Modulation of the biologic activities of IgE-binding factor. II. Physicochemical properties and cell sources of glycosylation-enhancing factor

T lymphocytes of rats treated with Bordetella pertussis vaccine (BP) formed a soluble factor that enhanced the glycosylation of IgE-binding factors during their biosynthesis, and provided the latter factors with the biologic activity to potentiate the IgE response. The present experiments demonstrated that pertussigen (leukocytosis-promoting factor) from BP induced normal rat spleen cells to form the glycosylation-enhancing factor. The same factor was obtained by incubation of normal spleen cells with 5 micrograms/ml, but not 2 micrograms/ml, concanavalin A. When normal rat mesenteric lymph node cells were incubated with the glycosylation-enhancing factor together with IgE, IgE-binding factors formed by the cells selectively potentiated the IgE response. The IgE-binding factors formed by the same cells upon incubation with IgE alone neither enhanced nor suppressed the IgE response. The glycosylation-enhancing factor changed the nature of IgE-binding factors formed by the rat-mouse T cell hybridoma, 23A4. IgE-binding factors induced by IgE alone lacked affinity for lentil lectin, whereas those induced by IgE in the presence of the glycosylation-enhancing factor had affinity for the lectin. The cell source of the glycosylation-enhancing factor appeared to be W 3/25+ Fc gamma R+ T cells. The glycosylation-enhancing factor was protein in nature and had a m.w. of about 25,000. The factor had affinity for acid-treated Sepharose and could be recovered from the beads by elution with lactose. The factor was different from interleukin 2 with respect to both its affinity for galactose and its isoelectric point.     

Presence of an antigen-specific T cell subset that forms IgE-suppressive factor and IgG-suppressive factor on antigenic stimulation

B6D2F1 mice were given three i.v. injections of ovalbumin (OA), and antigen-specific T cell clones were established from their spleen cells. One of the FcR+ T cell clones formed IgE-binding factors on incubation with OA-pulsed syngeneic macrophages. Neither soluble antigen nor macrophages alone induced factor formation. T cell hybridomas were constructed by fusion of the antigen-specific T cell clone with BW 5147 cells. Among 11 T cell hybridomas established, six clones produced IgE-binding factors on incubation with OA-pulsed BDF1 macrophages. Mouse IgE also induced the same hybridoma to form IgE-binding factors. The majority of IgE-binding factors formed by two T hybridomas and by those produced by the parent T cell clone had affinity for peanut agglutinin but for neither lentil lectin nor Con A. These hybridomas and the original T cell clone spontaneously released glycosylation-inhibiting factor, which inhibits the assembly of N-linked oligosaccharide(s) on IgE-binding factors. On antigenic stimulation, the T cell hybridomas produced both IgE-binding factors and IgG-binding factors. The IgE-binding factors consisted of three species with m.w. of 60,000, 30,000, and 15,000. Both the 60K and 15K IgE-binding factors selectively suppressed the IgE response of DNP-OA-primed rat mesenteric lymph node cells, whereas IgG-binding factors selectively suppressed the IgG response. The results indicate that antigen-primed FcR+ T cells produced IgE-suppressive factors and IgG-suppressive factors on antigenic stimulation. However, the T cell hybridomas were not committed to suppressive activity. When the hybridomas were stimulated by antigen in the presence of glycosylation-enhancing factor (GEF), the 60K, 30K, and 15K IgE-binding factors formed by the cells selectively potentiated the IgE response. IgG-binding factors formed by the cells in the presence of GEF failed to suppress the IgG response. It appears that antigen-specific FcR+ T cells regulate the antibody response through the formation of Ig-binding factors, but that the function of the cells could be switched from suppression to enhancement, depending on the environment of the cells.     

