Normal Rat Mast Cells Research Articles

Involvement of a serine protease in mast cell activation.

To examine the putative role of an endogenous serine esterase in mast cell activation, we have investigated the effect of inhibitors of, and substrates for, alpha-chymotrypsin in normal and permeabilized rat mast cells. These agents effectively blocked histamine release induced by anti-IgE, with an enhanced potency in permeabilized cells, but were ineffective against secretion evoked by compound 48/80. Activation of a chymotryptic enzyme, as evidenced by hydrolysis of a fluorescent substrate, was directly demonstrated following immunologic stimulation of permeabilized mast cells. No such activation was observed with compound 48/80. Immunologic stimulation also led to a significant increase in the total chymotryptic activity recoverable from the cells.

Supernormal Histamine Release and Normal Cytotoxic Activity of Beige (Chédiak-Higashi Syndrome) Rat Mast Cells with Giant Granules

The beige rat is an animal model of the Chédiak-Higashi syndrome. Since mast cells can be easily purified from the peritoneal cavity of rats, we investigated the function of beige rat mast cells with giant granules by using quantitative methods. Beige and normal rat mast cells were sensitized with anti-dinitrophenol (DNP) IgE antibodies and stimulated by DNP conjugated with human serum albumin. The proportion of histamine released to total histamine was significantly greater in beige rat mast cells than in normal rat mast cells. Since the supernormal histamine release of beige rat mast cells was observed after treatment with 12-O-tetradecanoylphorbol 13-acetate, calcium ionophore A23187, substance P or compound 48/80, it appeared to be attributable to the enlargement in granules in beige rat mast cells. Spontaneous cytotoxic activity of mast cells was assayed by incubating purified mast cells with 51Cr-labelled WEHI-164 cells. Both beige and normal rat mast cells showed significant cytotoxic activity, but no significant difference was detectable between beige and normal rat mast cells. Even after IgE-mediated stimulation, no significant difference in cytotoxic activity was detectable between beige and normal rat mast cells either. Giant granules of beige rat mast cells did not appear to influence the cytotoxic activity of mast cells.

Secretion of plasminogen activator inhibitor by normal rat pleural leukocytes in culture

The normal balance between coagulation and fibrinolysis in the pleural cavity is poorly understood despite the critical role of the pleura in the movement of the lungs. To determine the fibrinolytic activity and the interaction between plasminogen activators and their inhibitors in the normal pleural space, we tested normal rat pleural leukocytes, principally macrophages and mast cells, and their supernatants, for activity in an [125I]fibrin degradation assay. It was found that pleural leukocytes did not release plasminogen activator, but the leukocytes and their supernatants inhibited the plasminogen-dependent fibrinolysis caused by both alveolar leukocytes and mesothelial cells. Further experiments demonstrated that pleural leukocytes produce a protein inhibitor primarily against urokinase-induced fibrinolysis in culture and that macrophages are the main source of the inhibitor. The lysate of mast cell-enriched population exhibited high plasminogen activator activity while no such activity could be determined in macrophage-enriched lysate. These data show that normal rat pleural leukocytes contain plasminogen activator inside the cells and synthesize a urokinase-type plasminogen activator inhibitor in culture that may be important in the fibrinolysis/coagulation balance in the pleural space.

Determination of Cow Milk Formula Allergenicity in the Rat Model by in vitro Mast Cell Triggering and in vivo IgE Induction

The allergenicity of cow milk formulas was assessed in vitro by their ability to trigger prelabelled 3H-serotonin release from normal rat mast cells sensitized passively with rat reaginic anti-cow milk protein antisera. It was found that enzymatically hydrolyzed milk formulas had 1,000 (BEBAHA) to over 100,000 (ALFARE) times lower mast cell-triggering capacities than a standard milk formula (NAN). In vivo, these hydrolyzed formulas induced 100 (BEBAHA) to 10,000 (ALFARE) times less IgE antibodies than the standard milk formula.

