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.     

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.     

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.     

Monoclonal anti-Fc epsilon receptor antibodies with different specificities and studies on the expression of Fc epsilon receptors on human B and T cells

Three monoclonal antibodies, 1-7 (gamma 2b), 3-5 (gamma 1), and 8-30 (mu), specific to Fc epsilon receptors (Fc epsilon R) on human B cells were established. The two monoclonals (1-7 and 8-30) could inhibit the binding of IgE to Fc epsilon R in rosette formation assays, as well as FACS analysis, and were shown to recognize the same epitope of Fc epsilon R. The other monoclonal antibody (3-5) recognized the same molecule but a different epitope, and marginally inhibited the IgE binding. The molecules on RPMI 8866 cells recognized by these monoclonal antibodies had Mr of 46,000 and 25,000 to 30,000 daltons as determined by immunoprecipitation and SDS-PAGE analysis. By employing these monoclonal antibodies, the expression of Fc epsilon R on circulating lymphocytes was studied. Approximately 50% of B cells from normal, nonatopic individuals were found to express Fc epsilon R, and a remarkable increase in the expression of Fc epsilon R was observed in B cells of atopic patients. The expression of Fc epsilon R was not detected in T cells from atopic patients (including hyper IgE syndrome) as well as normal individuals. Incubation of B cells with PHA-conditioned medium plus IgE augmented the expression of Fc epsilon R in the Fc epsilon R+ B cell population but not in Fc epsilon R- population. PHA-conditioned medium plus IgE did not induce Fc epsilon R expression on T cells.     

Relationship between human IgE-binding factors (IgE-BF) and lymphocyte receptors for IgE

This study indicates that human IgE-binding factors (IgE-BF) found in the cellfree culture supernatant (CSN) of Fc epsilon R-bearing B cells are breakdown products of the surface Fc epsilon R. This conclusion is suggested by the following observations. 1) Fc epsilon R and IgE-BF share several antigenic determinants as shown by immunoprecipitation with several Mab to Fc epsilon R (MabER) and SDS-PAGE analysis of the precipitates. 2) Upon incubation at 37 degrees C, normal tonsillar lymphocytes lose their Fc epsilon R and this is associated in a time-related manner with the release in the CSN of molecules reacting with two MabER. 3) Surface radioiodinated tonsillar lymphocytes or RPMI 8866 cells release labeled IgE-binding molecules displaying the same antigenic composition and the same migration on SDS-PAGE as purified IgE-BF. 4) Peptide mapping of highly purified IgE-BF and Fc epsilon R reveals the presence of several identical fragments after digestion with either alpha-chymotrypsin, trypsin, or papain. Moreover, papain digestion of the 25-27 kD IgE-BF and of the affinity-purified Fc epsilon R, generated a 15 kD fragment reacting with two MabER and that is known to bind IgE. Although these data strongly suggest that IgE-BF may be directly derived from cell surface IgE receptors, they do not exclude the possibility that some IgE-BF may also be secreted without being first anchored in the cell membrane.     

The murine lymphocyte receptor for IgE. IV. The mechanism of ligand-specific receptor upregulation on B cells

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.     

Binding the low affinity Fc epsilon R on B cells suppresses ongoing human IgE synthesis

Our results support the hypothesis that binding the low affinity Fc epsilon R (Fc epsilon R-II, CD23) on IgE-secreting B cells, directly suppresses IgE production. IgE production from AF-10/U266 (a human IgE plasmacytoma) decreased upon incubation with anti-IgE mAb or IgE:anti-IgE immune complexes (IgE-IC). Synthesis was suppressed a maximum of 51% with 10 micrograms/ml of IgE-IC after a 24-h incubation. Spontaneous in vitro IgE synthesis from the B cells of highly atopic individuals was also inhibited in a similar fashion. This effect was isotype specific as IgA or IgG immune complexes did not alter IgE production from AF-10 nor did IgE-IC affect IgA or IgG synthesis from lymphoblastoid cell lines making IgG (GM1500 and RPMI 8866) or IgA (GM1056). U266/AF-10 cells displayed both membrane IgE (greater than 90%) and Fc epsilon R-II (23%). To evaluate the role of these membrane proteins in the observed suppression of IgE synthesis, we treated U266/AF-10 cells with IgE-IC that bound Fc epsilon R-II but could not bind membrane IgE, as the mAb used was directed against an idiotypic determinant on the myeloma IgE (PS) used to make the IgE-IC. Suppression was maximal (greater than 50%) with these complexes at 0.1 micrograms/ml and at a 1/1 ratio of mAb anti-IgE to human myeloma IgE. When IgE-IC were used that were constructed with heat denatured IgE or F(ab')2 fragments of IgE, suppression was abrogated indicating IgE-Fc epsilon R binding was required. Neither PS IgE nor mAb 5.1 (the components of IgE-IC) alone affected IgE synthesis. Furthermore, a mAb binding directly to CD23 suppressed IgE synthesis from AF-10 up to 60%. Using limiting dilution analysis, we determined that IgE production per AF-10 cell was constant (0.9 pg/cell/24 h), independent of cell density and cells incubated with IgE-IC were uniformly suppressed. To clarify the mechanism of IgE-IC-induced suppression on AF-10 cells, we assessed both the proliferative rate and cell cycle distribution upon incubation with IgE-IC. There was no correlation between IgE production and [3H]TdR incorporation by AF-10 cells incubated with IgE-IC or anti-CD23 mAb. The distribution of cells within the cell cycle was unaffected by these treatments, with 60% of the cells in G1. These results define a direct role for the Fc epsilon R-II on B cells in the regulation of ongoing IgE synthesis.     

