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Receptors for IgE of rat basophilic leukaemia (RBL) cells, maintained in different laboratories were isolated by means of various anti-receptor antisera, and characterized by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. Most antisera were shown to react with a receptor designated H and which, as shown previously, could be isolated by IgE-Sepharose but not by a combination of IgE and anti-IgE. Only two highly purified anti-receptor antibody preparations, which had been purified by adsorption to and elution from rat basophilic leukaemia cells, reacted primarily with a second kind of receptor molecule which had previously been designated R. Some indirect evidence was obtained which suggests that H is a highly immunogenic molecule. It was confirmed that the apparent molecular weight of H-like molecules varied on different RBL cell lines.

Human basophils were developed in suspension culture of mononuclear cells of cord blood in the presence of conditioned medium of phytohemagglutinin-stimulated human T cells, from which interleukin 2 had been eliminated. Approximately 50-90% of cells recovered after 2-4 wk of culture were basophil granulocytes, which bear receptors with high affinity for human IgE. Sensitization of the cells with human IgE followed by challenge with anti-IgE resulted in the release of histamine and arachidonic acid (AA). In both the cultured basophils and human lung mast cells, bridging of cell-bound IgE with anti-IgE induced a transient increase in phospholipid methylation and intracellular cyclic AMP (cAMP), and these processes were followed by Ca2+ uptake and release of both histamine and AA. As demonstrated in rat mast cells, evidence was obtained that the activation of a proteolytic enzyme and phospholipid methylation induced by the bridging of IgE receptors are involved in the subsequent increase in AMP, and are an essential step for transduction of triggering signals for mediator release.

Mast cells were isolated from human lung tissues by counter current centrifugation elutriation, followed by flotation through Percoll gradients. Purified human mast cells released histamine upon challenge with anti-IgE. An optimal concentration of anti-IgE for maximum histamine release from human lung mast cells was comparable to that required for histamine release from normal human basophil granulocytes. Human lung mast cells could be passively sensitized with mouse monoclonal IgE antibody for antigen-induced histamine release. Bridging of cell-bound IgE molecules on human mast cells by anti-IgE or its F(ab')2 fragments induced phospholipid methylation and an increase in intracellular cyclic AMP (cAMP). Incorporation of [3H]methyl groups into phospholipid reached a maximum at 30 sec after challenge with anti-IgE, whereas intracellular cAMP reached a maximum at 1 min. Both values declined to base line levels within 2 to 3 min. These biochemical events were followed by Ca2+ influx and histamine release. Ca2+ uptake and histamine release reached maximum at 2 to 3 min and 5 to 8 min, respectively. Neither phospholipid methylation nor initial rise in cAMP was inhibited by indomethacin, which indicates that these biochemical events are not the result of prostaglandin synthesis. However, inhibition of phospholipid methylation by inhibitors of S-adenosyl-L-methionine-mediated methylation, such as 3-deazaadenosine and S-isobutyryl 3-deazaadenosine, inhibited not only phospholipid methylation but also cAMP rise and subsequent Ca2+ uptake and histamine release. The results indicate that phospholipid methylation induced by bridging of IgE receptors on human mast cells is essential for Ca2+ influx and histamine release.

A fraction of isolated Raji cell membranes solubilized with 2m KBr which was capable of inhibiting C3-dependent rosettes was examined for its ability to inhibit the alternative pathway of complement. It was shown to decrease alternative pathway-dependent haemolysis of sheep erythrocytes and to accelerate decay of factor B from these cells. It had no effect on C2 decay, the classical pathway analog of factor B. Inhibitory activity solubilized from Raji cells was not removed by immunoadsorption with anti-factor H or anti-factor I, two well-characterized serum C3b-control proteins. It also differed in two functional respects from these proteins. Firstly, it failed to result in cleavage of a peptide bone in C3 which is characteristic of factor I; secondly, its inhibitory activity did not synergize with factor I in inhibiting the alternative pathway, unlike factor H. These results suggest that Raji cells contain a regulatory factor in their membranes for the alternative pathway of complement which is distinct from factors H and I.

Hybridoma antibodies directed against the Fc and Fab portions of rat IgE were produced by immunizing BALB/c mice with rat IgE and fusing the spleen cells with the nonsecreting plasmacytoma P3/X63Ag8.653. Two of the antibodies, designated as A2 and B5, were extensively characterized. Competitive binding experiments using rat IgE from the IR 162 and IR2 immunocytomas and rat IgG indicated that both A2 and B5 were epsilon-chain specific and not anti-idiotype. A2 also exhibited some cross-reactivity with mouse IgE. When IgE was treated with chymotrypsin so as to produce both F(ab')2 and Fab fragments, the enzyme-treated IgE retained reactivity with B5, but the reactivity to A2 was lost. Heat denaturation of IgE at 56 degrees C resulted in a progressive loss of reactivity of the IgE for both A2 and the Fc receptor on rat basophilic leukemia cells; the reactivity of B5 remained unchanged. A2 does not evidently interact with the same site on the Fc of IgE that is involved in binding to the rat basophilic leukemia cell Fc receptor; A2 exerted little influence on the binding of IgE to rat basophilic leukemia cells. Thus, the data indicate that the antigenic site for B5 is in the Fab region of the IgE molecule and that A2 reacts with the IgE Fc. Use of these antibodies to measure cell-bound IgE was also evaluated in dual label experiments, and potential problems in using divalent antibodies to quantitate cell surface antigens are discussed.