Monoclonal antibodies that inhibit IgE binding.

Four monoclonal antibodies were produced that inhibit IgE binding to the high affinity IgE receptor (Fc epsilon R) on rat basophilic leukemia cells. The four monoclonal antibodies (mAb) fall into two groups. The first group was comprised of 3 antibodies (mAb BC4, mAb CD3, and mAb CA5) that reacted with the Fc epsilon R at epitopes close or identical to the IgE-binding site. With 125I-labeled antibodies there was reciprocal cross-inhibition between the antibodies and IgE. The antibodies activated both RBL-2H3 cells and normal rat mast cells for histamine release. The 3 antibodies immunoprecipitated the previously described alpha, beta, and gamma components of the receptor. The number of radiolabeled Fab fragments of 2 of these antibodies bound per cell was similar or equal to the number of IgE receptors. In contrast, the mAb BC4 Fab bound to 2.1 +/- 0.4 times the number of IgE receptor sites. Therefore, the portion of the Fc epsilon R exposed on the cell surface must have two identical epitopes and an axis of symmetry. These 3 monoclonal antibodies recognize different but closely related epitopes in the IgE-binding region of the Fc epsilon R. The fourth monoclonal antibody (mAb AA4) had different characteristics. In cross-inhibition studies, IgE and the other 3 monoclonals did not inhibit the binding of this 125I-labeled monoclonal antibody. The number of molecules of this antibody bound per cell was approximately 14-fold greater than the Fc epsilon R number. This monoclonal antibody caused the inhibition of histamine release and it appears to bind to several cell components.

Studies of phosphorylation in rat mast cells (third report)--isolation of calcium-activated, phospholipid, diacylglycerol-dependent protein kinase from rat basophilic leukemia cells (RBL-1 cells)

Evidence is presented that RBL-1 cells, which are similar to normal rat mast cells in morphology and contain IgE receptors and histamine, contain a calcium-activated, phospholipid, diacylglycerol-dependent protein kinase. This enzyme is very similar in its activation requirements to the calcium-dependent enzyme termed protein kinase C in other tissues. The enzyme is activated by Ca2+. Diolein, but not other di, mono or triglycerides, substantially increases the enzyme activity. Among various phospholipids, phosphatidylserine is the most reactive activator; phosphatidylinositol, phosphatidic acid and phosphatidylethanolamine are less effective; and phosphatidylcholine is practically inactive. The enzyme is inhibited by chlorpromazine and local anesthetics such as dibucaine, tetracaine and procaine.

The fate of IgE bound to rat basophilic leukemia cells. III. Relationship between antigen-induced endocytosis and serotonin release.

We have previously shown that, unlike monomeric IgE, chemically derived dimers, trimers, and heavier oligomers of IgE were internalized efficiently. This finding suggested that endocytosis, like mediator release, is triggered by cross-linking of the cell surface receptors for IgE. In the present study, we analyzed the temporal and functional relationships between the two events. We used rat basophilic leukemia cells (RBL-HR+-2H3) and rat peritoneal mast cells, which were allowed to bind monomeric 125I mouse IgE hybridoma anti-dinitrophenyl (HI-DNP-E-26-82), and the polyvalent antigen 131I-dinitrophenylated human serum albumin (DNP15-HSA). We found that at 37 degrees C, 50% of the cell surface-bound immune complexes were internalized rapidly (t1/2 3 to 5 min) by RBL-HR+-2H3 cells with only minimal reduction (1/3) in the extent of internalization when very few of the receptors (approximately 5%) were saturated with IgE. Normal mast cells internalized cell surface-bound immune complexes at a similar rate (t1/2 4 to 5 min). Unlike serotonin release, internalization was independent of extracellular calcium and continued to increase as the ratio of DNP15-HSA to IgE increased 10- to 100-fold over the ratio required for optimal histamine release. In the RBL cells, internalization preceded serotonin release, reaching a peak at about 10 min, while the release (t1/2 13 to 19 min) continued for up to 60 min. Presumably, some of the cross-linked IgE internalized less effectively and continued to trigger serotonin release. The reverse relationship between the rates of internalization and release (t1/2 less than 1 min) was found in normal rat mast cells. We conclude that although cross-linking of two or more receptors triggered both endocytosis and exocytosis, the two events are not necessarily sequential.

