Immunologic properties of mast cells from rats infected with Nippostrongylus brasiliensis.

The concentration of IgE in the serum of Sprague-Dawley rats increased after infection with Nippostrongylus brasiliensis (NB). The IgE concentration in normal rats was less than 1 mug/ml. After re-infection with NB, the concentration increased in 100 to 300 mug/ml. Mast cells were purified from peritoneal cells of both normal and NB-infected animals. Purified mast cells from the infected animals released histamine upon exposure to NB antigen. The antibody specific for IgE released histamine from purified mast cells of both normal and infected animals. Dose-reponse curves of histamine release suggested that mast cells from NB-infected animals bear more IgE molecules than normal mast cells. Binding of 125I-labeled rat E myeloma protein with normal mast cells was demonstrated by autoradiography. Under the same experimental conditions, mast cells of infected animals were not labeled with 125I-IgE. Mast cells from both normal and infected animals failed to combine 125I-labeled IgG. The number of IgE molecules bound per mast cell was determined by incubating 125I-labeled IgE with purified mast cells. When mast cells were incubated incubated in 0.6 to 2 mug/ml of IgE, the number of IgE molecules combined with the mast cells from infected animals was about 10% of that bound with normal mast cells. The results indicated that a large proportion of IgE receptors on mast cells of infected animals was occupied by their own IgE. No significant difference was observed between normal mast cells and those of infected animals with respect to histamine content and intracellular levels of cyclic nucleotides.     

IgE-mediated release of histamine from human cutaneous mast cells

We investigated the ability of antigen-IgE interactions to stimulate histamine release from human infant cutaneous mast cells. Skin obtained at circumcision contained numerous perivascular mast cells, as assessed by light and electron microscopy. The histamine content of this tissue averaged 17.7 ng (+/- 1.5 SEM)/mg wet weight. Challenge of 200-microns thick sections of unsensitized skin with varying concentrations of monoclonal murine antibodies to human IgE caused no net release of histamine. After skin sections were incubated in the presence of 5 micrograms/ml of human myeloma IgE (S) for 120 min at 37 degrees C, monoclonal anti-IgE challenge resulted in 40.1% (+/- 6.0 SEM) histamine release. Similar passive sensitization with 1/20 dilutions of serum from humans expressing IgE to purified Juniperus sabinoides (JS) antigen rendered the tissue responsive to specific antigen challenge. Dose-related histamine release occurred over 30 min with optimal release of 12.6% (+/- 2.4 SEM) after stimulation with 100 ng/ml of JS antigen. This reaction required sensitization with serum containing IgE to JS and was antigen-specific. Optimal reactions to antigen occurred at 3 mM added Ca++, 34 degrees C to 37 degrees C, pH 7.2. Antigen-induced release was markedly influenced by the added Ca++ concentration; no release occurred in the absence of Ca++, 54% of the optimal response was observed at 2 mM Ca++, and 28% of the optimal response occurred at 4 mM Ca++. The addition of Mg++ did not influence antigen-induced release. The results of this study provide functional evidence that 1) human infant cutaneous mast cells express Fc-epsilon receptors; 2) these receptors are largely unoccupied in vivo; and 3) stimulation of passively sensitized infant mast cells with anti-IgE or specific antigen leads to immediate histamine release. This new system should permit detailed in vitro studies of immediate hypersensitivity reactions in human skin.     

Calcium-dependent activation of human neutrophils by a synthetic ionophore

Synthetic block copolymers composed of polyoxyethylene and poly-oxypropylene have been demonstrated to possess ionophore activity selective for monovalent cations and to cause histamine release from mouse mast cells and human basophils. We now report calcium-dependent release of granule contents from human neutrophils by the most active of these agents, TI30R2. At a concentration of 100 micrograms/ml (12.5 microM), net lysozyme release ranged from 17-40% after 30 minutes incubation at 37 degrees. Lysozyme release was dose-dependent over stimulus concentrations of 5-50 micrograms/ml (0.625-6.25 microM). Release was dependent upon the presence of extracellular calcium. T130R2 did not induce the release of superoxide anions over 30 minutes of incubation. As T130R2 induces sodium influx into cells, it is likely that a depolarizing influx of sodium ions in the presence of extracellular calcium constitutes a sufficient signal for granule release but not superoxide production by human neutrophils.     