REceptors for IgE of rat basophilic leukaemia (RBL) cells, maintained in different laboratories were isolated by means of IgE-Sepharose or IgE and anti-IgE, and characterized by SDS-polyacrylamide gel electrophoresis. All cell lines were found to be associated with a receptor molecule (R) which could be isolated either with IgE-Sepharose or IgE and anti-IgE and a second receptor (H) which could only be isolated with the aid of IgE-Sepharose. The relative amounts of these two molecules, as isolated from surface iodinated cells, varied from the RBL cell line to the other and their apparent molecular weights were not identical on all cell lines. Since comparisons were made on the same gel using receptors isolated from cells labelled with different isotopes of iodine, differences in molecular weight must be considered as being intrinsic and not due to methodological variations. These results provide an explanation why differences were observed among receptors for IgE as characterized in various laboratories. In spite of the fact that the various RBL cell lines originated from the same chemically-induced tumour they have, over the years, undergone changes which are reflected in the receptors for IgE.

The cDNA encoding the murine low-affinity receptor for IgE (Fc epsilon RII) has been isolated from a cDNA library prepared from B cells activated with lipopolysaccharide and interleukin 4. It encodes a 37-kDa protein of 331 amino acids with two potential N-linked glycosylation sites. Analogous to its human counterpart, there is no signal sequence and the putative transmembrane region is close to the amino terminus, indicating an inverse membrane orientation with the carboxyl terminus at the cell exterior. The predicted murine Fc epsilon RII amino acid sequence demonstrates a 57% identity with its human counterpart. The murine sequence has an additional internal repeat motif of 21 amino acids giving four repeats as compared to three in the human sequence. Furthermore, the murine Fc epsilon RII is truncated at the carboxyl terminus and the Arg-Gly-Asp sequence, a common recognition site of integrin receptors, which is found in the reverse configuration in human Fc epsilon RII, is missing. B cells activated with interleukin 4 and lipopolysaccharide have an increased amount of Fc epsilon RII mRNA as compared with resting or lipopolysaccharide-stimulated B cells. Con A-activated normal T cells, the TH-2 cell line D10, as well as the macrophage cell line J774 have no detectable Fc epsilon RII mRNA. Expression analysis using transiently transfected COS cells revealed that recombinant murine Fc epsilon RII binds anti-Fc epsilon RII as well as mouse and rat IgE but does not bind human IgE or mouse IgG. Fc epsilon RII expressed in COS cells has a molecular mass of 45 kDa whereas the Fc epsilon RII from B-cell lines is a 49-kDa protein.

The murine B lymphocyte Fc epsilon R is functionally multivalent. Radiolabeled rat IgE, when bound to the B cell Fc epsilon R will co-isolate with the Fc epsilon R on a rat IgE affinity column; examination of the affinity column eluate by SDS-PAGE reveals the component previously identified as the Fc epsilon R as well as E and L chains from IgE. At low levels of Fc epsilon R saturation, up to 30% of the Fc epsilon R bound IgE becomes bound to IgE-Affi-Gel. By using a biotin-avidin system, the coprecipitation of non-haptenated IgE with haptenated IgE was examined and the results suggest (but do not prove) a divalent receptor.

Cross-linking reagents were used to further characterize the murine B cell receptor for the Fc portion of IgE (Fc epsilon R) and compare this receptor to the well-characterized high-affinity Fc epsilon R on rat basophilic leukemia (RBL) cells. The disulfide cleavable and noncleavable reagents 3,3'-dithiobis(sulfosuccinimidyl) propionate (DTSSP) and bis(sulfosuccinimidyl) suberate (BS3) were used. With these reagents, efficient cross-linking of the alpha component of the high-affinity RBL Fc epsilon R to the membrane-buried beta and gamma components occurred only if the membrane was solubilized before the cross-linking reaction. In studies with purified murine B cells, IgE could be cross-linked to the Fc epsilon R on intact cells with either DTSSP or BS3. Under the same conditions, up to 10% of the B cell surface immunoglobulin (sIg) (both IgM and IgD) was also found to cross-link to a portion of the IgE/Fc epsilon R complex, suggesting that on the intact murine B cell the Fc epsilon R is frequently in close association with sIg. The B cell Fc epsilon R was also examined for the presence of receptor-associated proteins. Under conditions where the high-affinity RBL Fc epsilon R was substantially cross-linked to the alpha, beta, gamma complex, no evidence was seen for similar cross-linking of the B cell Fc epsilon R. Cross-linking experiments on affinity-purified Fc epsilon R preparations also gave no evidence for receptor-associated proteins with the B cell Fc epsilon R, although evidence for receptor-receptor association was seen. Thus, these data further support the concept that there may be little relationship between the high-affinity mast cell/basophil Fc epsilon R and the low-affinity lymphocyte Fc epsilon R.