On the secretory process of rat mast cells and rat basophilic leukaemia cells. Effects of aggregated IgGa on 45Ca influx and histamine secretion.

The biological role of heat-aggregated IgGa during the secretory process of rat mast cells and rat basophilic leukaemia (RBL) cells was analysed. Incubation of normal rat mast cells and rat basophilic leukaemia cells with heat-aggregated IgGa alone leads to a stabilization of the cells resulting in a reduction of mediator secretion and ion influx. When mast cells from Nippostrongylus brasiliensis-infected animals were incubated with heat-aggregated IgGa and subsequently challenged with anti-IgE, a dual effect with regard to ion influx and histamine release was observed, depending on the concentration of heat-aggregated IgGa or anti-IgE either a pronounced decrease or a marked enhancement was apparent. Our data suggest that heat-aggregated IgGa as well as immune complexes modulate the secretory response of mast cells and may thus play an important role in the allergic disease process.

Binding properties of IgE receptors on normal mouse mast cells.

Normal rat mast cells and mouse mast cells were purified, and the binding of rat IgE and mouse IgE to IgE receptors was measured. When normal rat mast cells were saturated with either rat IgE or mouse IgE, comparable numbers of IgE molecules bound to the cells. The number of the receptors on rat mast cells was approximately 3 X 10(5)/cell. The forward rate constant, K1, dissociation constant K-1, and equilibrium constant, KA, for rat IgE and mouse IgE were almost similar; KA for rat IgE and mouse IgE were 8 X 10(9) M-1 and 2.5 X 10(9) M-1, respectively. The results indicated that rat IgE and mouse IgE bind to the same IgE receptors on rat mast cells with comparable affinity. It was also found that IgE from the two species bound to mouse mast cells with high affinity. Forward rate constant, K1, for the binding of mouse IgE and rat IgE to IgE receptors on mast cells from BALB/c mice were 1.9 X 10(5) M-1 sec-1 and 1.5 X 10(5) M-1 sec-1, respectively. Mouse IgE and rat IgE dissociate from the receptors with comparable rate (approximately 10(-4) sec-1). However, mouse mast cells appear to have two distinct types of IgE receptors. One type binds both rat IgE and mouse IgE with comparable affinity, whereas the second type binds only mouse IgE. This type of receptor comprises about one-third to one-half of IgE receptors on mast cells of CBA/J mice, and about one-half to two-thirds of IgE receptors in BALB/c mice. Although the binding of rat IgE to these receptors was not detected, the presence of rat IgE along with 125I-mouse IgE interferred with the binding of the latter protein to the receptors. It was suggested that rat IgE might associate with the second type receptors with a forward rate constant comparable to those of mouse IgE, but dissociate rapidly from the receptors.

Bridging of IgE receptors activates phospholipid methylation and adenylate cyclase in mast cell plasma membranes.