Low density lipoprotein (LDL) inhibits histamine release from human mast cells

We examined the effect of low density lipoprotein (LDL) on histamine release from purified human lung mast cells. LDL inhibited anti-IgE- induced histamine release in a dose-dependent manner, with 100 micrograms/ml LDL-protein inhibiting histamine release by 53 +/- 8% (mean +/- SEM); half-maximal inhibition occurred at 40-80 micrograms/ml. LDL also inhibited calcium ionophore A23187-induced histamine release in a dose-dependent manner, with 1 mg/ml of LDL inhibiting histamine release by 83 +/- 9%; half maximal inhibition occurred at 220-280 micrograms/ml. Inhibition by LDL was time-dependent: half-maximal inhibition of anti-IgE- induced histamine release by LDL occurred at 30-50 minutes of incubation. The inhibitory effect of LDL was independent of buffer calcium concentrations (0-5 mM) or temperature (0-37 degrees C). These data are consistent with a newly defined immunoregulatory role for LDL.     

Magainin-2 releases histamine from rat mast cells.

The magainins are basic 23 amino acid peptides with a broad spectrum of antimicrobial activity. Their bactericidal effect has been attributed to their capacity to interact with lipid bilayer membranes. We observed histamine release by magainin-2 amide from rat peritoneal mast cells (ED50 = 13 micrograms/ml) but not from human basophils. This histamine-releasing reaction from peritoneal mast cells was due to a secretory rather than cytolytic effect, i.e., release occurred without concomitant liberation of lactic dehydrogenase. Furthermore, the pretreatment of mast cells with magainin-2 amide did not desensitize cells against subsequent challenge with other secretagogues. Maximum histamine release occurred in less than a minute at 25 and 37 degrees C. The addition of Ca2+ was not required for histamine release, although release was enhanced by the addition of 0.3-1 mM Ca2+. The addition of 3 mM Ca2+ or Mg2+ was markedly inhibitory. The presence of Na+ or Cl- ions in the medium was not required for release. Therefore, histamine release is not due to the formation of anion-selective channels in the membrane of mast cells. The results indicated that the characteristics of histamine secretion induced by magainin-2 amide were unlike IgE-mediated release but were similar to the mechanism of release attributed to some other basic peptides and to compound 48/80.     

Neutrophil defensins induce histamine secretion from mast cells: mechanisms of action.

Defensins are endogenous antimicrobial peptides stored in neutrophil granules. Here we report that a panel of defensins from human, rat, guinea pig, and rabbit neutrophils all have histamine-releasing activity, degranulating rat peritoneal mast cells with EC50 ranging from 70 to 2500 nM, and between 45 and 60% of the total histamine released. The EC50 for defensin-induced histamine secretion correlates with their net basic charge at neutral pH. There is no correlation between histamine release and antimicrobial potency. Degranulation induced by defensins has characteristics similar to those of activation by substance P. The maximum percent histamine release is achieved in <10 s, and it can be markedly inhibited by pertussis toxin (100 ng/ml) and by pretreatment of mast cells with neuraminidase. These properties differ from those for degranulation induced by IgE-dependent Ag stimulation and by the calcium ionophore A23187. GTPase activity, a measure of G protein activation, was induced in a membrane fraction from mast cells following treatment with defensin. Thus, neutrophil defensins are potent mast cell secretagogues that act in a manner similar to substance P and 48/80, through a rapid G protein-dependent response that is mechanistically distinct from Ag/IgE-dependent mast cell activation. Defensins may provide important pathways for communication between neutrophils and mast cells in defenses against microbial agents and in acute inflammatory responses.     

Mast cell histamine release induced by Portuguese man-of-war (Physalia) venom

Nematocyst venom from Portuguese Man-of War ($\text{Physalia}$ sp.) tentacles causes isolated rat peritoneal mast cells to release histamine. Extent of histamine release is dose-dependent (K_0.5 = 6.1 μg venom/ml) and attains 100% at high doses of venom. Release is independent of intra- and extracellular calcium levels and does not depend upon a cellular supply of ATP. The rate of histamine release is temperature-dependent and the extent of release is maximized broadly over the range of 10–30°C. The cytoplasmic marker lactate dehydrogenase, is released concomitantly with histamine but is more sensitive to the venom (K_0.5 = 2.1 μg/ml). Antimycin A, while it does not significantly affect venom-induced histamine release, increases the sensitivity of lactate dehydrogenase release (K_0.5 = 0.2 μg/ml). We conclude that $\text{Physalia}$ nematocyst venom induces the release of histamine from mast cells by a cytolytic mechanism and that this action is antagonized by an intracellular, energy-requiring process.     