The ontogenic appearance and lymphoid tissue distribution of the murine B cell IgE FcR (Fc epsilon R) was examined. Flow cytometry was utilized to study the expression of the Fc epsilon R on splenic B cells from mice of increasing age, as well as B cells from various lymphoid organs. A large panel of B cell tumors was also screened for the presence of the Fc epsilon R. The results demonstrate that the Fc epsilon R appears very late in B cell development, and is preceded in appearance even by IgD. In adult animals, the Fc epsilon R was found to be expressed on virtually all mature IgM, IgD bearing B cells, whether taken from the spleen, lymph nodes, or Peyer's patches. Further examination showed that B cells which had switched to express an isotype other than IgD, appeared to no longer display the Fc epsilon R. When surveying a variety of B cell tumors, the Fc epsilon R was found to be present on WEHI 279, an IgM, IgD-bearing lymphoma. The receptor was not found on pre-B cell, immature B cell, switched B cell, or secreting B cell tumors. Taken together, these results indicate that the B cell Fc epsilon R is expressed predominantly on mature, virgin B cells, and is lost after activation and switching.

IL-4 promotes IgG1 and IgE secretion by murine B cells stimulated with bacterial LPS. We show that stimulation of unprimed resting splenic B cells with LPS and 10(4) U/ml rIL-4 results in the expression of membrane (m) IgG1 and mIgE on 40 to 50% and 15 to 25% of the total B cell population, respectively, on day 4 of culture. The possibility of a significant contribution to cell surface staining by cytophilic, secreted Ig isotypes was eliminated by either the addition of anti-Fc gamma or anti-Fc epsilon R mAb during the culture or by acid treatment before staining. A similar proportion of IgE-expressing B cells are also found, after stimulation with LPS and 10(4) U/ml IL-4, by cytoplasmic staining using fluorescence microscopy. Cell sorting analysis further indicates that B cell populations that express mIgG1 and mIgE secrete these respective Ig isotypes. In addition, such cells show striking diminution in IgM secretion compared to mIgG1- or mIgE- sorted B cells. Stimulation with LPS and IL-4 (10(4) U/ml) induces co-expression of mIgG1 and mIgE on LPS-stimulated B cells; up to 75% of mIgE+ B cells co-express mIgG1 and up to 19% of mIgG1+ B cells express mIgE. This striking co-expression of mIgG1 and mIgE is mirrored by the co-expression of mIgG1 with mIgG3 and mIgG2b by B cells stimulated with LPS and 200 U/ml IL-4. Cell sorting analysis demonstrates that the B cell population that co-expresses mIgG1 and mIgE secretes both IgG1 and IgE. However, "two-color" cytoplasmic staining fails to demonstrate any B cells that simultaneously secrete both IgG1 and IgE.

Antisera directed at human C3 receptors were prepared by 2 different methods. In method I rabbits were immunized with EAC coated with C3 receptors of tonsil cells (AC3RS type I). In method II rabbits were immunized with insoluble aggregates of tonsil membrane constituents containing significant amounts of C3 receptors. After appropriate absorptions both types of AC3RS showed a selective reactivity with those cells known to express C3 receptors. With the EAC rosette inhibition test it is demonstrated that both types of AC3RS contain antibodies against the binding sites of C3b, C3bi and C3d receptors. The antibody titer was consistently higher in AC3RS of type II.

Th cell clones are known to induce an IL-4 dependent polyclonal IgE synthesis. Because IL-4 can induce the expression of the low affinity FcR for IgE (Fc epsilon RII) the ability of Th cell clones to induce Fc epsilon RII on purified splenic B cells was analyzed. It was found that a TH2 clone could cause a 50- to 100-fold superinduction of Fc epsilon RII after 2 days in culture; after 3 days, the Fc epsilon RII levels had almost returned to base line. The superinduction was inhibited by an anti-IL-4 antibody, 11B11, indicating its dependence on IL-4. A TH1 clone could cause a modest (four fold) induction of Fc epsilon RII, and this induction was not influenced by 11B11. A similar Fc epsilon RII induction was seen when using the supernatant from activated TH1 cells. The component(s) causing this relatively low level Fc epsilon RII induction is not known; a variety of known lymphokines were tested, and only IL-4 demonstrated any capacity for Fc epsilon RII induction on LPS-activated B cells. Addition of rIL-4 at concentrations of 400 U/ml or greater to the TH1 culture was sufficient to cause a Fc epsilon RII superinduction similar to that seen with the TH2 clone, while 40 U/ml was not. In order to determine a potential role for the Fc epsilon RII or its soluble fragment on the IgE synthesis mediated by TH2, a monoclonal anti-Fc epsilon RII, B3B4, was added to the culture. The addition of B3B4 did not have an influence on IgE levels in this system.