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.     

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.     

Influence of recombinant IL-4, IFN-alpha, and IFN-gamma on the production of human IgE-binding factor (soluble CD23)

This study documents the influence of rIL-4, IFN-gamma, and IFN-alpha on the production of IgE-BF and the expression of lymphocyte receptor for IgE or CD23 Ag (Fc epsilon R II) by human mononuclear cells. IL-4 increases the secretion of IgE-binding factor (BF) by highly purified B lymphocytes, adherent cells, and U937 monoblastic cells. The effect of IL-4 on purified B cells is augmented by costimulating the cells with F(ab')2 anti-IgM. IFN-gamma, IL-2, IL-1-alpha, or IL-1 beta and the low m.w. B cell growth factor have no effect on IgE-BF production by purified B cells even when they are used in combination with anti-IgM. Stimulation of purified T cells with IL-4 or IL-4 plus PMA leads to the production of very small amounts of IgE-BF that might well be derived from the contaminating non-T cells. IFN-gamma increases IgE-BF synthesis by unfractionated PBMC, T cell-depleted PBMC, adherent cells, and U937 cells suggesting that it induces monocytes to release IgE-BF, IFN-gamma suppresses the IL-4-induced Fc epsilon R II expression and IgE-BF production by highly purified B cells but not by PBMC or their T cell-depleted fractions. IFN-alpha inhibits IgE-BF production by IFN-gamma-stimulated PBMC and by IL-4-stimulated cells suggesting that it exerts its effect on B cells and on monocytes. Moreover IFN-alpha suppresses the IL-4-induced expression of Fc epsilon R II on B cells. Both IFN-alpha and IFN-gamma suppress the synthesis of IgE by PBMC in response to IL-4. Taken collectively the results indicate that: 1) IL-4 induces IgE-BF production by both B cells and monocytes, 2) IFN-gamma stimulates IgE-BF synthesis by monocytes but suppresses its production by IL-4-stimulated B cells, and finally 3) IFN-alpha inhibits IgE-BF synthesis in response to either IFN-gamma or IL-4.     

In vivo and in vitro regulation of IgE production in murine hybridomas

Normal BALB/c mice injected i.p. with the IgE-secreting hybridomas B53 (epsilon, kappa anti-DNP), SE1.3 (epsilon, kappa, anti-arsonate) or A3B1 (epsilon, kappa, anti-TNP) were monitored for serum IgE concentrations and frequencies of splenic T lymphocytes with surface membrane receptors for the Fc portion of IgE (Fc epsilon R+ T lymphocytes). Mice with B53 or SE1.3 hybridomas initially developed high concentrations of IgE and CD8+ Fc epsilon R+ T lymphocytes, followed by a progressive decline in both serum IgE and expression of cytoplasmic epsilon-chains in the hybridoma cells. Serum IgE concentrations in mice with A3B1 hybridomas progressively increased without development of Fc epsilon R+ T lymphocytes nor a subsequent decline in IgE or change in cytoplasmic epsilon-chain expression in the A3B1 cells. An in vitro system in which the IgE-secreting hybridoma cells were cocultured with spleen cells harvested from mice with established B53 tumors was used to investigate the mechanisms involved in the inhibition of IgE production by the hybridoma cells. The results of these studies indicate that: 1) the induction/upregulation of Fc epsilon R on CD8+ T lymphocytes in vivo requires factors in addition to high serum IgE concentrations; 2) in addition to CD8+ Fc epsilon R+ T lymphocytes and monocytes, another, as yet unidentified, splenic cell component appears to contribute to the process by which epsilon-chain expression in IgE-secreting hybridoma cells is suppressed, and 3) a hybridoma (A3B1) that fails to induce CD8+, Fc epsilon R+ T lymphocytes in vivo and is not inhibited in IgE expression in vivo, nonetheless is inhibited in IgE expression in vitro when cocultured with spleen cells from mice with B53 tumors.     

