Mechanism of histamine release from rat isolated peritoneal mast cells by dextran: The role of immunoglobulin E

In vivo passive sensitization of rat peritoneal mast cells withNippostrongylus braziliensis antiserum or rat monoclonal myeloma IgE greatly enhanced histamine releasein vitro by dextran or anti IgE, but did not alter release by compound 48/80 or A23187. Conversely, removal of IgE from the cells by acid pH abolished histamine release by dextran and anti IgE but did not impair release by compound 48/80. Whereas, histamine release from cells isolated from rats genetically resistant to dextran (NR rats) by anti IgE was potentiated by passive sensitization, dextran was unable to stimulate secretion from control or sensitized NR cells. The results suggest that dextran releases histamine by interaction with cell-fixed IgE and that the NR mast cell membrane lacks the ability to interpret this stimulus.     

On the mechanism of dextran-induced histamine secretion from rat peritoneal mast cells

The mechanism of histamine release induced from isolated rat peritoneal mast cells by clinical dextran was examined in detail. The putative involvement of cell-fixed IgE antibodies in the process was discounted by a number of experimental approaches. Instead, the characteristics of the release were found to be consistent with the interaction of the polysaccharide with specific glucoreceptors on the mast cell membrane.     

Histamine release from fawn-hooded rat mast cells is not potentiated by phosphatidylserine.

Histamine release from the peritoneal mast cells of the Fawn-hooded rat strain is not potentiated by phosphatidylserine (PS) or lysophosphatidylserine (Lyso-PS). When compared with Wistar rats the Fawn-hooded rats produce normal levels of IgE. Except for not being potentiated, the mast cells of Fawn-hooded rats release histamine similarly to Wistar rats when challenged with a variety of IgE and non-IgE mediated inducing agents. Mast cells of Fawn-hooded rats, however, do not respond significantly to dextran challenge which is not improved by PS of Lyso-PS. In the absence of PS the Ca2+ dependence of release from the Fawn-hooded mast cells is similar to the Wistar strain and both are similarly altered in their Ca2+ dependence by the presence of PS; with PS the mast cells of both rat strains release maximum percentages of histamine at levels of Ca2+ which are inhibitory in the absence of PS. The kinetics of release is similar in the two strains. However, when the mast cells are challenged with inducing agent in the absence of Ca2+ the rate of desensitization is slowed by PS in the Wistar strain but not the Fawn-hooded strain.     

Antigen-, anti-F(ab')2- and anti-IgE-induced histamine release from rat mast cells.

The existence of IgE antibody molecules on peritoneal mast cells from rats immunized with dinitrophenylated ascaris extract was demonstrated by double antibody immunofluorescence staining and by histamine release induced by anti-IgE antibodies. Cross-linking of the IgE molecules, which are fixed to mast cells, by interaction with either the homologous antigen or with antibodies to the Fab or Fc regions of IgE, triggered in the presence of calcium ions a chain of intracellular reactions which involve cyclic nucleotide modulation and energy-requiring processes leading ultimately to the release of histamine from the granules of these cells. Although there was no apparent difference in the mechanism(s) underlying the reactions triggered by any of these three agents, the extent of histamine release caused by antibodies to the Fc region of the IgE was significantly lower than that induced by cross-linking of IgE molecules through their Fab regions by reactions with either anti-Fab antibodies or antigens.     

Histamine release from human pulmonary mast cells

The enzyme collagenase was used to disperse human lung into its component cells. The resulting cell suspensions containedcirca 8% mast cells and were used for studies of mediator release without further purification. They exhibited a low (circa 7%) spontaneous release of histamine. They could be sensitized passively and released histamine upon challenge with anti-human IgE. They responded to concanavalin A but not to dextran. Phosphatidyl serine did not potentiate the release induced by these agents. The calcium ionophores, A23187 and ionomycin, both elicited histamine release. The cells were refractory to the action of the basic releasers 48/80 and peptide 401 (MCD-peptide). These results indicate marked differences between human pulmonary mast cells and the more widely used rat peritoneal mast cells.     

Histamine releasers and rat mast cells

The release of histamine from rat tissues rich in mast cells, induced by antigen, concanavalin A, dextran, compound 48/80, A 23187, phosphatidic acid and chlortetracycline, has been compared with that from isolated peritoneal mast cells of rats. Whereas most of the agents were more active than dextran in in vitro experiments, the reverse was found when they were injected intradermally into the skin, or subcutaneously into the paws. In fact, A 23187 and chlortetracycline (both calcium ionophores), as well as phosphatidic acid (another non-cytotoxic releaser), failed to release significant amounts of histamine when injected into the animal. Thus, the experimental conditions in which comparisons of the activities of histamine releasers are made should always be stated.     

