Binding properties and histamine release in variants of rat basophilic leukemia cells with changes in the IgE receptor.

Variants of the rat basophilic leukemia cell line were selected that had a decreased number of high affinity IgE receptors (Fc epsilon R). Cloned lines with fewer than 10,000 Fc epsilon R on their surface could release histamine following IgE-mediated stimulation. These variant lines were further characterized by binding studies with 125I-labeled IgE or a series of monoclonal antibodies (mAb) that inhibit IgE binding. Three of these mAb (mAb BC4, mAb CA5 and mAb CD3) bind competitively with IgE. A fourth mAb (AA4) inhibits IgE binding but its binding is not inhibited by IgE. In all the variant cloned lines, binding by the 3 mAb was highly correlated to IgE binding. Therefore, these epitopes are closely related. In contrast, there was poor correlation between mAb AA4 and IgE binding. However, even in these lines, mAb AA4 inhibited labeled IgE binding. Therefore, there is independent variation of the mAb AA4 epitope compared to the sites to which the other mAb and IgE bind.

Characterization of monoclonal antibodies produced by immunization with partially purified IgE receptor complexes.

A series of monoclonal antibodies (mAb) were produced following the immunization of mice with partially purified IgE receptors from the rat basophilic leukemia cell line (RBL-2H3). Twelve hybridoma cell lines were selected that secreted monoclonal antibodies capable of binding RBL-2H3 plasma membranes. These antibodies were all of the IgG1, or IgG2a subclass. All 12 antibodies bound to either intact or glutaraldehyde-fixed RBL-2H3 cells. Only one monoclonal (mAb 2AC3) inhibited 125I-labeled IgE binding (IC50 = 65 micrograms/ml compared to 1.0 microgram/ml for unlabeled IgE). This same mAb weakly precipitated the alpha component of the receptor from 125I-surface-labeled cells and directly triggered histamine secretion when incubated with RBL-2H3 cells. Therefore, this hybridoma most likely represents a low affinity anti-receptor antibody. Among the other 11 monoclonals, two caused direct histamine secretion from RBL-2H3 cells. These same two, as well as four others, released greater than 10% of total cellular histamine when rabbit anti-mouse antibody was added to cross-link mAb bound to the cell surface. One monoclonal (mA 1AD3) did not trigger histamine secretion but did inhibit IgE-mediated histamine release when incubated with pre-sensitized RBL-2H3 cells. Except for mAb 2AC3, none of the other monoclonal antibodies that caused or inhibited histamine secretion immunoprecipitated receptor or other protein components from either 125I-surface-labeled or intrinsically-labeled cells. However, two monoclonal antibodies (mAb 1CC4 and 1CD1) immunoprecipitated 45,000 and 55,000 proteins from 125I-surface-labeled cells. One hybridoma (mAb 2AA2) that failed to immunoprecipitate surface-labeled proteins did precipitate a 20,000 band from intrinsically-labeled cells. This band increased slightly in apparent mol. wt after reduction and, therefore, was not the previously described gamma component of the receptor. Because several mAb were capable of modulating histamine secretion, it appeared that some of the present monoclonal antibodies bound to undefined membrane components that are crucial to the secretory process of rat basophilic leukemia cells.

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.

Establishment of four mouse mastocytoma cell lines.

Four mastocytoma cell lines were isolated from four different mouse mastocytoma tumors. The tumors had been induced in mice treated with tetramethylpentadecane (pristane) and infected with Abelson murine leukemia virus. The cell lines have been carried in culture for over a year and can induce tumors when injected into the mouse strain in which the tumor originated. The cells contain histamine, have high affinity IgE receptors and release histamine by IgE, immune complex or ionophore A23187-induced reactions. This histamine release reaction requires Ca2+, is optimal at 37 degrees C, and is blocked by a number of metabolic inhibitors. There is no requirement for phosphatidylserine. Cloned sublines have been obtained which will be useful for Fc epsilon R, Fc gamma R; and histamine release studies.

Mouse mast cell activation and desensitization for immune aggregate-induced histamine release.

Immune aggregate-induced histamine release and desensitization were studied in mouse mast cells. Maximal histamine release was rapid, occurred at 37 degrees C, and required the addition of alpha-L-phosphatidyl-L-serine and Ca2+. The amount of histamine released varied with the composition of the immune aggregates and was dependent on the antibody concentration. Saturation of mast cell Fc epsilon receptors with rat or mouse IgE had no effect on subsequent immune aggregate-induced release. The incubation of mouse mast cells with immune aggregates in the absence of cations of alpha-L-phosphatidyl-L-serine did not stimulate the release of histamine but resulted in desensitization of the cells for release with the addition of the same or unrelated immune aggregates. Such cells are capable, however, of IgE-mediated histamine release. Mast cells desensitized for IgE-mediated histamine release by incubation with anti-IgE were capable of immune aggregate-induced release. These data suggest that IgE-mediated and immune aggregate-induced triggering of mouse mast cells occurs through separate receptors.

Spiroplasmas: serological grouping of strains associated with plants and insects.

Spiroplasma strains from plant and arthropod hosts, and from surfaces of flowers, were classified into three serological groups (designated I, II, and III) based on results from growth-inhibition tests. No significant cross reactions were observed among groups. The groupings were confirmed by ring-interface precipitin and microprecipitin tests, using membrane preparations as test antigens, and by organism-deformation tests. Serogroup I contained three subgroups: subgroup A (Spiroplasma citri strains Maroc R8A2 and C189), subgroup B (strain AS 576 and closely related strains from honeybee or flowers), and subgroup C (corn stunt spiroplasma strains). Serogroup II contained strains 23-6 and 27-31 isolated from flowers of the tulip tree (Liriodendron tulipifera L.) growing in Maryland. Serogroup III contained strains SR 3 and SR 9 isolated from flowers of the tulip growing in Connecticut. The subgroups of serogroup I were based on organism deformation, microprecipitin, and ring-interface precipitin tests. The data are consistent with the hypothesis that the three serogroups represent no less than three distinct spiroplasma species.