Surface proteins and glycoproteins of RBL cells were labelled enzymatically with 125I and then solubilized with Nonidet P-40. Analysis by polyacrylamide-gel electrophoresis in SDS on 10% gel revealed 10 distinctive peaks ranging in molecular weights from 17,000 to 200,000 daltons. Mainly components of higher molecular weights were bound by lentil-lectin Sepharose and could be eluted with alpha-methyl mannoside. The receptor for IgE was clearly shown to bind to the lentil-lectin. A second cell surface component which previously had been shown to bind to IgE-Sepharose as well, was found to bind only slightly to lentil-lectin. Thus, it can be concluded that the receptor for IgE is a glycoprotein with mannose and/or N-acetylglucosamine in the carbohydrate moiety(s).

Antigenic relationships between the low affinity Fc epsilon R present on murine B and T lymphocytes were studied. A rat mAb (B3B4) and two polyclonal antisera produced by immunizing with the murine B lymphocyte Fc epsilon RII were examined for their ability to inhibit binding of IgE to murine B or T lymphocytes, using an IgE-specific rosette assay. One polyclonal antiserum (goat-anti-mouse Fc epsilon R) inhibited binding of IgE to both B and T lymphocytes, whereas another polyclonal antiserum (rabbit-anti-mouse Fc epsilon R) and the rat mAb inhibited the binding of IgE to B lymphocytes but did not influence the binding of IgE to T lymphocytes. When lymphocytes were surface labeled with 125I, 49-kDa and 38-kDa IgE-binding proteins were immunoprecipitated from B lymphocyte lysates by B3B4 and from B and T lymphocyte lysates by the goat antiserum. Taken together, these results suggest that the Fc epsilon R present on murine B and T lymphocytes are structurally related receptors that share some, but not all, epitopes.

The synthesis and ligand-dependent regulation of the lymphocyte receptor for IgE (Fc epsilon R) has been studied. Using murine Fc epsilon R+ cell lines, a 44-kilodalton 35S-methionine-labeled Fc epsilon R precursor was isolated by immunoaffinity chromatography. In contrast to the final processed 49-kilodalton Fc epsilon R, this precursor cannot be isolated from IgE affinity columns, indicating the importance of this processing in the function of the Fc epsilon R. The IgE-mediated Fc epsilon R upregulation was also studied and it was demonstrated that the degradation rate of the Fc epsilon R was dramatically slowed by ligand occupation of the Fc epsilon R. This degradation involves the cell surface-mediated release of a 38-kilodalton Fc epsilon R fragment that can be isolated using monoclonal anti-Fc epsilon R antibodies. Thus, these results demonstrate that posttranslational processing is required for the lymphocyte receptor to gain significant IgE-binding capacity; once acquired its degradation is slowed by occupation of the Fc epsilon R with ligand. At least in the rodent model system, this slowing of the degradation helps explain the increased Fc epsilon R levels seen in the presence of high IgE levels.

Four growth studies were conducted to determine the signs, biochemical indices and histopathology of a thiamin deficiency and the thiamin requirement of young rainbow trout reared at 15°C on a semi-purified test diet. The major overt signs of a thiamin deficiency in rainbow trout are predominantly neurological: irritability and instability. Other signs include convulsions, feed refusal, dark pigmentation and finally mortalities. Growth reduction in the thiamin deficient trout appear to result from anorexia or feed refusal and not specifically to a thiamin deficiency. Although there were prominant neurological signs in the thiamin deficient trout, there were no histopathological signs in any tissues of the trout, including the brain and central nervous system, examined by light microscopic techniques. The tissue transketolase activity would appear to be a sensitive and specific indicator of the thiamin status in the trout. In addition, the levels of plasma lactate and serum pyruvate are also elevated in thiamin deficient trout. On the basis of the growth parameters, absence of deficiency signs and kidney and liver transketolase activity, the thiamin requirement of rainbow trout reared at 15°C on a semi-purified test diet is 1 mg/kg feed.

