IgE Myeloma Protein Research Articles

Cell-bound IgE and increased expression of Faɛ-receptors on dendritic cells in cutaneous infiltrates of mycosis fungoides

Skin biopsies of 31 non-atopic patients, 20 with mycosis fungoides, six with psoriasis and five with contact dermatitis, and of five non-atopic healthy controls were compared for the presence of cell-bound IgE and vacant IgE binding sites. IgE+ cells were demonstrated in the cutaneous infiltrate of nine (45%) patients with mycosis fungoides, two (33%) with psoriasis and one (20%) with contact dermatitis. Following pre-incubation of skin sections with IgE myeloma protein to saturate vacant IgE-binding sites, 14 out of 16 patients (88%) with stage I mycosis fungoides, five (83%) patients with psoriasis and one (20%) with contact dermatitis showed an increase in the number of IgE+ cells. While cell-bound IgE was positively related to serum IgE levels the expression of IgE-binding sites was not. All IgE+ cells were HLA-DR+ dendritic cells identified as either macrophages (CD68+, CD14+) or Langerhans cells (CD1+). Skin biopsies of non-atopic healthy controls or clinically uninvolved skin in mycosis fungoides had neither any IgE+ cells nor any vacant binding sites. Inhibition studies with IgG1, IgG4 and IgE myeloma proteins as well as with several enzymatic fragments of IgE demonstrated that IgE interacted with Fc epsilon-receptors through isotype-specific structures on the Fc epsilon-fragment. Four anti-CD23 monoclonal antibodies, however, were unable to stain vacant Fc epsilon-receptors nor could they block IgE-binding. We hypothesize that locally-secreted lymphokines, like IL-4 or interferon-gamma, induce Fc epsilon-receptors on dendritic cells in the cutaneous infiltrate and that these receptors become occupied in parallel with elevated serum IgE levels.

Do Lipocalins Play a Role in Mast Cell Physiology?.

Introduction. Aiming to identify molecular properties of allergens responsible for their “intrinsic allergenicity”, we focus on a subset of xenogeneic lipocalins (LCs) that comprise the major mammalian respiratory allergens. These structurally and functionally homologous molecules likely possess conserved molecular motifs promoting IgE-dependent allergy. We hypothesize that such LC “allergenicity” depends on non-IgE interactions of LCs with components of the innate immune system. Herein we describe a possible basis for such interactions between LCs and mast cells.Materials and Methods. Two sources of human mast cells were used; primary cultures derived from peripheral blood CD34+ progenitor cells; or the LAD-2 cell line. Cells were cultured in serum-free medium with recombinant human stem cell factor (SCF; 100 ng/ml). Monoclonal antibodies to human gp330/megalin (MAb E11) were a kind gift of Prof. Lars Rask (Uppsala University, Sweden). Cell surface protein expression was assessed by flow cytometry and gene transcription was measured by real-time PCR.Results. Monoclonal antibodies to an endocytic cell surface receptor (megalin, also known as low-density lipoprotein receptor-related protein (LRP)-2) known to bind multiple LCs stained the mast cell lines. This putative expression of megalin by the mast cells corresponded to their transcription of megalin mRNA as shown as by PCR. Moreover, mast cell megalin gene transcription could be induced (≥ 1000-fold) by overnight culture with monomeric IgE myeloma protein (100 ng/ml), and such induction of the megalin message correlated with both an increase in cell surface expression of the molecule and the specific binding of a purified human LC (a-1 microglobulin).Conclusion. Megalin, an LC-binding cell surface receptor, appears to be constitutively expressed by both progenitor and mature mast cells, and its expression seems to be strongly upregulated by culture with monomeric IgE. This is consistent with a role for direct mast cell-LC interactions in the development of IgE-dependent allergy. In addition, and also of potential clinical relevance, endogenous LCs may play a functional role in normal mast cell physiology.