Fibronectin and fibrinogen degradation products stimulate PMN-leukocyte and mast cell degranulation.

The ability of various peptides cleaved by plasmin from human fibrinogen and fibronectin or fibrinogen- and fibronectin- related synthetic peptides to induce histamine release from mast cells and collagenase and elastase from PMN-leukocytes was examined. Low molecular weight fibrinogen degradation products showed dose dependent secretion of collagenase. These peptides (mol. wt. 1.4 kD) at the concentration of 10(-5) M released about 47% of collagenase and 13% of elastase. Synthetic fibrinopeptides A and B had a similar strong collagenase releasing potency and also released histamine from mast cells. Peptides from plasmin digestion of fibronectin containing cell attachment site with sequence Arg-Gly-Asp-Ser and also synthetic peptide reproducing this amino-acid sequence at the concentration of 1000 micrograms/ml released about 50% of collagenase and 55% of elastase from PMN-leukocytes. Moreover peptides containing cell attachment and gelatin binding site induced histamine release from mast cells. The association of fibrinogen and fibronectin degradation with activation of mast cells may motivate the treatment with antihistaminic drugs of all pathological conditions where the intensive protein degradation takes place.     

Biochemical analysis of desensitization of mouse mast cells

Biochemical mechanisms of desensitization were explored by using peritoneal mouse mast cells saturated with monoclonal mouse IgE anti-DNP antibody. It was found that a 1-min incubation of the sensitized cells with 0.01 micrograms/ml DNP-HSA in the absence of Ca2+ was sufficient to desensitize the cells completely. The treated cells failed to release a detectable amount of histamine upon incubation with an optimal concentration (0.1 to 1.0 micrograms/ml) of DNP-HSA and Ca2+. Determination of the number of antigen molecules bound to mast cells revealed that only a small (less than 10%) fraction of cell-bound IgE antibody molecules reacted with desensitizing antigen, and that desensitized cells and untreated (sensitized) cells could bind comparable amounts of antigen upon incubation with rechallenging antigen. However, the binding of antigen molecules to desensitized cells failed to induce any of the early biochemical events, i.e., phospholipid methylation, cAMP rise, and 45Ca uptake, as well as histamine release. It was also found that intracellular cAMP levels in desensitized cells were comparable to those in sensitized cells. Desensitization by a suboptimal concentration of DNP-HSA was prevented by inhibitors of methyltransferases, such as 3-deaza adenosine plus L-homocysteine thiolactone. Sensitized cells pretreated with 0.01 micrograms/ml DNP-HSA in the absence of Ca2+ and in the presence of the methyltransferase inhibitors responded to an optimal concentration of antigen for histamine release when they were rechallenged in the presence of Ca2+. Inhibition of desensitization by methyltransferase inhibitors was reversed by the addition of S-adenosyl-L-methionine to the system. The results indicated that the activation of methyltransferases, induced by receptor bridging, is involved in the process of desensitization. Desensitization was inhibited by reversible inhibitors of serine proteases, such as p-aminobenzamidine, indole, and synthesized substrates of rat mast cell proteases. It was also found that diisopropylfluorophosphate (DFP), an irreversible inhibitor of serine proteases, completely blocked desensitization at the concentration of 10 to 40 nM. This concentration of DFP did not affect the antigen-induced histamine release, whereas 100- to 1000-fold higher concentrations of DFP did inhibit histamine release. The results suggest that serine proteases are involved in both the induction of histamine release and desensitization, and that the protease involved in desensitization is distinct from that involved in triggering histamine release.     

The human recombinant c-kit receptor ligand, rhSCF, induces mediator release from human cutaneous mast cells and enhances IgE-dependent mediator release from both skin mast cells and peripheral blood basophils.

