noted that the recovery process includes a reduction in cell size, the probable sealing of exocytotic cavities, a pronounced displacement of the cell nucleus and a resynthesis of histamine. During recovery, mast cells can entrap molecules from the extracellular fluid and later release these substances by a Ca-dependent mechanism. Our results suggest that microfilaments, calmodulin, Ca, and metabolic energy are necessary for recovery.     

Cytochemical localization of glycoconjugates in rat peritoneal mast cells during degranulation

Summary Cytochemical methods for the localization of glycoconjugates including concanavalin A-horseradish peroxidase (ConA-HRP) and dialysed iron were used to study the distribution of glycoconjugates in mast cell granules during degranulation. The ConA-HRP method revealed intense staining of discharged mast cell granules. Dialysed iron staining was seen at the granule periphery, with extruded granules exhibiting more intense staining than undischarged granules.Some of the work reported herein was performed in partial fulfillment of the requirements for a Ph.D. degree.     

Anaphylatoxin binding and degradation by rat peritoneal mast cells. Mechanisms of degranulation and control.

Incubation of radiolabeled human C3a with rat peritoneal mast cells resulted in high levels of uptake and extensive degradation of the ligand. Both cell-bound and free radiolabeled human C3a underwent extensive degradation by rat mast cells even at 0 degrees C. We examined several protease inhibitors for their ability to prevent degradation of radiolabeled human C3a by the rat mast cells. The inhibitors PMSF, chymostatin, and soybean trypsin inhibitor were most effective in preventing radiolabeled human C3a degradation. Degradation of the cell-bound ligand was totally inhibited only by PMSF. These compounds are effective inhibitors of a chymotrypsin-like enzyme (chymase) extracted from rat mast cells. Chemical cross-linking of radiolabeled human C3a to surface components on the rat mast cells, in the presence of PMSF, revealed one major and two minor bands. The mast cell component in both the major and minor bands proved to be chymase-associated based on a direct comparison with purified chymase isolated from rat mast cells. However, neither antichymase antibody nor chymase inhibitors influenced the degranulating activity of C3a on rat mast cells that occur independently of the C3a-chymase interactions. We conclude that there are neither specific C3a-binding sites on rat mast cells nor specific receptors whose occupancy leads to cellular activation. Although human C3ades Arg is inactive on guinea pig ileal and lung tissue, it binds to and induces degranulation of rat mast cells, as well as enhances vascular permeability in rat skin, at concentrations nearly identical to that of intact C3a. The fact that both C3a and C3ades Arg stimulated mast cell activation, at concentrations in excess of 10(-6) M, argues against specific binding sites for the anaphylatoxin on rat mast cells. It is proposed that the cationic C3a molecule activates rat mast cells in a secretory and nonlytic manner by a nonspecific mechanism similar to that of other polybasic compounds.     

Phosphatidylserine-specific phospholipase A1 stimulates histamine release from rat peritoneal mast cells through production of 2-acyl-1-lysophosphatidylserine

Lysophosphatidylserine (1-acyl-2-lyso-PS) has been shown to stimulate histamine release from rat peritoneal mast cells (RPMC) triggered by FcepsilonRI (high affinity receptor for IgE) cross-linking, although the precise mechanism of lyso-PS production has been obscure. In the present study we show that phosphatidylserine-specific phospholipase A(1), PS-PLA(1), stimulates histamine release from RPMC through production of 2-acyl-1-lyso-PS in the presence of FcepsilonRI cross-linker. The potency of 2-acyl-1-lyso-PS was almost equal to that of 1-acyl-2-lyso-PS. A catalytically inactive PS-PLA(1), in which an active serine residue (Ser(166)) was replaced with an alanine residue did not show such activity. sPLA(2)-IIA, another secretory PLA(2) that is capable of producing lyso-PS in vitro, was also a poor histamine inducer against RPMC. PS-PLA(1) significantly stimulated histamine release from crude RPMC, indicating that lyso-PS is mainly derived from cells other than mast cells. In agreement with this phenomenon, the enzyme stimulated the histamine release more efficiently when RPMC were mixed with apoptotic Jurkat cells. Under these conditions, lyso-PS with unsaturated fatty acid was released from the apoptotic cells treated with PS-PLA(1). Finally, heparin, which has affinity for PS-PLA(1), completely blocked the stimulatory effect of the enzyme. In conclusion, PS-PLA(1) may bind to heparan sulfate proteoglycan, efficiently hydrolyze PS appearing on plasma membranes of apoptotic cells, and stimulate mast cell activation mediated by 2-acyl-1-lyso-PS.     