Isolation and Characterization of a Novel IgD-Binding Protein fromMoraxella catarrhalis

A novel surface protein of the bacterial species Moraxella catarrhalis that displays a high affinity for IgD (MID) was solubilized in Empigen and isolated by ion exchange chromatography and gel filtration. The apparent molecular mass of monomeric MID was estimated to approximately 200 kDa by SDS-PAGE. The mid gene was cloned and expressed in Escherichia coli. The complete mid nucleotide gene sequence was determined, and the deduced amino acid sequence consists of 2123 residues. The sequence of MID has no similarity to other Ig-binding proteins and differs from all previously described outer membrane proteins of M. catarrhalis. MID was found to exhibit unique Ig-binding properties. Thus, in ELISA, dot blots, and Western blots, MID bound two purified IgD myeloma proteins, four IgD myeloma sera, and finally one IgD standard serum. No binding of MID was detected to IgG, IgM, IgA, or IgE myeloma proteins. MID also bound to the surface-expressed B cell receptor IgD, but not to other membrane molecules on human PBLs. This novel Ig-binding reagent promises to be of theoretical and practical interest in immunological research.

N-glycosylation influences epitope expression and receptor binding structures in human IgE

Although human IgE is relatively rich in carbohydrates, there are few studies concerning their structural and functional importance. The low serum concentration of IgE has limited carbohydrate characterisation to a few IgE myeloma proteins. Four to six of the seven potential N-glycosylation sites in the constant region of the epsilon chain seem occupied together with some residual microheterogeneity. We have used a panel of 28 anti-C ϵ2, 7 anti-C ϵ3 and 18 anti-C ϵ4 domain-specific anti-IgE mAbs, and rFc ϵRI α to examine the effect of N-glycosylation on epitope expression of human IgE. Myeloma proteins IgE(DES)- κ, IgE(ND)- λ and IgE(UD)- κ as well as polyclonal IgE were deglycosylated with PNGF and/or sialidase and tested in different ELISA. In all ELISA approaches, the reactivity of most domain-specific anti-IgE mAbs was independent of the glycosylation state of IgE(DES), except for one-third of the anti-C ϵ2 mAbs. These mAbs reacted better with deglycosylated IgE(DES) in the order of treatment PNGF/sialidase>PNGF≥sialidase>buffer control. In sharp contrast, the reactivity of IgE(DES) with rFc ϵRI α was not influenced by sialidase but markedly reduced following PNGF or PNGF/sialidase treatment. These findings were neither myeloma restricted nor caused by aggregation, since monomeric IgE demonstrated the same reactivity pattern. Thus, N-glycosylation seems to influence both structure and function of human IgE. The oligosaccharides modulate epitope expression, mainly in the C ϵ2-domain, as revealed by a subset of mAbs. They also promote subtle changes in the C ϵ3-domain, leading to a reduced Fc ϵRI α binding. These findings suggest physiological implications of carbohydrates in human IgE.

Endocytosis, storage, and release of IgE by human platelets: Differences in patients with type I allergy and nonatopic subjects

Platelets of atopic individuals differ in α-granular contents and in the amount of biologically active mediators released compared with platelets of nonatopic subjects. Because platelets carry the low-affinity IgE receptor (CD23), they may contribute to long-lasting IgE sensitivity by serving as a storage pool for IgE. We compared 45 atopic individuals with immediate-type allergies and 25 nonatopic control subjects with respect to storage and release of IgE by their platelets. Platelets of atopic individuals were characterized by a 10-fold higher median IgE content compared with those of nonatopic control subjects. The platelet IgE content correlated with the serum IgE level in the four atopic individuals with seasonal allergies who were followed up monthly over 1 year. Platelet stimulation with platelet activating factor, but not with thrombin or adenosine diphosphate, resulted in a release of 65% of the stored IgE. Conversely, platelet stimulation with monoclonal IgE/κ resulted in the release of the chemokine RANTES. Platelet α-granules were identified as the main storage compartment for IgE by postembedding immunocytochemistry. Although more than half of the α-granules showed gold labeling for IgE, additional labeling was found on the external face of the plasma membrane and within the open canalicular system, indicating endocytosis and exocytosis of IgE. Moreover, the detection of CD23 not only on the plasma membrane but also on membranes of the α-granules further supports the existence of an exchange of IgE between the blood plasma and an internal storage compartment. Endocytosis could be confirmed by the uptake of an IgE myeloma protein coupled to colloidal gold. We conclude that platelets of atopic individuals may contribute to allergic inflammation by serving as a storage pool for IgE and by their increased capacity to liberate further mediators of allergy in response to IgE stimulation. (J Allergy Clin Immunol 1997;100:235-41.)