Modulation of the biologic activities of IgE-binding factor. V. The role of glycosylation-enhancing factor and glycosylation-inhibiting factor in determining the nature of IgE-binding factors

Glycosylation-enhancing factor (GEF) and IgE-potentiating factor were detected in culture supernatants of rat mesenteric lymph nodes (MLN) cells obtained 14 days after infection with Nippostrongylus brasiliensis (Nb), but not in supernatants of MLN cells of 8-day Nb-infected rats. Both factors were also released from T cells upon antigenic stimulation of KLH + alum-primed spleen cells. The GEF from the Nb-infected rats and KLH + alum-primed spleen cells had affinity for p-aminobenzamidine agarose and were inactivated by phenylmethylsulfonylfluoride, an inhibitor of serine proteases. These results indicate that the GEF obtained in the two systems is a serine protease and is identical to that obtained by stimulation of normal T cells with lymphocytosis-promoting factor (LPF) from Bordetella pertussis. The concomitant formation of IgE-potentiating factor and GEF by Nb infection, by antigenic simulation of KLH + alum-primed spleen cells, and by treatment of rats with Bordetella pertussis vaccine suggests that the serine protease is involved in a common pathway leading to the selective formation of IgE-potentiating factor. In contrast, glycosylation-inhibiting factor (GIF) is always found during the selective formation of IgE-suppressive factor. IgE-suppressive factor and GIF were formed by MLN cells of 8-day Nb-infected rats and KLH-CFA-primed spleen cells. GIF was detected in culture supernatants of T cell hybridomas 23A4 and 23B6, which form unglycosylated IgE-binding factors upon incubation with IgE. GIF obtained from all of these sources bound to monoclonal anti-lipomodulin. These findings indicate that GIF or lipomodulin is involved in all systems, which leads to the selective formation of IgE-suppressive factor. However, the formation of GIF was not restricted to those conditions in which IgE-suppressive factor was selectively formed. The culture supernatants of MLN cells of 14-day Nb-infected rats and antigen-stimulated KLH + alum-primed spleen cells contained a small amount of GIF, which could be detected after inactivation of GEF. It appears that T cells from these sources formed GEF and GIF, but that GEF overcame the effect of GIF on glycosylation of IgE-binding factors. The results indicate that the nature and biologic activities of IgE-binding factors are decided by the balance between GEF and GIF formed by T cells.     

Monoclonal antibody specific for T cell-derived human IgE binding factors

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.     

Immunosuppressive effects of glycosylation inhibiting factor on the IgE and IgG antibody response

Glycosylation inhibiting factor (GIF) was purified from culture filtrates of a T cell hybridoma, 23A4, by affinity chromatography on anti-lipomodulin Sepharose. The factor exhibited phospholipase inhibitory activity upon dephosphorylation. Immunization of BDF1 mice with aluminum hydroxide gel (alum)-absorbed dinitrophenyl derivatives of ovalbumin (DNP-OA) resulted in persistent IgE and IgG antibody formation. However, repeated injections of the affinity-purified GIF into the DNP-OA-primed mice beginning on the day of priming prevented the primary anti-hapten antibody responses of both the IgE and the IgG1 isotypes. Treatment with GIF also diminished on-going IgE antibody formation in the DNP-OA-primed mice. The treatment changed the nature of IgE-binding factors formed by BDF1 spleen cells. Incubation of spleen cells from OA + alum-primed mice with OA resulted in the formation of IgE-potentiating factor, whereas spleen cells of OA-primed, GIF-treated mice formed IgE-suppressive factor upon antigenic stimulation. It was also found that Lyt-2+ T cells in the OA-primed, GIF-treated mouse spleen cells released GIF, which had affinity for OA and bore I-Jb determinant(s). Transfer of a Lyt-1+ cell-depleted fraction of the OA-primed, GIF-treated mouse spleen cells into naive syngeneic animals resulted in suppression of the primary anti-DNP IgE antibody response of the recipients to alum-absorbed DNP-OA, but failed to affect the anti-DNP antibody response to DNP-keyhole limpet hemocyanin. The results indicate that GIF treatment during the primary response to OA facilitated the generation of antigen-specific suppressor T cells.     