Inhibition of phospholipase C-independent exocytotic responses in rat peritoneal mast cells by U73122

The aminosteroid U73122 has been established as potent, selective, and cell-permeable inhibitor C-type phosphatidylinositol-specific phospholipases (PI-PLCs), and has been used to define a contribution of PI-PLCs as part of exocytotic signalling pathways in rat peritoneal mast cells (RPMCs). However, doubts have been raised regarding its PI-PLC selectivity of action. Therefore, in the present study, U73122 was tested in RPMCs under experimental conditions allowing to elicit exocytosis PI-PLC independently (streptolysin O [SLO]-permeabilised cells; stimulated by GTPgammaS; in the presence of low concentrations of free Ca2+). The release of [3H]5-hydroxytryptamine ([3H]5-HT) from [3H]5-HT-loaded RPMCs served as measure of secretion. U73122 potently inhibited the exocytotic response induced by 10 microM GTPgammaS (Ca2+: 10(-6) M) in permeabilised cells (IC50: 0.6 microM, n=5) in an insurmountable manner. In intact RPMCs, with a nearly equal potency (IC50: 4 microM, n=4), U73122 also inhibited the PI-PLC-dependent exocytotic response induced by concomitant application of nerve growth factor and lyso-phosphatidylserine (NGF/lyso-PS). CONCLUSION: U73122 exerts potent PI-PLC-independent secretostatic effects, limiting its use to define PI-PLC function within exocytotic processes.     

Ultrastructural characteristics of rat peritoneal mast cells undergoing differential release of serotonin without histamine and without degranulation

Summary Rat mast cells pretreated with the tricyclic antidepressant drug amitriptyline and stimulated with compound 48/80 secreted 60% of the total serotonin present in the cells, but only 15% of histamine, another amine stored in the same granules. Ultrastructural studies demonstrated that mast cells undergoing such differential release do not exhibit classical degranulation by compound sequential exocytosis. However, there were changes in granule shape and size, as well as alterations in many morphometric parameters consistent with secretion. Storage granules lost their homogeneity, exhibited greatly reorganized matrix and were surrounded by clear spaces which were often associated with small (0.1–0.01 m) cytoplasmic vesicles, some of which contained electron-dense material. Secretory granules often had bud-like protrusions or were fused together in series. Quantitative autoradiography localized ³H-serotonin outside the storage granules, close to small vesicles, while staining with ruthenium red demonstrated that vesicular structures associated with differential release were not endocytotic. These results suggest that amitriptyline may inhibit regular exocytosis and permit at least serotonin to be moved selectively from storage granules to the cytosol or small vesicles from which it is eventually released.     

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.     

Unresponsiveness of rat peritoneal mast cells to immunologic reactivation.