Disproportional distribution of isotype and non-isotype-specific IgG subclass anti-IgE autoantibodies in human cord serum.

The levels of naturally occurring IgG and IgG subclass anti-IgE autoantibodies (a-E Ab) were studied in 71 randomly collected cord sera with ELISA using solid-phase IgE-DES myeloma protein. IgG a-E Ab were present in all cord sera, and the range was 300-fold (1.8-540 arbitrary units/ML; median = 11.8). However, this activity is the sum of two major types of a-E Ab that we refer to as isotype-specific (IS) and non-isotype-specific (NIS) because they react with E-chain-specific and myeloma-restricted epitopes, respectively. These two types of a-E Ab were distinguished in IgG subclass a-E Ab inhibition ELISA for all samples using unheated IgE-PS and heated IgE-DES as inhibitors. IS and NIS a-E Ab were found among all four IgG subclasses though with different prevalences. No significant influence of gender was observed. Comparisons within each subclass indicate that NIS a-E Ab were more common than IS a-E Ab for the IgG1 (p < 0.00005), IgG2 (p = 0.07) and IgG3 (p < 0.005) subclasses while the reverse was true for IgG4 (p < 0.00005). In fact, only a minor part of the IgG1 (7%), IgG2 (13%) and IgG3 (9%) a-E Ab activity towards IgE-DES was IS while for IgG4 it was a major part (82%). Attempts to quantify IS and NIS IgG subclass a-E Ab using chimeric IgG subclass antihapten Ab suggested that the pool of IgG a-E Ab against IgE-DES was dominated by NIS a-E Ab particularly of the IgG1 subclass. The 75 percentiles for NIS IgG1, IgG2, IgG3 and IgG4 a-E Ab were 24, < 2, 2.1 and 0.27 ng/ml, respectively, whereas the corresponding figures for IS a-E Ab were 7, < 2, < 2 and 0.90 ng/ml. The findings raise questions on the definition of a-E Ab and suggest that caution should be exercised in the interpretation of any a-E Ab results that are detected with IgE myeloma proteins. The potentially more interesting IS a-E Ab may be overshadowed by the bulk of ubiquitous NIS a-E Ab and background. Consequently, discriminatory assays are necessary if the physiological implication of naturally occurring IS IgG subclass a-E Ab is to be elucidated and these considerations are not limited to studies of cord serum.

Structure and function of IgE myeloma protein VL from an atopic patient.

In a woman suffering from IgE myeloma, hay fever and polyvalent respiratory and skin allergy the IgE monoclonal protein VL was isolated and investigated with respect to structural and functional properties. The amino acid sequence of 22 isolated peptides--especially of the biologically significant C2-C3 part--corresponded with that originally described by Bennich et al. (Immunol Rev 1978;41:3-23; Prog Immunol 1974;13:49-58). However, in mass spectrometry the sugar residues on ASN 99 (219) and 252 (371) were deficient in sialic acids. The native IgE VL protein precipitated with high intensity all mannose-specific lectins as concanavalin A (Con A) and was able to release histamine after triggering by these lectins. The same lectins also elicited more histamine release and more positive skin reactions in atopic than in healthy persons. In sera from atopic patients the binding of IgE on Con A Sepharose 4B column was stronger than in normal persons. It is suggested that changes in the IgE glycosylation state may contribute to IgE-mediated pictures of clinical allergy by the nonimmunological pathway.