Bridging of IgE receptors on normal rat mast cells by divalent anti-receptor antibodies induced phospholipid methylation and an increase in intracellular cyclic AMP within 15 sec after the receptor bridging. These biochemical events were followed by Ca2+ influx and histamine release. When IgE receptors on isolated plasma membranes were bridged by the antibody, both the increase in the incorporation of [3H]methyl into lipid fraction and the synthesis of cyclic AMP were demonstrated. The synthesis of cyclic AMP in this system was enhanced in the presence of GTP. The results indicated that the bridged IgE receptors are linked to both methyltransferases and adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in the plasma membrane. An increase in cyclic AMP prior to receptor bridging suppressed phospholipid methylation in the plasma membrane, Ca2+ uptake, and subsequent histamine release. On the other hand, inhibition of phospholipid methylation by (S)-isobutyryl-3-deazaadenosine resulted in the suppression of cyclic AMP synthesis in the plasma membrane. These findings suggest that the activation of phospholipid methylation and the activation of adenylate cyclase are e, and subsequent histamine release. On the other hand, inhibition of phospholipid methylation by (S)-isobutyryl-3-deazadenosine resulted in the suppression of cyclic AMP synthesis in the plasma membrane. These findings suggest that the activation of phospholipid methylation and the activation of adenylate cyclase are e, and subsequent histamine release. On the other hand, inhibition of phospholipid methylation by (S)-isobutyryl-3-deazadenosine resulted in the suppression of cyclic AMP synthesis in the plasma membrane. These findings suggest that the activation of phospholipid methylation and the activation of adenylate cyclase are mutually regulated.

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

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

Larger oligomers of IgE are more effective than dimers in stimulating rat basophilic leukemia cells.

The relative activity of rat IgE oligomers in eliciting secretion from mast cells and tumor basophils was examined using stable oligomers prepared by cross-linking IgE with dimethyl suberimidate. Oligomers were tested in vivo using passive cutaneous anaphylaxis (PCA) and in vitro by measuring their capacity to induce histamine release from normal rat mast cells and [3H]-5-hydroxytryptamine release from rat basophilic leukemia (RBL) cells. The PCA results confirmed previous work that showed no large differences in the activity of dimers, trimers, and higher oligomers of IgE. The same was also true with normal rat mast cells; however, the RBL cells sharply discriminated between the variously sized oligomers. The stimulatory activity of all oligomers on the RBL cells was enhanced by D2O. The findings imply that the manner by which the clustering of the receptors for IgE leads to generation of signals in RBL cells involves a mechanism capable of discriminating between a variety of polymeric states of the receptors. In addition, our results suggest that no more than a few hundred trimers or an even smaller number of higher oligomers are capable of inducing a considerable amount of secretion from these cells.

Enzymatic formation of prostaglandin D 2 by rat basophilic leukemia cells and normal rat mast cells

It has been shown that the major cyclooxygenase product in rat basophilic leukemia (RBL-1) cells and in normal rat mast cells is prostaglandin D 2. In RBL-1 cells, prostaglandin D 2 isomerase activity was found in the 150 000 × g microsomal pellet as well as the supernatant fraction. Incubation of RBL-1 microsomes with arachidonic acid without cofactors yielded 17.5 ± 2% prostaglandin E 2 and 9.1 ± 1.4% prostaglandin D 2. The cyclooxygenase activity was enhanced (25%) by epinephrine and the addition of reduced glutathione led to a marked increase in prostaglandin D 2 synthesis (3-fold). Incubations with arachidonic acid, glutathione and epinephrine gave the maximum conversion to prostaglandin D 2, yielding 7 ± 0.4% prostaglandin E 2 and 35.6 ± 3.5% prostaglandin D 2. Incubations with [ 14C]prostaglandin H 2 to bypass cyclooxygenase confirmed the presence and glutathione dependence of the prostaglandin D 2 isomerase in the microsomal fraction and also revealed the presence of the same enzyme in the 150 000 × g supernatant. In contrast to RBL-1 cells, incubations of microsomes and supernatant from normal rat mast cells with [ 14C]arachidonic acid and [ 14C]prostaglandin H 2 localized the prostaglandin D 2 isomerase activity in the soluble fraction. Similar to the enzyme in the RBL-1 cells, the mast cell enzyme was glutathione dependent.

Stimulation of phospholipid methylation, Ca2+ influx, and histamine release by bridging of IgE receptors on rat mast cells.