Construction of antigen-specific suppressor T cell hybridomas from spleen cells of mice primed for the persistent IgE antibody formation

Attempts were made to generate Ag-specific suppressor T cells from Ag-primed spleen cells by using glycosylation inhibiting factor (GIF). BDF1 mice were primed with alum-absorbed OVA and their spleen cells were stimulated with OVA. Ag-activated T cells were then propagated in IL-2-containing conditioned medium. Incubation of the T cells with OVA-pulsed syngeneic macrophages resulted in the formation of IgE-potentiating factor and glycosylation-enhancing factor that has affinity for OVA, i.e., OVA-specific glycosylation-enhancing factor. However, if the same Ag-activated splenic T cells were propagated in the IL-2-containing medium in the presence of GIF T cells obtained in the cultures formed IgE-suppressive factors and OVA-specific GIF on antigenic stimulation. Thus we constructed T cell hybridomas from the Ag-activated T cells propagated by IL-2 in the presence of GIF. A representative hybridoma, 71B4, formed OVA-specific GIF on incubation with OVA-pulsed macrophages of BDF1 mice or C57B1/6 mice. However, if the same hybridoma cells were incubated with OVA alone or with OVA-pulsed macrophages of H-2k or H-2d strains, they produced GIF that had no affinity for OVA. The OVA-specific GIF bound to OVA-Sepharose but did not bind to BSA-Sepharose or KLH Sepharose. Intravenous injections of the OVA-specific GIF from the hybridoma suppressed the IgE and IgG1 anti-DNP antibody response of BDF1 mice to DNP-OVA, but failed to suppress the anti-hapten antibody responses of the strain to DNP-keyhole limpet hemocyanin, indicating that the factors suppressed the antibody response in a carrier-specific manner. However, the same OVA-specific GIF failed to suppress the anti-hapten antibody response of DBA/1 mice to DNP-OVA, suggesting that the immunosuppressive effects of the factors is MHC restricted.     

IgE-binding factors from mouse T lymphocytes. I. Formation of IgE-binding factors by stimulation with homologous IgE and interferon

Incubation of normal mouse spleen cells with homologous IgE resulted in the formation of soluble factors that inhibited rosette formation of mouse Fc epsilon R+ cells with IgE-coated ox erythrocytes. The soluble factors could be absorbed with mouse or rat IgE coupled to Sepharose and recovered from the beads by acid elution. However, the factors had no affinity for either human IgE or mouse IgG. The IgE-binding factors were derived from T cells. Production of the factors required Lyt1+ T cells and Fc gamma R+ cells, which suggests that the factors are derived from Fc gamma R+ Lyt 1+ T cells. The molecular size of IgE-binding factors was approximately 15,000 daltons. When IgE-binding factors were formed by BALB/c spleen cells, nearly one-half of the factors had affinity for lentil lectin, and the remaining half of the factors failed to bind to the lectin. The proportion of the two species of IgE-binding factors differed depending on mouse strains. The majority of the factors formed by B6D2F1 spleen cells had affinity for lentil lectin, but those formed by SJL spleen cells failed to bind to the lectin. The IgE-binding factors were also induced by incubation of normal spleen cells with polyinosinic-polycytidylic acid (pI:pC). The nucleotide stimulated splenic adherent cells to form "inducers" of IgE-binding factors, which in turn induced normal lymphocytes to form IgE-binding factors. The inducers of IgE-binding factors were inactivated (or neutralized) by antibodies specific for mouse Type I interferon. It was also found that purified mouse beta interferon could induce the formation of IgE-binding factors. IgE-binding factors induced by pI:pC consisted of two different molecules: one had a m.w. of 15,000 daltons, and another had a m.w. of between 40,000 and 60,000 daltons.     