Major differences in specificity among naturally occurring human IgG-subclass anti-IgE autoantibodies

Background: The specificity of naturally occurring IgG-subclass anti-IgE autoantibodies (a-IgE Ab) was studied by ELISA inhibition assay with a highly purified IgE-DES myeloma protein as a solid-phase antigen. Because the IgG isotypes differ in effector functions, detailed specificity studies of IgG subclass anti-IgE antibodies might clarify their putative role. Methods: Selected sera containing a high concentration of a single IgG a-IgE Ab subclass were allowed to react with purified immunoglobulins of all five classes including five different IgE myeloma proteins and papain-derived Fabϵ and Fcϵ fragments of IgE-DES. Results: The inhibition results indicated that IgG1, IgG2, and IgG3 a-IgE Ab reacted in a low-affinity reaction with IgE myeloma protein–restricted determinants because only IgE-DES was inhibitory. Moreover, the reacting epitopes were heat resistant (2 hours, 56° C) and localized in the Fabϵ-DES fragment. In sharp contrast, IgG4 a-IgE Ab reacted with high affinity to all five IgE myeloma proteins involving heat-susceptible epitopes confined to the Fcϵ fragment. Conclusions: It seems that a majority of IgG a-IgE Ab have a specificity for nonisotypic myeloma-restricted determinants whereas IgG4 a-IgE Ab were mainly isotype specific. These findings ought to be taken into account in the consideration of physiologic implications of IgG a-IgE Ab and in the interpretation of previous investigations in which intact IgE myeloma proteins have been used to detect IgG a-IgE Ab. (J A LLERGY C LIN I MMUNOL 1995;95:1011-9.)

Demonstration of specific high‐affinity Fcɛ‐receptors on the human basophil‐like leukemia cell line KU812 by flow cytometry

The specificity of IgE binding to a human basophil-like cell line (KU812) was studied by flow cytometry. Four IgE myeloma proteins, representing both light-chain types, one chimeric IgE protein, and polyclonal serum IgE blocked the direct binding of FITC-labeled IgE(DES) myeloma protein to KU812 cells in a dose-dependent and nearly equimolar way. Although not as efficiently as human IgE (from five to eight times less on a molar basis), both rat and mouse IgE blocked IgE(DES)-FITC binding to KU812 cells. In sharp contrast, all four human IgG subclasses, both IgA subclasses, and IgM myeloma proteins, as well as monomeric and heat-aggregated polyclonal human IgG, were unable to block significantly IgE(DES)-FITC binding to KU812 cells (< 0.5% on a molar basis). The cytophilic epitope on IgE was heat-susceptible (56 degrees C, 2 h), lost after reduction alkylation, and resident in the papain-derived Fc epsilon-fragment, but not in the papain-derived Fab epsilon- and Fc'epsilon-fragments nor in the pepsin-derived F(ab')2 epsilon- and Fc"epsilon-fragments. Washing and displacement experiments indicated that a major part of IgE reacted with high affinity to KU812 cells. The results indicate that the binding of IgE to KU812 cells is highly specific and involves the classical high-affinity Fc epsilon RI-receptor. Although the density of receptors is low, this human cell line offers a unique model to study IgE/Fc epsilon RI interactions.