Rat peritoneal mast cells cocultured with 3T3 fibroblasts (MC/3T3) were activated with Ag or with anti-IgE antibodies in the presence of Ca2+, and their responsiveness to a second similar challenge was evaluated. MC/3T3 were presensitized with IgE anti-DNP antibodies and activated with DNP-human serum albumin. When these MC/3T3 were reactivated with the same Ag 2 and 6 h later, they released only a minimal percentage of histamine. A gradual recovery of responsiveness was detected during the first 7 days after activation, and a full recovery was attained by days 14 to 21. A similar pattern of unresponsiveness was observed when MC/3T3 were challenged and rechallenged with cercarial Ag after presensitization with anticercarial serum. Activation of MC/3T3 with one Ag (DNP-human serum albumin or cercarial) and rechallenge 3 days later with the other Ag did not overcome the state of partial unresponsiveness. Challenging MC/3T3 with anti-IgE led to a subsequent unresponsiveness to rechallenge with the same ligand, regardless of whether or not the cells were presensitized with IgE antibodies. Cross-linkage with anti-IgE resulted in a more intense and prolonged state of unresponsiveness in comparison with that observed with Ag. When MC/3T3 activated with anti-IgE were rechallenged with various IgE-independent agents they released a percentage of histamine comparable to that of control cultures challenged with these secretagogues for the first time. MC/3T3 partially resynthesized their histamine content during the two-week period after activation. Our results suggest that MC undergo a temporary state of "physiologic" unresponsiveness after immunologic activation in the presence of calcium ions.     

Quercetin: A novel inhibitors of Ca2+ influx and exocytosis in rat peritoneal mast cells

The effect of the transport ATPase inhibitor, quercetin on histamine secretion from antigen sensitized mast cells was examined. At micromolar concentrations, quercetin had an immediate inhibitory effect on histamine secretion mediated by antigen, concanavalin A and ATP but it had little effect on release induced by the ionophores A23187 and X537A. Quercetin exerts its effect after the binding of the releasing ligands and the distinction between its effect on ligand induced and A23187 induced secretion suggests that it affects the normal path of Ca²⁺ entry into the cell.The inhibitory effects of quercetin were compared with those of the structurally related anti-allergic drugs cromoglycate and AH7725.     

Immunologically activated chloride channels involved in degranulation of rat mucosal mast cells.

Crosslinking of type I Fc epsilon receptors (Fc epsilon RI) on the surface of basophils or mast cells initiates a cascade of processes leading to the secretion of inflammatory mediators. We report here a correlation between mediator secretion and the activation of Cl- channels in rat mucosal-type mast cells (line RBL-2H3). Stimulation of RBL cells by either IgE and antigen or by a monoclonal antibody specific for the Fc epsilon RI, resulted in the activation of Cl- ion channels as detected by the patch-clamp technique. Channel activation occurred slowly, within minutes after stimulation. The channel has a slope conductance of 32 pS at potentials between 0 and -100 mV, and an increasing open-state probability with increasing depolarization. Activation of apparently the same Cl- channels could be mimicked without stimulation by isolating inside-out membrane patches in tyrode solution. Parallel inhibition of both Cl- channel activity and mediator secretion, as monitored by serotonin release, was observed by two compounds, the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and the anti-allergic drug cromolyn. NPPB inhibited both the antigen-induced Cl- current and the serotonin release, where half-maximal inhibition occurred at similar doses, at 52 microM and 77 microM, respectively. The drug cromolyn, recently found to inhibit immunologically induced mediator secretion from RBL cells upon intracellular application, also blocks Cl- channels (IC50 = 15 microM) when applied to the cytoplasmic side of an inside-out membrane patch. The observed Cl- channel activation upon immunological stimulation and the parallel inhibition of channel current and of serotonin release suggests a functional role for this Cl- channel in mediator secretion from the mast cells studied.     

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.     

Eicosanoid generation from antigen-primed mast cells by extracellular mammalian 14-kDa group II phospholipase A2.