IgE-binding factors from mouse T lymphocytes. III. Role of antigen-specific suppressor T cells in the formation of IgE-suppressive factor

BDF1 mice were given three i.v. injections of ovalbumin (OA) to induce antigen-specific suppressor T cells. Incubation of spleen cells of OA-treated mice with homologous antigen resulted in the formation of IgE-suppressive factor. This factor was not derived from antigen-specific suppressor T cells, but suppressor T cells were essential for determining the nature of IgE-binding factors formed. In the spleen cells of OA-treated mice, antigenic stimulation of antigen-primed Lyt-1+ (helper) T cells resulted in the formation of inducers of IgE-binding factor, whereas Lyt-2+, I-J+ T cells released glycosylation-inhibiting factor (GIF), and these two factors, in combination, induced unprimed Lyt-1+ T cells to form IgE-suppressive factor. The role of GIF is to inhibit the assembly of N-linked oligosaccharides on IgE-binding factors during their biosynthesis, and thereby provide them with a biologic activity: suppression of the IgE response. Under the experimental conditions employed, GIF was released spontaneously from antigen-specific suppressor T cells. However, antigenic stimulation of the cells enhanced the release of the factor. GIF from antigen-specific suppressor T cells has a m.w. of 25,000 to 30,000, as estimated by using gel filtration, binds to anti-I-J alloantibodies and to a monoclonal antibody specific for lipomodulin, and has affinity for specific antigen. The possible relationship between antigen-specific GIF and antigen-specific suppressor factors is discussed.     

Relationship among IgE-binding factors with various molecular weights

The rat-mouse T cell hybridoma 23B6 forms IgE-binding factors on incubation with IgE. The hybridoma cells incubated with IgE contained intracellular IgE-binding factors of 60K, 30K, 14K, and 10K daltons, and secreted the 60K, 30K, and 14K IgE-binding factors. Both intracellular and extracellular IgE-binding factors of 60K and 14K daltons selectively suppressed the IgE response, whereas the 30K and 10K factors failed to do so. Reduction and alkylation of the extracellular 60K IgE-binding factors yielded fragments of 30K, 14K, and 10K daltons with IgE-binding activity, and the same treatment of the 30K "inactive" IgE-binding factor yielded the 14K and 10K fragments. Among the fragments obtained by reduction and alkylation, only the 14K IgE-binding factors selectively suppressed the IgE response. Monoclonal antibody OX 3, which recognizes an Ia determinant, bound the 60K and 30K IgE-binding factor, and the 30K fragment obtained by reduction and alkylation. None of the 14K and 10K IgE-binding factors and fragments of comparable size obtained by reduction and alkylation contained the antigenic determinant. The results indicate that 30K, 14K, and 10K IgE-binding factors are derived from the 60K precursor molecules. Transfection of COS 7 monkey kidney cells with a single cloned cDNA, 8.3, which encodes rodent IgE-binding factor, resulted in the formation of IgE-binding factors of 60K and 11K daltons. Both species of the factors had affinity for lentil lectin and selectively potentiated the IgE response, but only the 60K IgE-binding factor bound to the monoclonal anti-Ia antibody. Reduction and alkylation of the 60K IgE-binding factors from the cDNA clone yielded an 11K fragment having the same properties as the 11K IgE-binding factor, with respect to the affinity for lentil lectin, and the biologic activities. Because the cDNA clone was constructed from mRNA of 23B6 cells, it appears that the 60K IgE-binding factor molecule from 23B6 cells is composed of a single peptide chain, and that the naturally formed 30K, 14K, and 10K IgE-binding factors are formed by post-translational modification of the 60K peptide.     

Antigen-specific T cells that form IgE-potentiating factor, IgG-potentiating factor, and antigen-specific glycosylation-enhancing factor on antigenic stimulation