Naturally occurring human IgA autoantibodies against IgE‐DES myeloma protein

The prevalence and specificity of naturally occurring human IgA anti-IgE autoantibodies (a-E Ab) were studied by ELISA with anti-IgA monoclonal antibodies (mAb) and a purified myeloma IgE as solid-phase protein, i.e., IgE-DES(kappa). Such detected IgA a-E Ab were common among adults, and significantly increased geometric means (GM) were found in patients with atopy (P = 0.006; n = 41; GM = 79.3 arbitrary units (AU)/ml) and filariasis (P = 0.02; n = 41; GM = 75.9 AU/ml), as compared with nonatopic controls (n = 42; GM = 48.8 AU/ml). No such difference was observed between age-matched nonatopic (n = 22; GM = 36.7 AU/ml) and atopic (n = 22; GM = 38.6 AU/ml) children. Children had significantly (P < 0.001) lower IgA a-E Ab concentrations than adults, probably as a result of age, because IgA a-E Ab concentrations and age of children were significantly correlated (n = 44; P < 0.05; r = 0.30). IgA a-E Ab concentrations were very low in cord serum (n = 32; median < 0.1 AU/ml). Sex did not influence IgA a-E Ab concentrations in any study group. The specificity of IgA a-E Ab in nine sera was studied by ELISA inhibition assay using IgE-DES myeloma as solid-phase protein and inhibitory proteins of the IgG, IgM, IgD, and IgE classes, including five different IgE myeloma proteins, as well as three enzymatic fragments of IgE-DES. The inhibitions indicated that all IgA a-E Ab tested reacted in a low-affinity reaction with determinants restricted to IgE-DES, i.e., the solid-phase protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet aggregation and histamine release by immunological stimuli

Platelet aggregation and histamine release were evaluated in normal and IgE pretreated human platelets exposed in vitro to IgE, anti-IgE and thrombin. The response of platelets from atopic donors directly stimulated with anti-IgE was also evaluated. Histamine release was measured by fluorimetric analysis of histamine content in platelets and in supernatants. The morphology of platelets exposed to immunological and non-immunological stimuli was recorded using an electron microscope. A detectable amount of histamine was measured in quiescent platelets. Their exposure to varying concentrations of thrombin produces a progressive aggregation which runs parallel to histamine release. The effects were significantly enhanced in platelets pretreated with IgE. Incubation of normal platelets with increasing concentrations of IgE myeloma protein, or with anti-human IgE antibody was ineffective on both aggregation and histamine release. However, incubation of platelets passively sensitized with IgE-myeloma protein with different concentrations of anti-human IgE antibody produces a concentration-dependent increase both in aggregation and histamine release. The same effects were obtained using platelets from atopic donors directly stimulated with anti-IgE. The electron microscopic pattern of platelet aggregation induced by thrombin was indistinguishable from that evoked by anti-IgE in IgE pretreated platelets. Loratadine, a non-sedative H1-receptor blocker, significantly abated platelet aggregation and histamine release induced by anti-IgE in IgE pretreated platelets.

Effect of loratadine, an anti-histamine drug, on cytosolic calcium elevation in rat peritoneal macrophages

Platelet aggregation and histamine release were evaluated in normal and IgE pretreated human platelets exposed in vitro to IgE, anti-IgE and thrombin. The response of platelets from atopic donors directly stimulated with anti-IgE was also evaluated. Histamine release was measured by fluorimetric analysis of histamine content in platelets and in supernatants. The morphology of platelets exposed to immunological and non-immunological stimuli was recorded using an electron microscope. A detectable amount of histamine was measured in quiescent platelets. Their exposure to varying concentrations of thrombin produces a progressive aggregation which runs parallel to histamine release. The effects were significantly enhanced in platelets pretreated with IgE. Incubation of normal platelets with increasing concentrations of IgE myeloma protein, or with anti-human IgE antibody was ineffective on both aggregation and histamine release. However, incubation of platelets passively sensitized with IgE-myeloma protein with different concentrations of anti-human IgE antibody produces a concentration-dependent increase both in aggregation and histamine release. The same effects were obtained using platelets from atopic donors directly stimulated with anti-IgE. The electron microscopic pattern of platelet aggregation induced by thrombin was indistinguishable from that evoked by anti-IgE in IgE pretreated platelets. Loratadine, a non-sedative H1-receptor blocker, significantly abated platelet aggregation and histamine release induced by anti-IgE in IgE pretreated platelets.