The Fc receptor for IgE (Fc epsilon R) on murine B lymphocytes was studied by using BALB/c mice infected 12 to 18 days previously with Nippostrongylus brasiliensis. B cells were enriched in the Sephadex G-10-passed lymphocytes by treating with anti-Thy-1.2 and complement (C). After stripping any cytophilic Ig with low pH, the B cells were 125I surface labeled; subsequently the membranes were solubilized with nonionic detergent, and putative Fc epsilon R components were allowed to bind to IgE-coated adsorbents. Bound radiolabel was eluted with low pH, and when examined by SDS-PAGE, was found to consist primarily of a relatively broad band centered at 49,000 m.w. (49K). Fluid-phase IgE could prevent the binding of the 49K component to the IgE solid-phase adsorbents. Rebinding studies further indicated that the 49K component exhibited a specificity for IgE, thus confirming that the 49K component was the murine B lymphocyte Fc receptor for IgE (Fc epsilon R). Some rebinding to rabbit IgG was observed, and by using 2.4G2, the monoclonal anti-Fc gamma 2b receptor (Fc gamma 2bR) antibody to isolate the IgG2b receptor, a clear distinction between the FC gamma 2bR and the 49K IgE receptor was demonstrated by SDS-PAGE analysis. Rabbit IgG was thus found to interact with both the 49K Fc epsilon R and the 59K FC gamma 2bR. The murine B lymphocyte Fc epsilon R was compared with the human B cell Fc epsilon R from the RPMI 8866 cell line and with the high affinity Fc epsilon R on rat basophilic leukemia cells by one- and two-dimensional gel analyses. The lymphocyte Fc epsilon R from mouse and human was found to be quite similar with respect to m.w. (45 to 50K) and isoelectric point (pI 4.5 to 5.0), whereas the basophil Fc epsilon R differed in both aspects.

Interest in the regulation of IgE synthesis has generated investigation of low-affinity Fc receptors for IgE (Fc epsilon R) and the related immunoregulatory IgE-binding factors. In an effort to facilitate biochemical analysis of the B lymphocyte Fc epsilon R, hybridoma technology has been used to create stable cell lines that maintain Fc epsilon R in high numbers. Fusion of the HAT-sensitive B lymphoma, M12.4.5, with murine B cells from Nippostrongylus brasiliensis infected BALB/c mice led to the formation of hybrid cells of B cell phenotype, all of which were Fc epsilon R+, including several that had greater than 50,000 Fc epsilon R/cell. The Fc epsilon R on these cells were biochemically identical to the Fc epsilon R on normal B cells with respect to binding affinity (approximately equal to 10(8) M-1), m.w. (49,000), and tryptic peptides. Each hybridoma cell line specifically increased its Fc epsilon R level between twofold and fourfold when cultured with rat or mouse IgE. Additional studies demonstrated that the increased IgE binding ability was due to an increase in receptor number rather than an affinity change, and the Fc epsilon R increase was seen on the entire cell population. Dose studies indicated that oligomeric IgE was 10-fold more effective than monomeric IgE in causing upregulation, and the effective concentrations required indicated that induction occurred only if IgE was present in saturating concentrations. Upon addition of IgE, peak Fc epsilon R levels were reached after 15 to 20 hr of culture; blocking protein synthesis with cycloheximide largely blocked the increase in Fc epsilon R levels. Additionally, the inductive signal IgE must constantly be present to maintain upregulated Fc epsilon R levels in that its removal from the culture resulted in a rapid decline of Fc epsilon R from induced to normal levels. Because Fc receptor upregulation is important to several systems describing Ig isotype-specific regulation, the ability to examine such receptor upregulation at a clonal level should aid in discerning the role of the Fc epsilon R in the regulation of IgE antibody synthesis.

Normal rat IgG-Sepharose binds the same two receptors from solubilized rat basophilic leukemia (RBL) cells as are bound by rat IgE-Sepharose. These receptors have previously been designated R and H, and have apparent mol. wts of 45,000 and 55,000, respectively. Inhibition studies indicate that, although soluble deaggregated IgG is capable of interacting with both receptors, its affinity is higher for H than for R. Conversely, soluble IgE preferentially interacts with R, but its affinity for H is somewhat higher than that of rat IgG. Control studies show that IgG receptor interaction to be specific in that it is significantly stronger than that obtained with F(ab')2 fragments or a variety of proteins having pIs approximating that of IgG.

The role of the carbohydrate portion of the receptor for IgE in the interaction with IgE was investigated. Membrane carbohydrates of rat basophilic leukemia (RBL) cells and rat mast cells (RMC) were labeled by treating the cells with galactose oxidase followed by [3H]-NaBH4. IgE receptors were separated from detergent solubilized membranes and examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Pretreatment with neuraminidase markedly increased the incorporation of 3H into both the total membrane extract and into the IgE receptors. SDS-PAGE analysis demonstrated the presence of galactose in all IgE-binding components of 2 RBL cell lines and the presence of sialic acid on the major IgE-binding component. Prior saturation of the cells with IgE did not prevent the carbohydrate labeling of the receptor, although it did block the labeling of its protein part, indicating that carbohydrates are not located in the binding site. Removal of terminal sialic acid residues with neuraminidase increased the affinity of the receptor for IgE without appreciably affecting the number of receptors per cell. In order to more drastically modify the receptor carbohydrates, RBL cells were grown in the presence of Tunicamycin (TN). TN was shown to markedly inhibit the incorporation of [3H]-glucosamine into the receptor. RBL cells grown in the presence of TN expressed fewer receptors at the cell surface, as judged both by ligand binding studies and external labeling procedures. These data cumulatively suggest that the carbohydrate moieties of the receptor are not directly located in the binding site of the IgE receptor; however, the TN studies suggest that receptor carbohydrate may play a role in transport of the receptor to the plasma membrane or in its orientation thereafter.