BDF1 mice were immunized with alum-absorbed OVA and T cell hybridomas were constructed from their splenic T cells. Many of the hybridomas constitutively produced glycosylation enhancing factor (GEF), which could switch the T cell hybridoma 23A4 cells from the formation of IgE-suppressive factors to the formation of IgE-potentiating factors. When one of the hybridoma clones, 12H5, was incubated with OVA-pulsed syngeneic or semi-syngeneic (H-2b) macrophages, the hybridoma produced GEF that have affinity for OVA, but not for either keyhole limpet hemocyanin or BSA. However, the same hybridoma constitutively produced nonspecific GEF, that lacked affinity for OVA. Upon incubation with OVA-pulsed macrophages, the same hybridoma produced both IgE-potentiating factors and IgG-potentiating factors which selectively enhance the IgE response and IgG response, respectively. Both Ag-specific GEF and nonspecific GEF from the hybridoma bind to p-aminobenzamidine-agarose, and are recovered by elution with benzamidine. It was also found that both OVA-specific GEF and nonspecific GEF from the hybridoma induced the release of arachidonic acid from phospholipids of mouse fibrosarcoma cell line, HSDM1C1 cells. GEF formed by the 12H5 hybridoma bound to alloantibodies reactive to the product(s) of the I-Ab subregion of major histocompatibility complex. The Ag-specific GEF consisted of two Mr species, of 70 to 90 kDa and 50 to 60 kDa, whereas nonspecific GEF consisted of 50 to 60 kDa and 25 to 30 kDa molecules. Reduction and alkylation treatment of the OVA-specific GEF resulted in the formation of nonspecific GEF, suggesting that Ag-specific GEF is composed of Ag-binding polypeptide chain and nonspecific GEF.     

Modulation of IgE synthesis by IgE-binding factors released by T cells of asthmatic patients with elevated serum IgE

The culture supernatants of unstimulated T cells (TCS) from asthmatic patients with elevated serum IgE were tested for IgE-binding factors (IgE-BFs) displaying the IgE-potentiating activity. The IgE-BFs were detected by their ability to inhibit the rosetting of RPMI 8866 cells with ox erythrocytes coupled with mouse monoclonal antibody (E-Mab) specific to Fc receptors for IgE (Fc epsilon R). TCS showing the rosette-inhibiting activity significantly enhanced the spontaneous IgE synthesis by B cells of allergic individuals. Interestingly, rosette-inhibiting factors could be removed by absorption with IgE-Sepharose from which they were subsequently eluated with acid buffer, indicating that the rosette inhibition was indeed mediated by IgE-BFs. In addition, such IgE-BFs had affinity for concanavalin A and lost their IgE-potentiating activity after treatment with trypsin and neuraminidase. In contrast, T cells treated with tunicamycin released IgE-suppressing factors capable of inhibiting the IgE-potentiating activity of TCS derived from untreated T cells. On the other hand, the culture supernatants from subpopulations depleted of Fc epsilon R+ T cells but not of Fc gamma R+ T cells contained neither rosette-inhibiting factors nor IgE-potentiating factors, suggesting that IgE-BFs were released by in vivo pre-activated Fc epsilon R+ T cells. With regard to circulating Fc epsilon R+ T cells determined by E-Mab, they were significantly higher in asthmatic patients with elevated serum IgE (0.77 +/- 0.15%) than in normal subjects (0.17 +/- 0.07%) in spite of a very small proportion of T cells bearing Fc epsilon R.     

Modulation of the biologic activities of IgE-binding factors. VII. Biochemical mechanisms by which glycosylation-enhancing factor activates phospholipase in lymphocytes

Cells of the T cell hybridoma 23A4 produce IgE-binding factors lacking N-linked oligosaccharides (unglycosylated form) when they are incubated with IgE alone. In the presence of glycosylation-enhancing factor (GEF) or bradykinin, however, the same cells produce IgE-binding factors with N-linked oligosaccharides (glycosylated form). Switching the cells from the formation of unglycosylated IgE-binding factors to the formation of glycosylated factors was accompanied by the release of both glycosylation-inhibiting factor (GIF) in its phosphorylated form, i.e., phosphorylated lipomodulin, and arachidonate from the cells. Analysis of the biochemical processes for the release of GIF from 23A4 cells showed that affinity-purified GEF or bradykinin induced transient phospholipid methylation and diacylglycerol (DAG) formation, and enhanced 45Ca uptake into the cells. Inhibitors of methyltransferases, i.e., 3-deaza-adenosine plus L-homocysteine thiolactone, inhibited not only phospholipid methylation but also DAG formation and GIF release. Exogenously added 1-oleoyl-2-acetyl glycerol, i.e., a DAG that is permeable to the plasma membrane, induced the release of GIF from the cells. It was also found that 12-O-tetradecanoyl-phorbol 13-acetate (TPA) switched 23A4 cells and normal lymphocytes to the selective formation of N-glycosylated IgE-binding factor, and induced the release of GIF from the cells. 32PO4-labeled lipomodulin was detected in the extract of 23A4 cells 3 to 5 min after the addition of GEF, bradykinin, or TPA. These results indicate that GEF and bradykinin induced the activation of methyltransferases and phospholipase C for the formation of DAG, which in turn activated Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) for the phosphorylation of lipomodulin. Because lipomodulin loses phospholipase inhibitory activity after phosphorylation, increased phospholipase A2 activity would be expressed by this process.     