The effect of soft mineral water on urinary stone risk factors

Platelet aggregation and histamine release were evaluated in normal and IgE pretreated human platelets exposed in vitro to IgE, anti-IgE and thrombin. The response of platelets from atopic donors directly stimulated with anti-IgE was also evaluated. Histamine release was measured by fluorimetric analysis of histamine content in platelets and in supernatants. The morphology of platelets exposed to immunological and non-immunological stimuli was recorded using an electron microscope. A detectable amount of histamine was measured in quiescent platelets. Their exposure to varying concentrations of thrombin produces a progressive aggregation which runs parallel to histamine release. The effects were significantly enhanced in platelets pretreated with IgE. Incubation of normal platelets with increasing concentrations of IgE myeloma protein, or with anti-human IgE antibody was ineffective on both aggregation and histamine release. However, incubation of platelets passively sensitized with IgE-myeloma protein with different concentrations of anti-human IgE antibody produces a concentration-dependent increase both in aggregation and histamine release. The same effects were obtained using platelets from atopic donors directly stimulated with anti-IgE. The electron microscopic pattern of platelet aggregation induced by thrombin was indistinguishable from that evoked by anti-IgE in IgE pretreated platelets. Loratadine, a non-sedative H1-receptor blocker, significantly abated platelet aggregation and histamine release induced by anti-IgE in IgE pretreated platelets.

Anti-human IgG causes basophil histamine release by acting on IgG-IgE complexes bound to IgE receptors.

We have reexamined the ability of anti-human IgG antibodies to induce histamine release from human basophils. A panel of purified murine mAbs with International Union of Immunological Societies-documented specificity for each of the four subclasses of human IgG was used. Of the 24 allergic subjects studied, the basophils of 75% (18/24) released greater than 10% histamine to one or more anti-IgG1-4 mAb, whereas none of the 13 nonatopic donor's basophils released histamine after stimulation with optimal amounts of anti-IgG mAb. The basophils of 85% (11/13) of the nonatopic donors did respond to anti-IgE challenge, as did 92% (22/24) of the atopic donor cells. Histamine release was induced most frequently by anti-IgG3, and 10/18 anti-IgG responder cells released histamine with mAb specific for two or more different subclass specificities. The rank order for induction of histamine release was anti-IgG3 greater than anti-IgG2 greater than IgG1 greater than anti-IgG4. As in our previous study using polyclonal anti-IgG, 100- to 300-micrograms/ml quantities of the anti-IgG mAb were required for maximal histamine release, about 1000-fold higher than those for comparable release with anti-human IgE. Specificity studies using both immunoassays and inhibition studies with IgE myeloma protein indicated that anti-IgG induced histamine release was not caused by cross-reactivity with IgE. Ig receptors were opened by lactic acid treatment so that the cells could be passively sensitized. Neither IgE myeloma nor IgG myeloma (up to 15 mg/ml) proteins could restore the response to anti-IgG mAb. However, sera from individuals with leukocytes that released histamine upon challenge with anti-IgG mAb could passively sensitize acid-treated leukocytes from both anti-IgG responder and nonresponder donors for an anti-IgG response. The only anti-IgG mAb that induced release from these passively sensitized cells were those to which the serum donor was responsive. Sera from non-IgG responders could not restore an anti-IgG response. These data led to the hypothesis that the IgG specific mAb were binding to IgG-IgE complexes that were attached to the basophil through IgE bound to the IgE receptor. This was shown to be correct because passive sensitization to anti-IgG could be blocked by previous exposure of the basophils to IgE. We conclude that anti-IgG-induced release occurs as a result of binding to IgG anti-IgE antibodies and cross-linking of the IgE receptors on basophils.