The gene product of the steel locus of the mouse represents a growth factor for murine mast cells and a ligand for the c-kit proto-oncogene receptor, a member of the tyrosine kinase receptor class of oncogenes (for review, see O. N. Witte. 1990. Cell 63:5). We have studied the effect of the human recombinant c-kit receptor ligand stem cell factor (rhSCF) on the release of inflammatory mediators from human skin mast cells and peripheral blood basophils and compared its activity to that of rhIL-3, rhSCF (1 ng/ml to 1 microgram/ml) activated the release of histamine and PGD2 from mast cells isolated from human skin. Analysis by digital video microscopy indicated that purified human skin mast cells (84 +/- 5% pure) responded to rhSCF (0.1 to 1 microgram/ml) challenge with a rapid, sustained rise in intracellular Ca2+ levels that was accompanied by secretion of histamine. A brief preincubation (10 min) of mast cells with rhSCF (0.1 pg/ml to 1 ng/ml) significantly enhanced (100 +/- 35%) the release of histamine induced by anti-IgE (3 micrograms/ml), but was much less effective on IgE-mediated release of PGD2. In contrast, a short term incubation with rhSCF did not potentiate the secretion of histamine activated by substance P (5 microM). A 24-h incubation of mast cells with rhSCF did not affect the release of mediators induced by anti-IgE (3 micrograms/ml), probably due to receptor desensitization, rhSCF (1 ng/ml to 3 micrograms/ml) neither caused release of histamine or leukotriene C4 (LTC4) release from leukocytes of 14 donors, nor induced a rise in intracellular Ca2+ levels in purified (greater than 70%) basophils. Brief preincubation (10 min) of leukocytes with rhSCF (1 ng/ml to 3 micrograms/ml) caused an enhancement (69 +/- 11%) of anti-IgE-induced release of histamine that was significant at concentrations as low as 3 ng/ml (p less than 0.05), whereas it appeared less effective in potentiating IgE-mediated LTC4 release. In contrast, a prolonged incubation (24 h) with rhSCF (0.1 pg/ml to 100 ng/ml) did not enhance the release of histamine or LTC4 induced by anti-IgE (0.1 microgram/ml), whereas rhIL-3 (3 ng/ml) significantly potentiated the release of both mediators.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Corticosteroids inhibit prostaglandin release from perfused mesenteric blood vessels of rabbit and from perfused lungs of sensitized guinea pig.

Infusion of norephinephrine (NE) (1 - 3 mug/ml/min) into the isolated mesenteric vascular preparation of rabbit resulted in a rise in perfusion pressure, which was associated with the release of prostaglandin E-like substance (PGE) at a concentration of 2.81 +/- 0.65 ng/ml in terms of PGE2. Indomethacin (3 mug/ml) abolished the NE-induced release of PGE. Arachidonic acid (0.2 mug/ml) in the presence of indomethacin did not restore the NE-induced release of PGE. Hydrocortisone (10 - 30 mug/ml) and dexamethasone (2 - 5 mug/ml) also inhibited the NE-induced release of PGE. The inhibitory action of both corticosteroids was abolished by arachidonic acid (0.2 mug/ml). Antigen-induced release of a prostaglandin-like substance (PGs) (43.1 +/- 3.8 ng/ml in terms of PGE2 and a rabbit aorta contracting substance (RCS) from perfused lungs of sensitized guinea pigs was completely abolished by indomethacin (5 mug/ml) or by hydrocortisone (100 mug/ml). Indomethacin, however, increased histamine release up to 280% of the control level, which was 470 +/- 54 ng/ml, while hydrocortisone diminished histamine release down to 30% of the control level. A superimposed infusion of arachidonic acid (1 mug/ml) into the pulmonary artery reversed the hydrocortisone-induced blockade of the release of RCS and PGs. It may be concluded that corticosteroids neither inhibit prostaglandin synthetase nor influence prostaglandin transport through the membranes but they do impair the availability of the substrate for the enzyme.     

Protein L. A bacterial Ig-binding protein that activates human basophils and mast cells.