Phagocytosis of sheep erythrocytes (E) bearing rabbit immunoglobulin and rat C3 by rat peritoneal mast cells was quantitated by using 51Cr-labeled E and was confirmed by electron microscopy. The relative importance of IgG Fc receptor interaction in C3-mediated phagocytosis was assessed. Removal of any traces of IgG antibody by absorption of IgM antibody with protein A-Sepharose and absorption of the other reagents with sheep E had no effect on the phagocytosis of C3b- and C3bi-coated cells. IgG antibody enhanced the phagocytosis of intermediates prepared with IgM, purified components and rat C3 (EaIgMClgp4hu oxy2hu3brat) with a graded dose response, but was only additive and not synergistic with C3. The independence of the C3- and Fc-mediated signals was confirmed by using chemically produced polymers of rabbit IgG to inhibit phagocytosis. These polymers, especially tetramers and higher aggregates, completely blocked ingestion of EAIgG, but not that of EAIgMC1423b or -3bi. When IgG was substituted for IgM in the C3b intermediate, the IgG polymers inhibited about 50% of the phagocytosis. Cumulatively, the data demonstrated that in the case of rat mast cells, the stimuli to phagocytosis induced by C3 and IgG are independent; either is sufficient by itself.

The similarity between murine and rat IgE was examined in terms of their fixation to target cells and interaction with monospecific antibodies to rat epsilon-chain (anti-epsilon). Purified rat monoclonal IgE (IgEr) was found to block the fixation of murine reagin (IgEm) to mouse and rat skin and to rat basophilic leukemia (RBL) cells. The capacities of mouse reaginic serum (MRS), rat reaginic serum, and IgEr to inhibit the binding of radiolabeled 125I-IgEr to RBL cells were shown to be similar. These results suggest that the binding of IgE of either species occurs on the same or on adjacent receptor sites of mast cells and RBL cells. The antigenic cross-reactivity between IgEm and IgEr was established by depletion of the reaginic activity from MRS by treatment of MRS with anti-epsilon. The reaginic activity of MRS could be recovered by the addition of IgEr to anti-epsilon:IgEm complexes. From these findings it may be inferred that i) IgEm and IgEr share some antigenic determinants and ii) the regions of the immunoglobulins responsible for fixation to receptors on mast cells and RBL cells are identical or similar.

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.

The ability of IL-4 to influence the developmental expression of the murine B cell IgE Fc receptor (Fc epsilon R) was examined. Spleen cells from neonatal mice of increasing age were incubated overnight with IL-4 and subsequently examined with multicolor flow cytometry. The results demonstrate that IL-4 can significantly increase the number of maturing B cells which express the Fc epsilon R. This effect was only seen however, on those neonatal B cells which already displayed surface IgD. Splenic B cells which were IgM+, IgD- failed to express the Fc epsilon R when treated with IL-4, even though they responded by increasing their level of class II Ag expression. Further experiments showed that the inability of IgD- immature B cells to express the Fc epsilon R could not be entirely explained by their assignment to the Ly-1 lineage. Taken together, these results indicate that IL-4 can accelerate the developmental expression of the B cell Fc epsilon R, but only on those B cells that are mature enough to express IgD.

Selective growth of human basophilic granulocytes was obtained in suspension cultures of mononuclear cells from umbilical cord blood. Approximately 50 to 80% of nonadherent cells recovered from 2- to 3-wk-old cultures contained metachromatic granules, and these cells were identified as human basophilic granulocytes by electron microscopy. Histamine content of cultured human basophils was comparable to that in peripheral blood basophils. Cultured basophils bear 2.7 to 3.7 X 10(5) IgE receptors per cell that bind both human IgE and rodent IgE with comparable affinity. Average equilibrium constants of the receptors for human IgE and mouse IgE were 2.56 +/- 0.88 X 10(9) M-1 and 1.85 +/- 0.86 X 10(9) M-1, respectively. The cell-surface component of the IgE receptors on cultured basophils has a m.w. of 64,000. Cultured basophils could be passively sensitized with human IgE and mouse IgE monoclonal antibody, and sensitized basophils released characteristic cytoplasmic granules and both histamine and arachidonate upon challenge with either anti-human IgE or antigen. Incubation of cultured basophils with ionophore A23187 or F-Met-Leu-Phe resulted in histamine release. However, compound 48/80 failed to induce histamine release from the cells.