Monoclonal antibodies defining epitopes on human IgE

Twelve monoclonal antibodies (mAb) were isolated that bound to six clusters of epitopes on the constant region of the epsilon chain of human IgE. Four of the mAb bound to the C ϵ 1 or early C ϵ 2 regions; three of these bound to the IgE myeloma protein PS and to serum IgE but not to the IgE myeloma protein ND. These mAb probably recognize an allotyplc marker. Another mAb reacted with heatdenatured, but not native IgE. Four of the mAb failed to release histamine; the epitopes recognized by these mAb are in the C ϵ1, C ϵ 2 and C ϵ3–4 regions of IgE. Three of these non-histamine releasing mAb did not bind to IgE on the basophil surface. These mAb recognize epitopes in C ϵ2 and C ϵ3–4 that are not accessible when IgE is bound to its receptor. Four mAb inhibited IgE binding to basophils; two of these did not release histamine, and two others that bind to epitopes in the C ϵ2–4 domain, released histamine and therefore blocked IgE binding by steric hindrance. Inhibition of IgE binding by different mAb suggest that the Fc ϵRI and Fc ϵRII bind to partly overlapping regions of the IgE molecule although the sites do not appear to be identical. A number of sites on C ϵ 1 and C ϵ3–4 were accessible when IgE is bound to its basophil receptor. The data support the concept that only part of the Fc portion of IgE is hidden in the receptor and that portions of C ϵ 1–4 are accessible on the cell surface. These mAb should be useful in determining the domains of IgE that are critical for its biological activity.

Responsiveness to human mononuclear cell-derived histamine-releasing factor. Studies of allergic status and the role of IgE.

Human mononuclear cell-derived histamine-releasing factor (MNC-HRF) has m.w. of 29,000 and 23,000 and two charge species isoelectric at pH 6.9 and 7.3. Minor forms of HRF are also seen at m.w. of 12,000 to 15,000 and 60,000 to 80,000. In this manuscript we have further defined the response of human basophils to MNC-HRF in terms of atopic status of cell donors and the role of IgE antibody. We found that basophils of allergic persons, nonallergic controls, or donors who have positive skin tests but no symptoms, cannot be distinguished based upon responsiveness to HRF. Further, the response of basophils that have been desensitized to anti-IgE is retained when they are subsequently stimulated with HRF derived from human MNC. Thus an interaction with cell-surface IgE to cause histamine release is not evident. Nevertheless, one of two IgE myeloma proteins tested were capable of binding HRF and such binding could be utilized as a purification step. We conclude that MNC-HRF is heterogeneous in terms of size, charge, and affinity for a subpopulation of IgE. Histamine release is more prominent in atopic individuals but does not appear to require an interaction with IgE.

Leukotriene C 4 generation from human eosinophils stimulated with IgG- Aspergillus fumigatus antigen immune complexes

Sepharose beads coated with IgG stimulate eosinophils to produce leukotriene C 4 (LTC 4). This observation has been extended with specific immobilized IgG/antigen immune complexes to elicit mediator generation. An extract of Aspergillus fumigatus was covalently coupled to Sepharose beads and incubated with the IgG fraction of immune serum from patients with allergic bronchopulmonary aspergillosis. These beads elicited generation of 7.72 ± 1.7 pmol of LTC 4 immunoreactive material (n = 5) from 1 × 10 6 normal eosinophils of >86% purity, and significantly less LTC 4 (0.73 ± 0.19 pmol per 10 6 cells; n = 3) was produced by eosinophils after incubation with beads treated with IgG from normal nonimmune serum. The maximum antibody-dependent release achieved represented approximately 20% of that induced by the calcium ionophore (A23187). LTC 4 was measured by radioimmunoassay and validated by reverse-phase high-performance liquid chromatography. The amount of LTC 4 generated was dependent on the concentration of A. fumigatus-specific IgG, and mediator release was completely abolished by prior adsorption of the IgG fraction onto Sepharose-protein A ( Staphylococcus aureus). Grass pollen-specific IgG antibody/antigen complexes, in combination with Sepharose beads, also triggered generation of LTC 4 immunoreactive material. There was no evidence to suggest that IgE/ A. fumigatus immune complexes triggered LTC 4 generation, although IgE myeloma protein, in association with Sepharose beads, was a weak stimulus. The efficacy of the IgG immune complex-dependent stimulation of eosinophils suggests a possible physiologic mechanism whereby these cells could participate in the inflammatory changes associated with allergic bronchopulmonary aspergillosis and similar allergic disorders.