Relationship between T cell receptors and antigen-binding factors. I. Specificity of functional T cell receptors on mouse T cell hybridomas that produce antigen-binding T cell factors

Upon antigenic stimulation with OVA-pulsed syngeneic macrophages, the mouse T cell hybridoma 231F1 produced glycosylation inhibiting factor (GIF) having affinity for OVA and IgE-suppressive factors, whereas another T cell hybridoma, 12H5, cells produced OVA-binding glycosylation enhancing factor (GEF) and IgE-potentiating factor. The OVA-binding GIF from the 231F1 cells is an Ag-specific Ts cell factor, whereas OVA-binding GEF from the 12H5 cells is an Ag-specific augmenting factor. Both hybridomas express CD3 complex and functional TCR-alpha beta. Cross-linking of TCR-alpha beta or CD3 molecules on the hybridomas by anti-TCR-alpha beta mAb or anti-CD3 mAb and protein A resulted in the formation of the same factors as those obtained by the stimulation of the cells with OVA-pulsed syngeneic macrophages. It was also found that both the 231F1 cells and 12H5 cells formed IgE-binding factors upon incubation with H-2d and H-2b APC, respectively, with a synthetic peptide corresponding to residues 307-317 in the OVA molecules (P307-317). Six other synthetic peptides, including those containing the major immunogenic epitope, i.e., P323-339, failed to stimulate the hybridomas in the presence of APC. Indeed, all of the 10 T cell hybridoma clones, which could produce either OVA-binding GIF or OVA-binding GEF, responded to P307-317 and APC for the formation of IgE-binding factors. In contrast, GIF/GEF derived from six other hybridoma clones, whose TCR recognized P323-339 in the context of a MHC product, failed to bind to OVA-coupled Sepharose. The results indicate the correlation between the fine specificity of TCR and the affinity of GIF/GEF to the nominal Ag. The amino acid sequence of P307-317 suggested that TCR on the cell sources of Ag-binding factors are specific for an external structure of the Ag molecules.     

Regulatory effects of human IgE-binding factors on the IgE response of rat lymphocytes

Normal human peripheral blood T cells were propagated in the presence of human interleukin 2, and activated cells were incubated with human IgE-dimer to induce IgE binding factor formation. The cells were then fused with a mutant of the human T cell line CEM. Five of the T cell hybridomas formed IgE binding factors upon incubation with human IgE-dimer. Because IgE binding factors formed by the human T cell hybridomas had affinity not only for human IgE but also for rat IgE, the biologic activities of the factors were evaluated by using antigen-primed rat mesenteric lymph node (MLN) cells. When parent T cells were propagated with crude IL 2, which contained glycosylation enhancing factor (GEF), IgE binding factors formed by all of the five hybridomas had affinity for Con A, but only a fraction of the factors bound to lentil lectin. The 60,000 and 15,000 IgE binding factors formed by two representative hybridomas, i.e., 166A2 and 166G11, selectively potentiated the IgE-forming cell response of rat MLN cells. When parent T cells were obtained by propagation with purified IL 2, which did not contain GEF, and the cells were incubated with IgE-dimer in the presence of glycosylation inhibiting factor (GIF), T cell hybridomas constructed from the cells formed IgE binding factors that lacked affinity for Con A but bound to peanut agglutinin (PNA). The 30,000 IgE binding factors formed by two of such hybridomas, 398A3 and 400G2, selectively suppressed the IgE response of rat MLN cells. It was also found that the biologic activities and carbohydrate moieties of human IgE binding factors could be switched by changing the culture conditions of the hybridomas. When the 166A2 hybridoma was cultured with human IgE in the presence of bradykinin, essentially all of the IgE binding factors that were formed by the cells bound to lentil lectin, and the factors that were formed in the presence of bradykinin exerted higher potentiating activity than those obtained in the absence of bradykinin. On the other hand, IgE binding factors formed by the same cells in the presence of GIF had affinity for PNA, and selectively suppressed the IgE response of rat MLN cells.     