Peptostreptococcus magnus strain 312 (10(6) to 10(8)/ml), which synthesizes a protein capable of binding to kappa L chains of human Ig (protein L), stimulated the release of histamine from human basophils in vitro. P. magnus strain 644, which does not synthesize protein L, did not induce histamine secretion. Soluble protein L (3 x 10(-2) to 3 micrograms/ml) induced histamine release from human basophils. The characteristics of the release reaction were similar to those of rabbit IgG anti-Fc fragment of human IgE (anti-IgE): it was Ca2(+)- and temperature-dependent, optimal release occurring at 37 degrees C in the presence of 1.0 mM extracellular Ca2+. There was an excellent correlation (r = 0.82; p less than 0.001) between the maximal percent histamine release induced by protein L and that induced by anti-IgE, as well as between protein L and protein A from Staphylococcus aureus (r = 0.52; p less than 0.01). Preincubation of basophils with either protein L or anti-IgE resulted in complete cross-desensitization to a subsequent challenge with the heterologous stimulus. IgE purified from myeloma patients PS and PP (lambda-chains) blocked anti-IgE-induced histamine release but failed to block the histamine releasing activity of protein L. In contrast, IgE purified from myeloma patient ADZ (kappa-chains) blocked both anti-IgE- and protein L-induced releases, whereas human polyclonal IgG selectively blocked protein L-induced secretion. Protein L acted as a complete secretagogue, i.e., it activated basophils to release sulfidopeptide leukotriene C4 as well as histamine. Protein L (10(-1) to 3 micrograms/ml) also induced the release of preformed (histamine) and de novo synthesized mediators (leukotriene C4 and/or PGD2) from mast cells isolated from lung parenchyma and skin tissues. Intradermal injections of protein L (0.01 to 10 micrograms/ml) in nonallergic subjects caused a dose-dependent wheal-and-flare reaction. Protein L activates human basophils and mast cells in vitro and in vivo presumably by interacting with kappa L chains of the IgE isotype.     

Synergistic stimulation of histamine release from rat peritoneal mast cells by 12-O-tetradecanoylphorbol 13-acetate (TPA)-type and non-TPA-type tumor promoters

Thapsigargin, a non-TPA (12-O-tetradecanoylphorbol 13-acetate)-type tumor promoter, provoked histamine release from rat peritoneal mast cells at concentrations above 30 ng/ml, but not at 10 ng/ml. TPA-type tumor promoters such as TPA, teleocidin and aplysiatoxin released very little, if any, histamine even at 100 ng/ml. When mast cells were incubated in medium containing thapsigargin at 10 ng/ml and varying concentrations of TPA-type tumor promoters, histamine release was increased synergistically. Maximum synergistic effects were observed at 10 ng/ml of each TPA-type tumor promoter. Palytoxin, another non-TPA-type tumor promoter, having no effect on histamine release at up to 10 pg/ml, also induced histamine release in the presence of 10 ng/ml of each TPA-type tumor promoter. However, no synergistic effect on histamine release was observed when mast cells were incubated in medium containing two different non-TPA-type tumor promoters, e.g., 10 ng/ml thapsigargin and 10 pg/ml palytoxin, or in medium containing two different TPA-type tumor promoters, e.g., TPA and teleocidin, TPA and aplysiatoxin, or teleocidin and aplysiatoxin (all at 10 ng/ml). These results suggest that the release of histamine from mast cells is stimulated synergistically under the mutual influence of TPA-type tumor promoters and non-TPA-type tumor promoters.     

Modulation of cyclic AMP in purified rat mast cells. II. Studies on the relationship between intracellular cyclic AMP concentrations and histamine release.

Changes in intracellular and extracellular rat mast cell adenosine 3':5' monophosphate (cAMP) concentrations during stimulation of histamine release by 48/80 were studied. There was a rapid and progressive fall in intracellular cAMP beginning within 10 sec after the addition of 48/80. The lowest cAMP values were obtained at 10 min, with return to control levels by 30 min. The fall in cAMP was dose-related with progressive decreases in 10-min cAMP measurements as the 48/80 concentration was increased from 0.25 to 1.00 mug/ml. There was a graded increase in histamine release over the same concentration range. Attempts to demonstrate significant amounts of cAMP in the medium during 48/80 stimulation were unsuccessful, indicating that the changes in cAMP intracellularly are not due to altered cellular permeability. There was a general correlation between the ability of pharmacologic agents to sustain high intracellular levels of cAMP in the presence of 48/80, and inhibition of histamine release. Theophylline (20 mM) which increased cAMP levels 2- 3-fold prevented a detectable decrease in cAMP after 1 mug/ml 48/80 (measured at 10 min) and almost completely inhibited histamine release. Prostaglandin E1 (27 muM) also raised cAMP levels, decreased the 48/80-induced fall in cAMP (by 42%). Epinephrine increased mast cell cAMP levels, but did not prevent the subsequent 48/80-induced decrease in cAMP and did not inhibit histamine release. Carbamylcholine (1 nM), adenine (1 muM), and diazoxide (10 muM) lowered mast cell cAMP and potentiated 48/80 induced release. In view of previous studies from this laboratory indicating that 48/80 stimulates mast cell phosphodiesterase, it seems likely that the 48/80-induced fall in cAMP is due, at least in part, to increased cAMP destruction. Since agents which prevent the fall in cAMP inhibit histamine release, it is apparent that cAMP is an important part of the control mechanism of histamine secretion. On the other hand, it cannot be concluded that a decrease in cAMP alone is sufficient to produce a response since carbamylcholine, diazoxide, and adenine which lower cAMP do not alter histamine release unless 48/80 is also present.     