Purified beta1H globulin (beta1H) was shown to bind to C3b coated cells by both immunofluorescent and radioactive tracer techniques. With EAC43, the amount of beta1H bound was directly proportional to the amount of C3 used to prepare the cells; EA, EAC14 and EAC14oxy2 bound very small amounts of beta1H. The C3b binding site on beta1H was labile in that not all of the purified 125I-beta1H was capable of binding to C3b, even when an excess of cell-bound C3b was present. Scatchard analysis of binding of beta1H to C3b-coated cells indicated an equilibrium constant of 10(9) L/M. Deviations from linearity were regularly found on Scatchard analyses. This was consistent with the hypothesis that the beta1H binding sites exhibit negative cooperativity in that as more sites become occupied, it becomes more difficult to fill the remaining sites. The stoichiometry of the reaction between C3b and beta1H was examined using EAC14oxy23 prepared with 131I-C3 and beta1H labeled with 125I. Between 0.5--0.8 beta1H molecules were bound per C3b molecule. Other alternative pathway components influenced the binding of 125I-beta1H to cell bound C3b. Both C3b and native C3 inhibited binding of labeled beta1H at an efficiency approximately 1/1,000 that of unlabeled beta1H. Factor B inhibited binding with 1/280 the efficiency of unlabeled beta1H. Properdin caused a dose-dependent increase in the binding of beta1H; this enhancement was abrogated if B was also present in the reaction mixture. Scatchard analysis indicated that the enhancement of beta1H binding by P resulted in an increased number of available binding sites rather than an increase in the affinity of binding.

The post-translational processing and maturation of the receptor for IgE (Fc epsilon R) on murine hybridoma B cells were studied to determine the carbohydrate content and the importance of processing events in cell surface expression and ligand (IgE) binding ability. Endo and exoglycosidase treatment demonstrated that the mature receptor is composed of two to three complex-type N-linked oligosaccharides and contains sialic acid. Pulse-chase experiments indicated that the receptor is synthesized as a 44,000 dalton precursor that begins to be processed by 1 hr to the mature 49,000 dalton form, and the latter is expressed at the cell surface by 2 hr. It was determined that the processing included the conversion of N-linked oligosaccharides to the complex type as well as an additional processing event, because in the presence of tunicamycin, the receptor is synthesized as a 36,000 dalton precursor that is processed to a 38,000 dalton species. Analysis of the effects of tunicamycin treatment and endo F digestion on soluble Fc epsilon R isolated from cell supernatants demonstrated the existence of several m.w. species of Fc epsilon R fragments, and indicated that only the higher m.w. fragments were N-glycosylated. The use of several inhibitors of the N-linked carbohydrate processing pathway demonstrated that the addition of core N-linked side-chains, but not their processing to the complex type, is required for cell surface expression of Fc epsilon R. Also, processing of N-linked carbohydrate is not required for ligand binding activity. Finally, IgE affinity chromatography indicated that the 49,000 and 38,000 dalton (tunicamycin) Fc epsilon R bind IgE more effectively than their precursor forms, 44,000 and 36,000 daltons, respectively, indicating that a processing event independent of N-linked glycosylation is necessary for optimal ligand binding activity.

A rat hybridoma producing a high-affinity IgG2a monoclonal antibody (B3B4) directed against against the murine lymphocyte IgE receptor (Fc epsilon R) was established by using purified Fc epsilon R from Fc epsilon R+ murine hybridoma B cells as immunogen. The monoclonal and polyclonal anti-Fc epsilon R inhibited the binding of IgE to the murine lymphocyte Fc epsilon R and were also used to isolate the Fc epsilon R. B3B4 specifically recognized only the 49-Kd Fc epsilon R on murine B lymphocyte as determined by immunoprecipitation and SDS-PAGE analysis. In addition to its reaction with intact Fc epsilon R, B3B4 also recognized Fc epsilon R fragments that were present in the culture media of Fc epsilon R+ hybridoma cells. The predominant fragments isolated were 38 Kd and 28 Kd by SDS-PAGE analysis. When tested for reactivity with other cell types, B3B4 was highly specific for murine B lineage cells in that it did not significantly react with Fc epsilon R on macrophages and T cells and, in addition, did not react with the high affinity mast cell Fc epsilon R. B3B4 completely blocked IgE rosetting, and a reciprocal inhibition of binding was seen in a dose-dependent fashion between IgE and B3B4, indicating a close proximity of the IgE and B3B4 binding sites. Saturation binding analysis indicated that the Fab' fragment of B3B4 bound to twice as many sites/cell as IgE, suggesting that there are two identical B3B4 determinants per 49-Kd Fc epsilon R or that the IgE binding site is formed by the association of at least two 49-Kd Fc epsilon R. However, unlike IgE, neither B3B4 nor F(ab')2-B3B4 nor Fab'-B3B4 were very effective in causing Fc epsilon R upregulation on murine hybridoma B cells; in fact, B3B4 prevented this upregulation when added in combination with IgE. These results suggest that a site-specific interaction provided only by IgE may be essential for ligand-specific upregulation. Both polyclonal and monoclonal antibodies will be useful in further studies concerning the functional relationship between the membrane Fc epsilon R and the soluble Fc epsilon R fragments.