Effect of sensitization and non-antibody IgE on 48/80 induced histamine release from isolated rat mast cells

Histamine release from isolated rat mast cells from non-immunized and immunized Hooded Lister rats was induced by compound 48/80. The histamine release was decreased with a lower maximum at the optimal concentration of 48/80 when using cells from immunized rats compared to non-immunized control rats. The stimulation of IgE antibody production, after immunization using B. pertussis as an adjuvant was also accompanied by an elevation of total serum IgW. The 48/80 induced histamine release from Sprague Dawley mast cells was not inhibited by immunization. Non-antibody IgE showed a non-competitive inhibition of 48/80 induced histamine release when myeloma IgE was incubated with mast cells from both Hooded Lister and Sprague Dawley rats. The results indicate the existence of different receptors for IgE and 48/80.     

Complement dependence of histamine release in chronic urticaria.

BACKGROUND: IgG autoantibodies directed to the alpha-subunit of the IgE receptor have been identified in 30% to 45% of patients with chronic urticaria. However, the exact mechanism by which histamine secretion is initiated is uncertain. OBJECTIVE: Histamine release from cutaneous mast cells may occur by cross-linking the IgE receptor or by activation of complement. Our goal is to distinguish these 2 possibilities. METHODS: We incubated human cutaneous mast cells with patient sera, decomplemented sera, or purified patient IgG. The IgG was also added to pooled normal serum or to sera deficient in either C2 or C5, and its ability to activate mast cells was assessed. Mast cells were incubated with human IgE myeloma to saturate alpha-subunits to determine the effect on histamine release. RESULTS: Patient sera released histamine (18.26% +/- 4.39%), but purified IgG from patients (5.5% +/- 4.3%) did not. Addition of the patient IgG to normal sera rendered the sera positive for histamine-releasing activity (18.4% +/- 4.3%), whereas control IgG or patient IgG added to C2- or C5-deficient sera did not release histamine. Histamine release with decomplemented patient sera was also diminished (8.34% +/- 4.3%). Preincubation of mast cells with a C5-blocking peptide decreased histamine release but was not statistically significant; there was a significant decrease after preincubating mast cells with IgE myeloma. CONCLUSION: The degranulation of mast cells by IgG autoantibodies in patients with chronic urticaria requires binding to the IgE receptor and activation of the classical complement cascade. Saturation of the IgE receptor with IgE inhibits such degranulation, presumably by preventing binding of the requisite IgG.     

Modulation of tryptase and histamine release from human lung mast cells by protease inhibitors

Inhibition of IgE dependent histamine release from human mast cells by protease inhibitors has been observed in skin, tonsil and synovial tissues. However, little is known about the actions of protease inhibitors on tryptase release from human lung mast cells. We therefore examined the ability of protease inhibitors to modulate tryptase and histamine release from human lung mast cells. IgE dependent tryptase release from dispersed lung mast cells was inhibited to a maximum of approximately 53.8% and 44.5% by N-a-tosyl-L-lysine chloromethyl ketone (TLCK) and N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK), respectively. A similar degree of inhibition of calcium ionophore A23187 (CI) induced tryptase release was also observed with these two inhibitors. Preincubation of TLCK or TPCK with the mast cells at 37 degrees C for 20 minutes before addition of anti-IgE or CI did not improve their ability to inhibit anti-IgE and CI induced tryptase release. At a concentration of 10 microg/ml, protamine inhibited anti-IgE or CI induced tryptase release; but at 100 microg/ml, it increased anti-IgE and CI induced release of tryptase from lung mast cells. A concentration dependent inhibition of anti-IgE and CI induced release of histamine from lung mast cells was also observed with TLCK, TPCK and protamine. The maximum inhibition of anti-IgE induced histamine release was approximately 40.7%, 40.2% and 33.4% with TLCK, TPCK and protamine, respectively. At the concentrations tested, TLCK and TPCK by themselves did not stimulate tryptase and histamine release from lung mast cells. A specific inhibitor of aminopeptidase, amastatin, had no effect on anti-IgE induced tryptase and histamine release and was used as control. In conclusion, it was demonstrated that protease inhibitors are able to inhibit IgE dependent tryptase and histamine release from human lung mast cells, which suggested that they could be developed to a novel class of anti-inflammatory drugs to treat allergic conditions in man.     