Concerted stimulation of PI-turnover, Ca2+-influx and histamine release in antigen-activated rat mast cells

Phospholipid metabolism in rat mast cells activated by antigen was examined with reference to phosphatidylinositol (PI) turnover. Upon antigen stimulation, histamine release from passively sensitized mast cells with IgE was potentiated by adding phosphatidylserine (PS). The addition of antigen to [3H]glycerol-prelabeled and sensitized mast cells induced a marked loss of radioactivity of PI and a concurrent accumulation of 1,2-diacylglycerol (DG) and phosphatidic acid (PA) within 5 to 60 sec. Furthermore, this antigen-induced PI breakdown was enhanced in the presence of Mg2+. Histamine release occurred in parallel with PI breakdown. On the other hand, the transient Ca2+ influx into mast cells, as measured by uptake of 45Ca2+, was found to occur quickly after cells were activated by antigen, which was concerted with PI breakdown. These results suggest that enhanced PI turnover may be an important step in the biochemical sequence of events leading to release of histamine, and that not only Ca2+ but also Mg2+ appears to take a part in stimulus-response coupling in rat mast cells.     

Characterization of the anti-inflammatory effect of FK-506 on human mast cells.

We have examined the effects of FK-506 and of the struturally related macrolide rapamycin, which bind with high affinity to a specific binding protein (FKBP), to evaluate the involvement of this protein in the release of preformed (histamine) and de novo synthesized inflammatory mediators (sulfidopeptide leukotriene C4 and prostaglandin D2) from mast cells isolated from human lung parenchyma. FK-506 (0.1 to 300 nM) concentration dependently inhibited histamine release from lung parenchymal mast cells activated by anti-IgE. FK-506 was more potent in lung mast cells than in basophils (IC50 = 1.13 +/- 0.46 nM vs 5.28 +/- 0.88 nM; p less than 0.001), whereas the maximal inhibitory effect was higher in basophils than in lung mast cells (88.4 +/- 2.5% vs 76.4 +/- 3.8%; p less than 0.01). FK-506 had little or no inhibitory effect on histamine release from lung mast cells challenged with compound A23187, whereas it completely suppressed A23187-induced histamine release from basophils. FK-506 also inhibited the de novo synthesis of 5-lipoxygenase (sulfidopeptide leukotriene C4) and cyclo-oxygenase (prostaglandin D2) metabolites of arachidonic acid from mast cells challenged with anti-IgE. Unlike in basophils, Il-3 (3 to 30 ng/ml) did not modify anti-IgE- or A23187-induced histamine release from lung mast cells nor did it reverse the inhibitory effect of FK-506. Rapamycin (3 to 300 nM) had little or no effect on the release of histamine from lung mast cells, but it was a competitive antagonist of the inhibitory effect of FK-506 on anti-IgE-induced histamine release from human mast cells with a dissociation constant of about 12 nM. These data indicate that FK-506 is a potent anti-inflammatory agent that acts on human lung mast cells presumably by binding to a receptor site (i.e., FKBP).     