Normal rat mast cells were stimulated by antibodies against IgE receptors (anti-RBL) or by anti-IgE, and [3H]methyl group incorporation into phospholipids, 45Ca uptake, and histamine release were examined. Anti-RBL or its F(ab')2 fragments and anti-IgE induced an increase in the incorporation of [3H]methyl into phospholipids, in 45Ca influx, and in histamine release. By contrast, Fab' monomer fragments of anti-RBL induced none of these reactions. The transient increase of [3H]methyl incorporation in lipids peaked within 15 sec after the addition of either anti-RBL or anti-IgE and fell to basal level in 30 sec. This was then followed by an influx of 45Ca that increased to a maximum in 2 min and by histamine release that reached a maximum in 3 min. Inhibition of phospholipid methylation resulted in an inhibition of 45Ca influx and histamine release. These findings demonstrate that phospholipid methylation in rat mast cells is induced by bridging of IgE receptors on the cell surface and that increased methylation of phospholipids sets the stage for an influx of Ca2+ and subsequent release of histamine.

Further studies on the structural requirements for polypeptide-mediated histamine release from rat mast cells.

Structure-activity studies have been performed on a series of naturally occurring and 'tailor-made' polypeptides, by measurement of ability to induce selective histamine release from normal rat peritoneal mast cells in vitro. Compounds investigated include corticotropin and melittin derivatives, mast-cell-degranulating peptide from bee venom, polymyxin B, bradykinin and various synthetic poly(amino acids) and short-chain peptides. It was confirmed that a cluster of four basic residues (lysine or arginine) was optimal for histamine release by corticotropin and melittin polypeptides, provided that the C-terminal carboxyl group was substituted (by, for instance, amidation). In contrast, the presence of a free C-terminal carboxyl group or nearby dicarboxylic acid residues led to a considerable diminution in histamine-releasing activity. Likewise, polypeptides comprised essentially of acidic amino acids were inactive. On the basis of these observations it has been possible to predict that synthetic peptides comprising a particular sequence within the Fc region of human immunoglobulin E, the immunoglobulin class particularly involved in mediation of allergic reactions of the immediate type, would possess potent histamine-releasing activity when similarly made to react with normal rat mast cells. The further study of such a structure should throw new light on the molecular basis of allergen-antibody triggering of mast cells.

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′) 2 fragments 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.

Histamine Release from Rat Mast Cells by Antibodies against Rat Basophilic Leukemia Cell Membrane

Abstract Attempts were made to prepare antibodies against receptors for IgE. Rat basophilic leukemia (RBL) cells were saturated with IgE and solubilized in Nonidet P-40, and IgE receptor complexes were precipitated with anti-IgE. A rabbit was immunized with the precipitates and antibodies against cell membrane components were specifically purified by using RBL cells. After absorption, the antibody preparation (anti-RBL) was specific for mast cells but did not contain anti-IgE. Anti-RBL inhibited the binding of 125I-IgE with RBL, normal rat mast cells, and solubilized receptors. Immunofluorescence staining of RBL and mast cells with anti-RBL was significantly inhibited, if their receptors had been saturated with IgE. These results indicated that anti-RBL contained antibodies against receptor molecules. Both anti-RBL and anti-IgE induced immediate skin reactions in normal rats, whereas anti-lymphocyte serum, which cross-reacts with mast cells but lacks anti-receptors, failed to induce the reaction. Infection of rats with Nippostrongylus brasiliensis, which enhanced IgE synthesis and resulted in saturation of receptors on mast cells with IgE, increased the sensitivity of their skin to anti-IgE and abolished the reactivity to anti-RBL. The results suggested strongly that anti-receptor antibodies were responsible for the skin reactions by anti-RBL. Incubation of peritoneal mast cells from inbred Hooded Lister rats with anti-RBL resulted in histamine release without changing the viability of the cells. It appears that anti-RBL induced non-cytotoxic histamine release without participation of cell-bound IgE.