An intestinal galactose-specific lectin mediates the binding of murine IgE to mouse intestinal epithelial cells.

A number of lactose-binding lectins have recently been identified in the rat and mouse intestine, one of which corresponds to the C-terminal domain of IgE-binding proteins, originally identified in rat basophilic leukemia (RBL) cells and mouse 3T3 fibroblasts. In the present report, we describe the affinity purification of a rat intestinal lactose-specific lectin which binds murine IgE antibodies. This binding most likely occurs via the immunoglobulin carbohydrate chains, as it is inhibited by lactose. This intestinal lectin molecule is also immunologically related to the previously described IgE-binding protein (epsilon BP) isolated from RBL cells, since it is recognized by antibodies raised against recombinant epsilon BP. This intestinal form of epsilon BP has a molecular mass of 17.5 kDa, which is much lower than that of its RBL cell analogue (31 kDa). The attachment of IgE to the mouse intestinal epithelium was demonstrated by immunohistochemistry, along with the presence of a corresponding mouse intestinal epsilon BP. The carbohydrate-dependent nature of this attachment was established by demonstrating that IgE binding to mouse epithelium was specifically abolished by lactose (4 mM) and by a blood-group-A-active tetrasaccharide (0.2 mM), but not by mannose (10 mM). Finally, the association of IgE with the mouse intestinal epithelium was prevented by competition with the purified IgE-binding lectin isolated from rat intestine. Although the physiological function of this intestinal protein is still unknown, the finding that IgE binds to a lectin in the intestinal epithelium pinpoints a possible novel mechanism for the regulation of IgE-mediated disorders, such as food allergy.     

Fine specificity, structure, and proteolytic susceptibility of the human lymphocyte receptor for IgE

Properties of the Fc receptor for IgE (FC epsilon R) on cultured human B lymphoblastoid cells (RPMI 8866) were studied. Specificity for human IgE (hIgE) was demonstrated by inhibition studies with both Fc epsilon R+ intact cell and detergent-solubilized receptor preparations. No interaction of the FC epsilon R with other hIg classes or with rodent IgE was seen. In other studies, 3,3-dithiobis(sulfosuccinimidyl) propionate was used to cross-link hIgE to 125I surface-labeled 8866 cells. After detergent solubilization, the 125I receptor components were isolated by immunoprecipitation, and receptor peptides of 83 and 46 kilodalton kD were demonstrated by SDS-PAGE in the presence of reducing agents. Cross-linking performed after detergent solubilization gave identical results. Tryptic maps of the 83 and 46 kD polypeptides were identical with respect to surface-iodinated peptides; this indicates a structural homology between these components. The 83 kD component was more difficult to elute from IgE affinity columns, potentially because of an increased number of IgE binding sites per FC epsilon R molecule. Limited proteolysis studies of the purified FC epsilon R with papain and V8 protease from Staphylococcus aureus demonstrated that a 16 kD FC epsilon R fragment was rapidly produced. This component was also seen after papain treatment of intact cells, and it retained the ability to interact with anti-FC epsilon R antisera and, at least in the absence of detergent, with hIgE affinity columns. Potential relationships between the FC epsilon R and lymphokines that modulate the IgE response (IgE-binding factors) are discussed.     