Biochemical analysis of glucocorticoid-induced inhibition of IgE-mediated histamine release from mouse mast cells

Pretreatment of mouse mast cells with 10(-7) to 10(-6) M dexamethasone (DM) during overnight sensitization with mouse IgE antibody resulted in inhibition of antigen-induced histamine release and degranulation. The inhibition of both degranulation and histamine release increased linearly with the duration of the treatment; maximal inhibition was obtained after approximately 16 hr with DM. The addition of DM to sensitized mast cells immediately before antigen challenge did not affect the antigen-induced histamine release. DM interacted directly with mast cells by binding to DM-specific cytoplasmic receptors. The treatment of mast cells with DM did not affect the binding of IgE to mast cells or intracellular cAMP levels. Bridging of cell-bound IgE anti-DNP antibody on mouse mast cells either by multivalent DNP-HSA or by anti-IgE induced phospholipid methylation at the plasma membrane and Ca++ influx into the cells. Pretreatment of mast cells with DM inhibited the antigen-induced phospholipid methylation and Ca++ uptake but failed to affect histamine release by Ca++ ionophore A23187. The results suggest that DM treatment inhibits histamine release by the inhibition of the early stage of biochemical processes leading to opening Ca++ channels but does not affect the process distal to Ca++ influx or the binding of IgE molecules to IgE receptors.     

Mast cells are not required for anaphylatoxin-induced ileal smooth muscle contraction

The complement-derived anaphylatoxin peptides, C3a and C5a, have long been considered to manifest their spasmogenic activities primarily through stimulation of mast cells. Although mast cells represent the major non-circulating repository for histamine, these cells also elaborate a number of additional, highly potent spasmogenic mediators derived from arachidonic acid. The same lipid mediators can be released by many other cell types. As a result, evaluation of the role of mast cells in anaphylatoxin-dependent responses cannot be based exclusively upon an analysis of the mediators released. We evaluated the role of mast cells in anaphylatoxin-induced ileal smooth muscle contractions by testing isolated segments of ileal tissues derived from genetically mast cell-deficient mice and their congenic normal (+/+) littermates. Isolated tissues from either congenic normal (+/+) or mast cell-deficient Sl/Sld mice responded similarly to acetylcholine, histamine, serotonin, prostaglandin E2, and the thromboxane A2 analog, U-46619. At 1 microgram/ml, histamine induced contractions of greater magnitude in tissues from mast cell-deficient animals; however, this mediator also desensitized the tissues to repeat challenge with histamine at the same concentration. C5a at 1 nM resulted in contractions equivalent to approximately 50% of the maximal KCl response; normal and mast cell-deficient tissues responded in a similar manner. C5a also released histamine from the normal mouse ileum, in addition to causing contraction of the tissues. C3a at 200 nM also produced similar contractile responses in both +/+ and S1/S1d tissues. These studies show that the anaphylatoxin peptides C3a and C5a are capable of contracting smooth muscle-containing tissues by a mechanism completely independent of mast cells. In addition, we also demonstrated that mast cell degranulation does not necessarily provoke ileal contraction. Thus compound 48/80, a mast cell degranulating agent unrelated to the anaphylatoxins, did not induce contractions in ileal tissues, even when used at concentrations as high as 100 micrograms/ml. Compound 48/80 did release histamine from the +/+ ileum, however, indicating that the agent was able to cause degranulation of ileal mast cells. Taken together, these data indicate that spasmogenic responses to anaphylatoxins (and possibly other agents) that are associated with mast cell degranulation need not necessarily require mast cell mediator release for their expression.     

Release of arylsulfatase A but not B from rat mast cells by noncytolytic secretory stimuli.

The net percentage of release of arylsulfatase activity from purified rat mast cells induced by rabbit anti-rat F(ab')2 was consistently only about 1/3 that of histamine. Isoelectric focusing of the released and residual arylsulfatase activities demonstrated specific release of the A type without B and a net percentage of immunologic release of arylsulfatase A equivalent to that of histamine. When the net percentage of histamine and arylsulfatase A release were nearly maximal (88 and 76%) in response to the calcium ionophore A23187, specific release of arylsulfatase B did not occur. Thus, arylsulfatase A and not B was associated with the secretory granule released from the rat mast cell by reversed anaphylaxis or the calcium ionophore. In contrast, subcellular fractionation of water-lysed mast cells yielded arylsulfatase B with the heparin- and chymase-containing granule fraction and arylsulfatase A in the aqueous fraction comprised of cell sap and granule water eluate. It may be that arylsulfatase B resides in a minor second granule, whereas arylsulfatase A is loosely associated with the predominant secretory granule of the rat mast cell.