Oestradiol enhances in vitro the histamine release induced by embryonic histamine-releasing factor (EHRF) from uterine mast cells.

The relationship between maternal hormones and factors secreted by the implanting embryo is still controversial. We have analysed the in-vitro effect of oestradiol and human embryo-derived histamine-releasing factor (EHRF) on histamine release from rat uterine mast cells. Rat uterine mast cells which were preincubated with oestradiol and then challenged with human EHRF gave histamine release values two- to threefold higher than those without preincubation. The enhancement observed was time- and temperature-dependent. A similar enhancement was obtained with human sensitized basophils but not with rat peritoneal mast cells. Oestradiol, used as a direct challenge, did not induce any histamine release from either rat uterine or peritoneal mast cells, or from human sensitized basophils. Oestradiol preincubation also enhanced the histamine release induced by anti-IgE but did not enhance the histamine release induced by substance P or compound 48/80, two secretagogues that are not mediated by IgE. Moreover, uterine fragments derived from rats at various oestrus phases, with different amounts of endogenous oestrogen, were challenged in vitro with EHRF. The release of histamine by mast cells was higher at the proestrus and preimplantation phases than at dioestrus. All these findings suggest that the interaction of oestradiol with rat uterine mast cells was capable of enhancing in vitro the histamine releasing effect of EHRF.     

IgE receptors, IgE content and secretory response of mast cells in athymic rats.

Athymic, nude (Lewis rnu/rnu) and normal (Lewis +/+) rats were used to study the T-cell dependence of the regulation of IgE receptors and IgE content on mast cells in vivo. We estimated the number of IgE receptors and the IgE content on peritoneal mast cells using a cytofluorometric technique. The secretory capacity of the mast cells was measured in terms of histamine release as a function of anti-IgE concentration by incubation with antibodies in vitro. Two groups of rats, aged 6 and 13 weeks, were examined. The mast cells of the rats belonging to the older age group (both normal and athymic) had a higher total protein (equivalent to dry mass or size) and mediator content (heparin, histamine and 5-hydroxytryptamine) in accordance with previous findings. Mast cells of the athymic rats of both age groups contained similar levels of IgE receptors and IgE and did not differ in these respects from those of the normal rats. Of the IgE receptors available for binding, about 80% were occupied by IgE in mast cells of normal and of athymic rats in both age groups. The anti-IgE-induced histamine release of the mast cells was, however, significantly lower in athymic rats compared to the normal controls. A change in housing from barrier-maintained to conventional conditions for 2 weeks enhanced the IgE-receptor and IgE content, as well as the releasability of histamine of the mast cells of both athymic and normal rats. The basal level of IgE occupancy of the receptors on mast cells was therefore not impaired in the athymic rats, as might be inferred from previous findings of a T-cell dependence of the IgE immune response. The results further indicate that T-lymphocyte-derived cytokines appear not to be required for either the expression of IgE receptors or for their ability to acquire IgE on mast cells, but such factors seem to enhance the release of histamine following the cross-linkage of the IgE receptor on mast cells in normal conditions.     

Immunological modulation of cholinergic histamine release in isolated rat mast cells

Isolated purified rat mast cells release histamine when exposed to acetylcholine according to different patterns of sensitivity.The degree of histamine release is correlated with the levels of reaginic antibodies presumably bound to the mast cell membrane. In fact, mast cells passively sensitized with mouse myeloma IgE against egg albumin or DNP₂-lysine, react to acetylcholine with a release of histamine, which is proportional to the IgE concentration in the incubation medium.The histamine release induced by acetylcholine is due to the stimulation of a musearinic receptor. Accordingly, acetylthiocholine is unable to evoke histamine release and preincubation of sensitized cells with atropine fully inhibits the cholinergic histamine release. The histamine release evoked by acetylcholine is potentiated by the exposure of sensitized cells to the specific antigen.The present results suggest that sensitization of mast cells is a crucial factor in modulating their sensitivity to acetylcholine.     