Thymus-dependent in vivo suppression of IgE synthesis in a murine IgE-secreting hybridoma

In previous studies we demonstrated that BALB/c mice bearing ascitic tumors of the IgE-secreting hybridoma B53 (epsilon, kappa, anti-dinitrophenyl) developed large numbers of Lyt-1-2+ Fc epsilon R(+) T lymphocytes (T cells with membrane Fc receptors) in response to the elevated serum IgE concentration. The development of Fc epsilon R(+) T lymphocytes was followed by a progressive decrease in the levels of serum IgE in spite of continued proliferation of the hybridoma cells. This sequence of events suggested that the IgE-secreting hybridoma triggered a suppressive immunoregulatory circuit of the host that inhibited IgE expression by the hybridoma cells. The present studies were undertaken to investigate the basis for the subsequent decline in serum IgE levels in mice with B53 tumors and to identify host factors that might be involved in this process. We observed that ascitic B53 cells recovered at increasing time points from BALB/c mice exhibited a selective decline in steady state levels and rates of synthesis of epsilon-heavy chain protein and mRNA. The expression of kappa-light chain protein and mRNA appeared relatively unchanged. The decrease in epsilon-heavy chain gene expression did not occur when B53 tumors were passaged in nu+/nu+ mice or in BALB/c mice depleted of Lyt-2+ cells (suppressor/cytotoxic cell lineage), but did occur in nu+/nu+ mice reconstituted with neonatal BALB/c thymus and in BALB/c mice depleted of L3T4+ cells (helper/inducer cell lineage). That Fc epsilon R(+) T lymphocytes were directly involved in the inhibition of IgE expression was supported by the earlier and more pronounced inhibition of B53 IgE in mice infused with Fc epsilon R(+) T lymphocytes. We conclude from these findings that: 1) the decline in serum IgE levels that occurs toward the end of each generation of in vivo passage of the B53 hybridoma is due to decreased production of IgE by the hybridoma cells, 2) the decreased production of IgE is due to a selective loss of epsilon mRNA expression, 3) the decrease production of IgE by B53 cells is dependent on the presence of Lyt-2+ cells, and 4) Fc epsilon R(+) T lymphocytes participate in the mechanism by which IgE production is suppressed.     

Fc receptors and immunoglobulin binding factors

Receptors for the Fc portion of Ig (Fc receptors, FcR) are found on all cell types of the immune system. Three types of FcR react with IgG: Fc gamma RI is a high-affinity receptor binding IgG monomers whereas Fc gamma RII and Fc gamma RIII are low-affinity receptors binding IgG immune complexes; the three types of Fc gamma R are members of the Ig superfamily. Two FcR react with IgE:Fc epsilon RI is a multichain receptor binding IgE with high affinity; it is composed of an IgE-binding alpha chain, homologous to Fc gamma RIII, and of gamma and beta chains that are necessary for receptor expression and signal transduction. The low-affinity Fc epsilon RII is the only FcR described so far that is not a member of the Ig superfamily but resembles animal lectins; it is composed of a transmembrane chain with an intracytoplasmic NH2 terminus. Fc alpha R has homology with Fc gamma R and is a member of the Ig superfamily. Receptors for IgM and IgD are not characterized yet. Finally, Ig transport is made by FcR-like molecules such as the poly-Ig receptor or an MHC-like receptor found on neonatal intestine. A remarkable property of most FcR is the fact that they are released in cell supernatants and circulate in biological fluids as immunoglobulin binding factors (IBF) generated either by cleavage at the cell membrane or by splicing of FcR transmembrane exon. Immunoglobulin binding factors may interfere with Ig-mediated functions and have direct immunoregulatory activities. Involvement of FcR or IBF has been postulated in several diseases, and monoclonal antibodies to FcR are beginning to be used in therapeutics, particularly to target cytotoxic effector lymphocytes and monocytes to tumor cells.     

The murine lymphocyte receptor for IgE. III. Use of chemical cross-linking reagents to further characterize the B lymphocyte Fc epsilon 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.     