Modulation of the biologic activities of IgE binding factor. VI. The activation of phospholipase by glycosylation enhancing factor

Glycosylation enhancing factor (GEF) from rat T cells is a kallikrein-like enzyme and enhances the assembly of N-linked oligosaccharides to IgE binding factors during their biosynthesis, whereas another T cell factor, i.e., glycosylation inhibiting factor (GIF), is a fragment of phosphorylated lipomodulin (i.e., phospholipase inhibitor), which when dephosphorylated inhibits phospholipase and the glycosylation process. The two T cell factors compete with each other when they are added to normal mesenteric lymph node cells during the formation of IgE binding factors. The addition of GEF to T cell hybridoma 23A4 cell switches the cells from the formation of unglycosylated IgE binding factor to the formation of N-glycosylated IgE binding factor. However, GEF neither inactivated GIF nor inhibited the formation of GIF by the T cell hybridoma. Stimulation of the T cell hybridoma with either affinity-purified GEF or bradykinin resulted in the release of GIF from the cells. GIF released by GEF stimulation had a m.w. of approximately 15,000 and bound to monoclonal antibody against lipomodulin. GEF and bradykinin also induced normal mesenteric lymph node cells to release GIF. Incorporation of 14C-arachidonic acid into 23A4 cells, followed by stimulation of the cells with GEF, resulted in the release of 14C-arachidonate. The results suggest that lipomodulin, a phospholipase inhibitory protein, is present in lymphocytes, and indicate that GEF and bradykinin induce the activation of phospholipase by stimulating cells to release lipomodulin.     

Characterization of a monoclonal antibody directed against the murine B lymphocyte receptor for IgE

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.     

FcR epsilon+ lymphocytes and regulation of the IgE antibody system. V. Preliminary physicochemical characterization of the T cell-selective IgE-induced regulant EIRT

We previously showed that murine lymphoid cells exposed to elevated levels of IgE exhibit the de novo expression of Fc receptors for IgE (FcR epsilon), and the production of soluble mediators, which we have termed IgE-induced regulants (EIR). Described herein is the preliminary physicochemical characterization of one such regulant, that being the EIR responsible for the Lyt-2+ T cell-dependent expression of FcR epsilon and secretion of an IgE-binding factor (IgE-BF) which can potentiate IgE synthesis; the former activity has been denoted EIRT for its selectivity of action on T cells, and the latter activity has been termed enhancing effector molecule (EEM) for its presumed potentiating influence on IgE antibody synthesis. Characterized in parallel was the conventional lymphoid cell-derived cEIRT and a murine monoclonal T cell hybridoma-(MBI-2)-derived mcEIRT. EIRT from either source was shown to exhibit the characteristics of a protein with a molecular mass of 45 to 60 kd. Once enriched by gel filtration, neither cEIRT nor mcEIRT preparations displayed any other EIR-like activity except that of EIRT, as evidenced by the ability of these preparations to act selectively to induce the Lyt-2 T cell-dependent expression of FcR epsilon and the production of EEM, the lack of detectable SFA activity that could induce Lyt-1+ T cells to produce the IgE-BF denoted suppressive effector molecule (SEM), and the lack of detectable levels of the B cell-selective EIRB, as indicated by the incapacity of either preparation to induce B cell FcR epsilon expression. Neither cEIRT nor mcEIRT displayed IgE-binding affinity, in contrast to the EEM produced in response to stimulation with these regulants. The only EIR-like activity detected in the unfractionated supernatant fluid from cultures of the monoclonal T cell hybridoma MBI-2 was that of EIRT. Careful in vitro analysis established that such preparations did not contain enhancing factor of allergy (EFA), SFA, EIRB, or IgE-BF. Thus, the enhancement of IgE synthesis observed in animals given this mcEIRT preparation was most likely due to the activity of EIRT known to be present. During the course of these studies, clues as to the physicochemical nature of other EIR activities was obtained. Thus, upon molecular sieve analysis, two distinct molecular mass species of EIRB (one 15 to 20 kd and the other 30 to 35 kd) were demonstrated to be present in conventional lymphocyte-derived cEIR.(ABSTRACT TRUNCATED AT 400 WORDS)     

Murine B cell hybridomas bearing ligand-inducible Fc receptors for IgE

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.