The extracellular form of 14-kDa group II phospholipase A2 has been found to accumulate at various types of inflammatory sites. In the present paper, we have studied the possible role of the extracellular 14-kDa group II phospholipase A2 in the process of prostaglandin production in activated rat mast cells. When mast cells obtained from the peritoneal cavity of rats were sensitized with IgE, challenged with antigen and then exposed to extracellular 14-kDa group II phospholipase A2, appreciable release of prostaglandin D2 was observed. Generation of prostaglandin D2 was dependent on the concentration of the phospholipase A2 as well as that of the antigen, while no appreciable prostaglandin D2 generation was observed with cells in the absence of the antigen. No histamine release was observed under the same conditions. Phosphatidylcholine in mast cell membranes was appreciably hydrolyzed to liberate free arachidonic acid when mast cells were incubated with 14-kDa group II phospholipase A2 added exogenously in the presence of the antigen. Both the generation of prostaglandin D2 and the release of arachidonic acid were retarded by inhibitors specific to 14-kDa group II phospholipase A2. Thus, 14-kDa group II phospholipase A2 may function in the process of inflammation by acting on IgE-antigen-primed mast cells, which are not fully activated, to generate eicosanoids.     

Group II phospholipase A2 mRNA synthesis is stimulated by two distinct mechanisms in rat vascular smooth muscle cells.

Two potent inflammatory mediators, interleukin 1 (IL-1) and tumor necrosis factor (TNF) as well as lipopolysaccharide (LPS) increased group II phospholipase A2 (PLA2) mRNA levels, which resulted in enhanced secretion of the PLA2 enzyme from rat smooth muscle cells. cAMP-elevating agents also stimulated the release of PLA2 and increased the mRNA, but IL-1, TNF and LPS did not affect cAMP levels. Furthermore, the effects of TNF and cAMP-elevating agents were not additive but synergistic. Therefore, we concluded that the level of rat group II PLA2 mRNA is controlled at least by two distinct mechanisms, one involves cAMP and the other is mediated by TNF, IL-1 and LPS. This study also suggests important roles of group II PLA2 in pathogenesis of vascular inflammation.     

Characterization of primate bronchoalveolar mast cells. II. Inhibition of histamine, LTC4, and PGD2 release from primate bronchoalveolar mast cells and a comparison with rat peritoneal mast cells

As described in the preceding companion paper, bronchoalveolar lavage (BAL) of the primate Macaca arctoides infected with the nematode Ascaris suum yields a population of cells containing a high proportion of mast cells (21%). Nedocromil sodium, a new drug undergoing clinical evaluation for the treatment of reversible obstructive airways disease, inhibited the release of histamine, LTC4, and PGD2 from these cells challenged with antigen (with IC30 values of 2.1 X 10(-6) M, 2.3 X 10(-6) M, and 1.9 X 10(-6) M, respectively) and with anti-human IgE (IC30 values of 4.7 X 10(-6) M, 1.3 X 10(-6) M, and 1.3 X 10(-6) M, respectively). Cromolyn sodium was essentially inactive. Histamine release from rat peritoneal mast cells induced by anti-rat IgE was, however, inhibited by both nedocromil sodium and cromolyn sodium with IC30 values of 1.1 X 10(-6) M and 5.5 X 10(-7) M, respectively. Both compounds induce phosphorylation of a 78,000 m.w. protein in the rat peritoneal mast cell in the absence of any stimulus at the same concentrations as those required to inhibit histamine release stimulated by anti-IgE. This event may be part of a feedback mechanism to limit degranulation. Nedocromil sodium and cromolyn sodium were equipotent in their ability to inhibit anti-IgE-induced histamine release from rat peritoneal mast cells, but differed markedly in their ability to inhibit histamine release from macaque BAL cells.     

Immunologic release of heparin from purified rat peritoneal mast cells.

High m.w. [35S]heparin, labeled in vivo or in vitro, was released from purified rat mast cells by challenge with rabbit anti-rat F(ab')2, guinea pig anti-rat IgE, or calcium ionophore. The released and the residual heparin were isolated by Dowex 1 chromatography and were of comparable size by Sepharose 4B gel filtration. The majority of the released heparin was found by differential centrifugation to be granule-associated. Net percentage of mast cell heparin release, quantitated by metachromasia after isolation on Dowex 1 chromatography, correlated in a linear fashion with net percentage of histamine release, with heparin exhibiting a threshold requirement for onset of release. The correlation of histamine and high m.w. heparin release provides chemical support for the conclusion of others from ultrastructural studies that mast cell activation by immunologic means or by the calcium ionophore results in secretion of the whole granule.     