Degranulation of in vitro differentiated mast cells stimulated by two monoclonal IgE specificities

Confluent spread of mature nondividing mast cells is obtained after 1 month's growth of lymph node cells taken from mice immunized with horse serum and plated on embryonic fibroblast monolayers. The degranulation capacity of these mast cells and histamine release stimulated by monoclonal IgE antibodies and their antigens were studied. The mast cells were first incubated with either anti-2,4-dinitrophenyl (DNP) IgE or with anti-ovalbumin (OVA) IgE and then with the other IgE to study the ability of one IgE specificity to saturate the receptors and block the ability of the other IgE to bind and evoke degranulation. Saturation of the receptors and maximal histamine release (86-92%) were obtained within 2-3 h incubation with excess IgE (1-10 micrograms/ml). No histamine was released after incubation for 3-4 h with the other IgE (0.38-4.0% histamine release). Seventeen days after the excess, unbound, saturating IgE anti-DNP was washed away, 75% of the histamine was still released after antigen DNP-bovine serum albumin was added. However, these mast cells were still effectively blocked from sensitization with IgE anti-OVA (1.71% histamine release). The blocking could be broken if the second IgE molecules were allowed to stay longer than 4 h in culture. From 10 h onwards, the degranulation capacity steadily increased (from 59.6% histamine release after 10 h to 79.5% after 42 h). In studies with 125I-labeled IgE, there was a direct correlation between the rate of binding of the second IgE to the mast cells (from 1.7% binding after 3 h to 75% after 48 h) and the increase in degranulation rate with the second antigen (from 2.40% histamine release to 65.4%). In contrast, only slight binding of the 125I-labeled IgE of the saturating specificity occurred (4.2% after 3 h to 12.7% binding after 48 h). Incubation of mast cells, previously saturated with 125I-labeled IgE, with cold IgE of either specificity did not proportionally reduce the cell-bound label. This suggests that no substitution of IgE molecules on the receptors occurred. When the mast cells saturated with anti-OVA were incubated with IgE anti-DNP together with tunicamycin, the development of the degranulation capacity by DNP-bovine serum albumin was inhibited. The results suggest that IgE molecules of the other specificity stimulated the appearance of new receptors on the mast cell surface.     

A new concept of triggering mechanisms of IgE-mediated histamine release.

It is generally accepted that an initial step of reaginic hypersensitivity reactions is a bridging of mast cell--bound IgE antibody molecules by antigen. Since IgE molecules are firmly bound to receptors on mast cells, bridging of cell-bound IgE molecules probably brings receptor molecules into close proximity. A hypothesis was therefore presented that such a local change in membrane structure and/or possible interaction between adjacent receptor molecules may be triggering mechanisms of IgE-mediated histamine release. The hypothesis was tested by use of antibodies against "exposed portion" of receptor molecules on rat basophilic leukemia cells. It was found that antireceptor antibodies and its F(ab')2fragments induced noncytotoxic histamine release from normal rat mast cells without participation of IgE, while the monovalent Fab' fragments of the antibody failed to do so. However, sensitization of normal rat skin with the Fab' fragments followed by an intravenous injection of antirabbit IgG induced skin reactions. These findings support the concept that bridging of receptors rather than polymerization of IgE molecules is responsible for the activation of membrane-associated enzymes which in turn leads to histamine release.     

Monoclonal murine anti-DNP IgE:in vitro histamine release of rat mast cells in the presence of reaginic rat and mouse sera

Mast cells from normal rats and animals reinfected withNippostrongylus brasiliensis (N.b.) were sensitizedin vitro with monoclonal anti-DNP mouse IgE, reaginic mouse serum with ovalbumin (OA) specificity and reaginic rat serum against N.b. The sensitized cells were triggered for the release of histamine with DNP-bovine-serum-albumin (DNP-BSA), OA, N.b.-homogenate and guinea-pig antimouse IgE. The histamine release from normal mast cells sensitized with monoclonal mouse IgE was inhibited either with N.b.-reaginic rat serum or OA-reaginic mouse IgE (30g/10⁵ mast cells) completely densensitized mast cells from reinfected rats for specific histamine release in the presence of either N.b.-antigen or DNP-BSA. Anti-mouse IgE which was prepared by monoclonal mouse IgE did not bind to rat mast cells sensitized with rat IgE as was revealed by immunofluorescence experiments. Consequently we observed that anti-mouse IgE failed to trigger the histamine release from mast cells of reinfected rats.     