Development of Mast Cells in Vitro

Abstract Mast cells were obtained by long term culture of rat thymus cells on rat embryonic fibroblast monolayers. Pure mast cell preparations obtained by culture were incubated with 125I-labeled rat E myeloma protein to study receptors for IgE on their surface. When the cells were obtained after 35 to 45 days culture, the average number of receptors per mast cell was 100,000 to 400,000. An equilibrium constant of the binding reaction between their receptor and rat IgE was in the order of 108 M-1. The histamine content of the cultured mast cells was 0.2 to 5 µg/106 cells. The measurement of histamine content in mast cells recovered after different periods of culture suggested that the histamine content increased with maturation. Even after 45 to 50 days culture, the histamine content of cultured mast cells was significantly lower than that in rat peritoneal mast cells. The cultured mast cells were passively sensitized in vitro with rat IgE antibody against Nippostrongylus brasiliensis. The sensitized cells released histamine upon incubation with the antigen. It was also found that cultured mast cells released histamine upon exposure to compound 48/80. These results indicated that cultured mast cells have physiologic functions similar to those of normal rat mast cells, but they have not reached full maturation.

Disparity of IgE Binding Between Normal and Tumor Mouse Mast Cells

The binding constants of rat and mouse IgE to normal and neoplastic rat and mouse mast cells have been measured. None of the cells distinguishes between rat and mouse IgE. Rat normal and neoplastic mast cells and mouse mastocytoma cells show very similar binding properties: k1 approximately 10(5), k-1 approximately 10(-5) and, therefore, KA approximately 10(10). Normal mouse mast cells also show a forward rate constant, k1, of 10(5) but the dissociation rate constant, k-1, is 50 times higher (5 X 10(-4)). The detergent-solubilized receptors reflect the properties of the cell-bound receptors. The discrepancy in binding by normal and neoplastic mouse mast cells remains unexplained but appears to be a reproducible finding for the cells from three strains of normal mice and three different mastocytomas studied.

Binding of rat IgE with the subcellular components of normal rat mast cells

Attempts were made to obtain subcellular components of rat mast cells which contain receptors for IgE. Rat peritoneal cells were treated with 125I-labeled rat IgE to label mast cells, and disrupted by ultrasonication. More than 90% of cell-bound IgE was recovered in the 20,000 g supernatant fraction which had the ability to block passive cutaneous anaphylaxis with mouse and rat reaginic antibodies. Gel-filtration of the supernatant fraction through a Sepharose 6B column showed that both radioactivity and PCA blocking activity were associated with subcellular components which were eluted in the void volume. When the 20,000 g supernatant fraction of normal peritoneal cells was incubated with 125I-rat IgE, the protein combined with the subcellular components. Incubation of the 20,000 g supernatant with an IgE-rich rat serum inhibited the binding of 125I-IgE with the components, whereas normal rat serum failed to do so. When the sepharose void volume fraction was obtained from purified mast cells and labeled with 125I, approximately 25% of radioactive material was coprecipitated with IgE-anti-IgE complexes. Evidence was obtained that subcellular components of macrophages were not coprecipitated with the antigen-antibody complexes. These results indicated that the components eluted in the void volume fraction contained receptors for IgE. In another experiment, the 20,000 g supernatant fraction was obtained from rat peritoneal cells or purified mast cells whose membrane was labeled with 125I, and fractioned by gel filtration followed by DEAE cellulose chromatography. More than 2 3 of the radioactivity and PCA-blocking activity associated with the sepharose void volume fraction were recovered in the 3 M NaCl fraction which contained 12–15% of total protein. When the 3 M fraction obtained from purified mast cells was labeled with 125I and incubated with an IgE-rich serum, a significant amount of radioactive material in the fraction was precipitated with IgE-anti-IgE complexes. The results indicate that blocking of the PCA reaction by this fraction is due to the presence of receptors for IgE.