Release of 14-kDa group-II phospholipase A2 from activated mast cells and its possible involvement in the regulation of the degranulation process.

Group II phospholipase A2 was detected in appreciable amounts in rat peritoneal mast cells. The effect of several inhibitors specific to 14-kDa group-II phospholipase A2, including two proteinaceous inhibitors and a product of microorganisms with a low molecular mass, on mast-cell activation was examined. When rat peritoneal mast cells were sensitized with IgE and then challenged with antigen, the specific phospholipase-A2 inhibitors suppressed histamine release in a concentration-dependent manner. By contrast, these inhibitors showed no effect on prostaglandin generation under the same conditions. Histamine release from rat peritoneal mast cells subjected to non-immunochemical stimuli, such as concanavalin A, the Ca2+ ionophore A23187, compound 48/80 and substance P was also suppressed. When rat peritoneal mast cells were treated with 14-kDa-group-II-phospholipase-A2-specific inhibitors, washed and stimulated, histamine release was not affected appreciably. Similar suppressive effects of the inhibitors on histamine release were observed with mouse cultured bone-marrow-derived mast cells. When bone-marrow-derived mast cells were activated, they secreted both a soluble and an ecto-enzyme form of 14-kDa group-II phospholipase A2, although appearance of the enzyme associated with the external surface of cells was observed transiently. An appreciable amount of membrane phospholipids was degraded during activation of mast cells, which was decreased by treatment with 14-kDa-group-II-phospholipase-A2 inhibitor. These observations suggest that degranulation and eicosanoid generation in mast cells are regulated independently by discrete phospholipases A2 and that the 14-kDa group-II phospholipase A2 released from mast cells during activation may play an essential role in the progression of the degranulation process.     

Secretory phospholipase A2 receptor-mediated activation of cytosolic phospholipase A2 in murine bone marrow-derived mast cells

The current study examined the signal transduction steps involved in the selective release of arachidonic acid (AA) induced by the addition of secretory phospholipase A2 (sPLA2) isotypes to bone marrow-derived mast cells (BMMC). Overexpression of sPLA2 receptors caused a marked increase in AA and PGD2 release after stimulation of BMMC, implicating sPLA2 receptors in this process. The hypothesis that the release of AA by sPLA2 involved activation of cytosolic PLA2 (cPLA2) was next tested. Addition of group IB PLA2 to BMMC caused a transient increase in cPLA2 activity and translocation of this activity to membrane fractions. Western analyses revealed that these changes in cPLA2 were accompanied by a time-dependent gel shift of cPLA2 induced by phosphorylation of cPLA2 at various sites. A noncatalytic ligand of the sPLA2 receptor, p-amino-phenyl-alpha-D-mannopyranoside BSA, also induced an increase in cPLA2 activity in BMMC. sPLA2 receptor ligands induced the phosphorylation of p44/p42 mitogen-activated protein kinase. Additionally, an inhibitor of p44/p42 mitogen-activated protein kinase (PD98059) significantly inhibited sPLA2-induced cPLA2 activation and AA release. sPLA2 receptor ligands also increased Ras activation while an inhibitor of tyrosine phosphorylation (herbimycin) inhibited the increase in cPLA2 activation and AA release. Addition of partially purified sPLA2 from BMMC enhanced cPLA2 activity and AA release. Similarly, overexpression of mouse groups IIA or V PLA2 in BMMC induced an increase in AA release. These data suggest that sPLA2 mediate the selective release of AA by binding to cell surface receptors and then inducing signal transduction events that lead to cPLA2 activation.