Histamine release from rat mast cells sensitized with mouse antiserum

The characteristics of the antigen-induced and nonantigen-induced histamine release from rat peritoneal mast cells sensitized in vitro with mouse anti-ovalbumin serum were investigated. The effects of some antiallergic drugs on these release reactions were also studied.Besides antigen-specific IgE antibody, heat-labile factor(s) responsible for the non-antigen-induced histamine release were found in mouse antiserum. Such factors were also present in normal mouse serum. In the absence of antigen, the combination of phosphatidyl serine and Ca⁺⁺ induced some extent of histamine release from mast cells treated with these factors.From the present results it is suggested that quercetin selectively and verapamil primarily act to block calcium-gate opening resulting from antigen-antibody interaction on the mast cell membrane, while theophylline and disodium cromoglycate selectively inhibit the passage of calcium through open calcium channels.     

Isolation and some properties of mast cells from the mesentery of the rat and guinea pig

A number of enzymes were screened for their ability to dissociate mesenteric tissues from the rat and guinea pig into their component cells. The bacterial enzyme collagenase was found to be the most satisfactory agent and a procedure based on the use of this protease was developed. The resulting suspensions contained 1–2% free mast cells and exhibited a low (ca. 5%) spontaneous release of histamine. The tissue cells contained less histamine than rat peritoneal mast cells and the guinea pig cells were smaller in size. Cells obtained from actively sensitized animals responded to antigenic challenge more strongly than the chopped tissue indicating that they were functionally intact. Rat mesenteric cells could be passively sensitized with homologous reaginic antibody and also responded to anti-rat IgE. The immunologically induced releases from rat mesenteric and peritoneal cells showed differing sensitivities to potentiation by phosphatidyl serine but the responses were directly comparable in the absence of this effect. Rat mesenteric cells also responded, but less effectively than the peritoneal cells, to the ionophore A23187, concanavalin A, ATP and basic secretagogues. They were, however, essentially refractory to the action of dextran. In contrast, guinea pig mast cells responded strongly only to the ionophore and weakly or not at all to the other agents. These results indicate marked inter-and intra-species differences in the reactivity of mast cells and suggest that rat peritoneal cells should not be used as the sole model for studying histamine secretion.     

The effect of rat mast cell sensitization with mouse IgE antibody on anaphylactic histamine release by anti-mouse IgE and Con A

The peritoneal mast cells of rat (Wistar X August) F1 were incubated with purified mouse IgE antibodies and then challenged (in the presence of 50% D2O) with antigen, anti-mouse IgE and concanavalin A. It was found that incubation of mast cells with IgE antibodies led to different level of cell sensitization (in mast cells from different rats), expressed in antigen-induced histamine release (0-52%). Moreover, a) significant antigen-induced histamine release was usually accompanied by high Con A- and anti-IgE-induced histamine release from these cells; the magnitude of release was comparable to Con A- and anti-IgE-induced release from control, nonsensitized cells of the same rat; b) low antigen-induced histamine release was accompanied by the decrease of Con A- and anti-IgE-induced release, as compared to the release from control cells. This fall of reactivity to Con A and anti-IgE was statistically significant and was irreversible during 120 min.     

The human adenoidal mast cell. Susceptibility to different secretagogues and secretion inhibitors

Mast cells from adenoid tissues of man do not form a homogeneous population but differ in size and ultrastructure. They degranulate on stimulation with specific antigen, ionophore A 23187, Concanavalin A and, under certain conditions, antihuman IgE or acetylcholine. Compound 48/80, dextran, purified C3a or C5a are ineffective. Adenosine exerts either an enhancing or inhibiting effect on the stimulated histamine release, depending on the actual conditions of the mast cells. The study of a range of known mast cell degranulation inhibitors revealed important differences between human adenoidal mast cells and human basophils or animal mast cell systems. The efficacies of the drugs or drug combinations so far tested correspond well with the established therapeutic effects in man.     

The loss of sensitization of mouse and rat mast cells by acid pH treatment

Short incubation of sensitized mouse and rat peritoneal mast cells at 37 degrees C in well defined acid pH (3.5 for mouse and 4.5 for rat mast cells) induces the loss of sensitization, as indicated by antigen-induced histamine release. The acid pH pretreatment of normal mouse and rat mast cells seems to facilitate their subsequent sensitization with mouse IgE antibodies. The possibility, that acid pH treatment removes non-specific IgE from the cells and thus facilitates the binding of specific IgE antibodies is discussed.