Comparison of the Fc receptors for IgE on human lymphocytes and monocytes

Fc receptors for IgE (Fc epsilon R) on human peripheral blood lymphocytes and monocytes and cultured lymphoblastoid and macrophage-like cell lines were compared with respect to: 1) binding affinity for radiolabeled IgE, 2) inhibition of IgE-specific rosette formation and inhibition of binding of radiolabeled IgE by an antiserum raised against Fc epsilon R isolated from a lymphoblastoid cell line, and 3) m.w. of radiolabeled cell surface proteins precipitated with the anti-Fc epsilon R serum. Scatchard analysis of 125I-IgE binding to lymphocytes, monocytes, and their corresponding cell lines showed biphasic binding curves with all cell types, from which 2 binding affinities were calculated to be KA = 6.2 +/- 1.1 and 2.0 +/- 0.5 x 10(7) M-1. The anti-Fc epsilon R serum inhibited both IgE rosette formation and binding of radiolabeled IgE by lymphocytes and monocytes but did not inhibit IgE rosettes formed by basophils. The inhibitory activity of the anti-Fc epsilon R serum could be absorbed with Fc epsilon R(+) but not with Fc epsilon R(-) cell lines. The anti-Fc epsilon R serum precipitated 2 peptides having m.w. of approximately 47,000 and 23,000 daltons from lysates of both cell surface-labeled lymphocyte and macrophage cell lines. These data indicate that Fc epsilon R on normal lymphocytes and monocytes, as well as on cultured lymphoblastoid and macrophage-like cells, are related structurally, since they share antigenic determinants, bind IgE with a similar affinity, and have similar m.w. However, they differ in all 3 parameters from Fc epsilon R on basophilic granulocytes.     

Recombinant human immune interferon induces increased IgE receptor expression on the human monocyte cell line U-937

Human recombinant gamma interferon (IFN-gamma), which is free from other lymphokines, significantly increased expression of receptors for IgE (Fc epsilon R) on the human monocyte cell line U-937. Fc epsilon R were measured by assaying specific (saturable) binding of 125I-labeled or fluorescein isothiocyanate (FITC)-labeled human IgE (Sha) to U-937 cells. Cell-bound IgE was analyzed by gamma counting and by flow cytometry. IFN-gamma-induced enhancement in IgE binding was a consequence of an increase in the number and density of Fc epsilon R, as cell size did not change significantly after treatment. Scatchard analysis of 125I-IgE binding curves revealed the presence of a homogeneous population of binding sites for IgE in control and in IFN-gamma-treated cells. IFN-gamma treatment did not change the value of the dissociation constant of Fc epsilon R for 125I-IgE. IFN-alpha and IFN-beta had only slight effects on the expression of Fc epsilon R. Dexamethasone (200 nM) diminished the IFN-gamma-induced enhancement in the number of Fc epsilon R by about 50%, the same extent as in control cells. IFN-gamma treatment did not cause a significant alteration in cell number, cell cycle kinetics, or macromolecular synthesis, and enhanced expression of Fc epsilon R was probably not mediated through the cyclic AMP system.     

Possible orientational constraints determine secretory signals induced by aggregation of IgE receptors on mast cells.

Three biologically active monoclonal antibodies (mAbs) specific for the monovalent, high-affinity membrane receptor for IgE (Fc epsilon R) were employed in analysing the secretory response of mast cells of the RBL-2H3 line to crosslinking of their Fc epsilon R. All three mAbs (designated F4, H10 and J17) compete with each other and with IgE for binding to the Fc epsilon R. Their stoichiometry of binding is 1 Fab:1 Fc epsilon R, hence, the intact mAbs can aggregate the Fc epsilon Rs to dimers only. Since all three mAbs induce secretion, we conclude that Fc epsilon R dimers constitute a sufficient 'signal element' for secretion of mediators for RBL-2H3 cells. The secretory dose-response of the cells to these three mAbs are, however, markedly different: F4 caused rather high secretion, reaching almost 80% of the cells' content, while J17 and H10 induced release of only 30-40% mediators content. Both the intrinsic affinities and equilibrium constants for the receptor dimerization were derived from analysis of binding data of the Fab fragments and intact mAbs. These parameters were used to compute the extent of Fc epsilon R dimerization caused by each of the antibodies. However, the different secretory responses to the three mAbs could not be rationalized simply in terms of the extent of Fc epsilon R dimerization which they produce. This suggests that it is not only the number of crosslinked Fc epsilon Rs which determines the magnitude of secretion-causing signal, but rather other constraints imposed by each individual mAb are also important.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of Fc epsilon RII/CD23 on phytohemagglutinin-activated human peripheral blood T lymphocytes. I. Enhancement by IL-2 and IL-4.