Rodent B cells respond to culture with IgE by increasing their IgE-specific Fc receptors (Fc epsilon R). The mechanism of this upregulation was characterized on Fc epsilon R+ murine B cell hybridoma lines. Measurements of [35S]methionine incorporated into the Fc epsilon R over time indicated that IgE did not appreciably increase the rate of Fc epsilon R synthesis. In contrast analysis of Fc epsilon R decay from surface radioiodinated B hybridoma cells demonstrated that IgE acted to slow the rate of Fc epsilon R degradation. Very little endocytosis of monomeric IgE was seen; this, combined with the observation that lysomotropic agents failed to inhibit Fc epsilon R degradation suggested that decay occurs at the cell surface. A soluble receptor immunoassay was developed, using monoclonal anti-Fc epsilon R, and this assay demonstrated that cell-bound IgE inhibited the release into the culture media of soluble immunoreactive Fc epsilon R. Examination of the soluble Fc epsilon R by SDS-PAGE after isolation with monoclonal anti-Fc epsilon R demonstrated that it was 10,000 m.w. smaller than the cell-associated Fc epsilon R. IgE affinity columns failed to bind the Fc epsilon R fragment, indicating that the ligand binding activity was largely lost. Thus this study demonstrated that IgE-dependent Fc epsilon R induction on B cells occurs because IgE upon binding to the B cell surface, inhibits the proteolytic cleavage and release of the Fc epsilon R into the surrounding medium, and it is this inhibition of degradation that causes the higher Fc epsilon R levels.

A B cell hybridoma secreting monoclonal antibody against human IgE binding factors was obtained by immunization of BALB/c mice with partially purified IgE binding factors, and fusion of their spleen cells with SP-2/0-AG14 cells. The monoclonal antibody bound all of the 60,000, 30,000, and 15,000 dalton IgE binding factors from two T cell hybridomas and those from activated T cells of a normal individual. The antibody bound both IgE-potentiating factors, which had affinity for lentil lectin, and IgE-suppressive factors, which had affinity for peanut agglutinin. However, the monoclonal anti-IgE-binding factor bound neither Fc epsilon R on RPMI 8866 cells nor IgE binding factors from the B lymphoblastoid cells. A monoclonal antibody against Fc epsilon R on B cells (H107) bound the 60,000 and 30,000 dalton IgE binding factors from both T cell hybridomas and RPMI 8866 cells but did not bind the 15,000 dalton IgE binding factors from either T cells or B cells. The results indicate that T cell-derived IgE binding factors have a unique antigenic determinant that is lacking in both Fc epsilon R on B cells and B cell-derived IgE binding factors. The anti-IgE binding factor and anti-Fc epsilon R monoclonal antibodies both failed to stain cell surface components of IgE binding factor-producing T cell hybridomas. However, both antibodies induced the T cell hybridoma to form IgE binding factors. The results suggest that the T cell hybridomas bear low numbers of Fc epsilon R that share antigenic determinants with IgE binding factors secreted from the cells.

A monoclonal mouse anti-rat IgE, ELIVB5 (B5) was used in a solid-phase radioimmunoassay (RIA) to detect antigen specific IgE in antisera from rats infected with a larval nematode. Binding of 125I-labeled B5 was specific for rat IgE and did not crossreact with human IgE. The RIA was used to demonstrate crossreactivity between antigens of closely related larval nematodes.

Previous resonance energy-transfer measurements have suggested that immunoglobulin E (IgE) may bend near the junction of its Fc and Fab segments in order to bind to its high-affinity receptor on rat basophilic leukemia cells. In order to test this possibility, two monoclonal antibodies were employed that bind specifically to rat IgE (IgER) when IgER is in solution and when it is bound to receptors on the plasma membrane. The F(ab')2 fragment of one monoclonal (B5) that is specific for the Fab region of IgER was labeled with donor probes and bound to IgER, and the quenching of the fluorescence of these donors due to simultaneous binding of the Fab' fragment of an anti-Fc monoclonal (A2) that was labeled with an acceptor probe at its interchain disulfide bond was measured. Significantly less energy transfer between these probes was observed when IgER was bound to its receptor on membrane vesicles than when it was free in solution, and this result is interpreted in light of other energy-transfer measurements using A2 and B5 that were preferentially labeled near their combining sites with donors and acceptors, respectively, as well as measurements of the distance of closest approach between these sites and the membrane surface. These results along with previous energy-transfer measurements and other biochemical information form the basis for a working model of the conformation and orientation of receptor-bound IgE. This study demonstrates the use of fluorescently labeled monoclonal antibodies as highly selective energy-transfer probes in assessing structures of macromolecular complexes on the plasma membrane.