Despite evidence for the expression of low affinity Fc receptor for IgE (Fc epsilon RII)/CD23 in T cell lines and pathologic T cells, Fc epsilon RII/CD23 in normal human T cells is still unclear. We studied the expression of Fc epsilon RII/CD23 on T cells in short-term culture of normal human PBMC stimulated with 15 micrograms/ml PHA. PHA stimulation also resulted in the release of soluble Fc epsilon RII/CD23 (IgE binding factor). Using two-dimensional flow cytometry, more than 10% of the Fc epsilon RII/CD23+ cells were found to co-express CD3 Ag. Both CD4+ and CD8+ T cells expressed Fc epsilon RII/CD23. The induction of Fc epsilon RII/CD23 on PHA-activated T cells was enhanced by IL-2 as well as IL-4. Both IL-2 and IL-4 also augmented PHA-induced production of soluble Fc epsilon RII/CD23. The enhanced expression of Fc epsilon RII/CD23 on T cells by both lymphokines was suppressed by rabbit anti-IL-4 antiserum, suggesting the involvement of an IL-4-dependent process even in the IL-2-dependent Fc epsilon RII/CD23 expression on T cells. The expression of mRNA for Fc epsilon RII/CD23 on PHA and IL-4-stimulated PBMC was examined by Northern blot analysis. Fc epsilon RII/CD23 mRNA was detected in RNA prepared from the T cell fraction depleted of B cells and macrophages (Fc epsilon RII+CD3+ = 6.2%, Fc epsilon RII+CD3- = 0.8%). The expression of the mRNA for Fc epsilon RII/CD23 on CD3+ T cells was also confirmed by in situ hybridization with Fc epsilon RII/CD23 cDNA combined with CD3 rosette formation at the single cell level.     

Production and characterization of a monoclonal antibody specific for the human lymphocyte low affinity receptor for IgE: CD 23 is a low affinity receptor for IgE

In the present study a gamma 1 kappa monoclonal antibody, Mab 25, specific for the receptor for the Fc fragment of IgE on lymphocytes (Fc epsilon RL) was established. This antibody was generated after fusion of spleen cells from mice immunized with the EBV-transformed lymphoblastoid cell line RPMI 8866, which is known to express Fc epsilon RL at high density. Mab 25 inhibits strongly the binding of IgE to RPMI 8866 cells and to other Fc epsilon RL-positive EBV-transformed lymphoblastoid cell lines. A 50% inhibition of IgE binding was observed at a Mab 25 concentration of 10 ng/ml. The binding of IgE was also inhibited by Fab fragments of Mab 25, suggesting that the inhibition is not simply due to steric hindrance or to an eventual binding through its Fc portion. Mab 25 only binds to cell lines expressing Fc epsilon RL. Mab 25 immunoprecipitated a single polypeptide with an apparent m.w. of 42 Kd, pI 4.9. The membrane molecule bound to and eluted from a Mab 25 immunoabsorbent had the same apparent m.w. and pI as the Fc epsilon RL purified from an IgE immunoabsorbent. Additionally, when RPMI 8866 cell lysates were cleared with Mab 25, no Fc epsilon RL could be bound to or eluted from an IgE immunoabsorbent. Mab 25 was found to weakly bind to a minor proportion of blood (1 to 4%), tonsil (2 to 9%) and spleen (4 to 5%) mononuclear cells with a low intensity. By double fluorescence analysis, most of the Fc epsilon RL-positive cells were found to be CD 20-positive B lymphocytes. The staining pattern of Mab 25 and the biochemical characteristics of the antigen detected by Mab 25 were comparable to those of the CD 23 Mab. The four CD 23 Mab MHM 6, PL 13, HD 50, and Tü 1 were found to inhibit the binding of IgE. PL 13 was found to totally inhibit the binding of Mab 25 to RPMI 8866 cells, whereas Tü 1 and MHM 6 only partially inhibited Mab 25 binding. HD 50 was unable to block the binding of Mab 25. The finding that different CD 23/Fc epsilon RL-specific monoclonal antibodies recognizing distinct epitopes have in common the capacity of inhibiting the binding of IgE suggests that upon binding they induce a conformational alteration of the Fc epsilon RL resulting in a loss of the IgE binding capacity. In conclusion, our data demonstrate that the CD 23 antigen is a low affinity receptor for IgE on lymphocytes.