IgE pyroglobulinemia in multiple myeloma

A new case of IgE/K myeloma in a 38-year-old male is described. The clinical course, bone marrow picture and serum electrophoretic pattern were typical of multiple myeloma. Heating of the serum at 56°C induced an irreversible gel formation or pyroglobulin that was isolated and proved to be identical to the IgE paraprotein, by biochemical and immunochemical methods. The thermoprecipitability was abolished by treatment with urea, sodium dodecyl sulphate and neutral salts and not inhibited by sugars, alcohols, 2-β-mercaptoethanol and guanidine HC1. The association of an IgE myeloma protein with a pyroglobulin is a finding not previously reported in a myeloma of this class.

Quantitative and qualitative analysis of the Fc receptor for IgE (Fc epsilon RII) on human eosinophils.

In order to characterize the Fc receptor for IgE (Fc epsilon RII) on human eosinophils, we have compared the binding of human IgE myeloma protein to that of a monoclonal antibody (mAb BB10) directed against a common antigenic determinant of the Fc epsilon RII present on eosinophils, platelets and macrophages. Scatchard analysis of the binding to human eosinophils of the BB10 mAb revealed a linear monophasic binding curve, with a binding affinity of 1.17 x 10(7) M-1 and a number of 10(5) binding sites per cell. Biochemical analysis of the human eosinophil Fc epsilon R, performed by immunosorbent chromatography with either BB10 mAb or IgE, showed under nonreducing conditions a major component of 200 kDa. Under reducing conditions, 3 peptide fragments were obtained, with molecular masses of 45-50, 23 and 15 kDa. Finally, comparative analysis suggested that the Fc epsilon RII of human eosinophils and of a human macrophage cell line (U937) are structurally related and differ from the high-affinity Fc epsilon RI present on basophilic granulocytes.

IgE-immunotoxins. II. IgE-ricin A-chain

In order to develop a reagent capable of killing cells with high-affinity IgE Fc receptors, such as mast cells and basophils, ricin A-chain (the toxic portion of ricin) was conjugated to rat IgE myeloma protein, IR 162, via derivatization of the IgE by n- -succinimidyl-3-(2-pyridyldithio)propionate (SPDP) thus creating an IgE-immunotoxin. Monensin (10−7−10−8 M), a carboxylic ionophore, facilitated IgE-ricin A-chain (3 × 10−7 M) toxicity in a dose-related fashion with significant reductions in [3H]leucine incorporation compared to cells exposed only to monensin. This enhanced toxicity could be inhibited by the addition of both anti-ricin A-chain or anti-IgE, suggesting that different routes of intracellular processing may play a role in determining the toxicity of the IgE-ricin A-chain conjugate. Ricin B-chain (5 × 10−7 and 5 × 10−8 M) added to free ricin A-chain (10−6−10−8 M) reproducibly facilitated toxicity, and this toxicity could be inhibited (30–90%) by lactose (50 mM). Ricin B-chain also facilitated IgE-ricin A-chain (2.75 × 10−8 M) toxicity; however, this toxicity was not affected by lactose. The data suggest that ricin B-chain potentiates the cytosolic access of internalized IgE-immunotoxin and that the binding and internalization of the toxin was mediated via the IgE Fc receptor. A second type of IgE-ricin A-chain conjugate was synthesized whereby both IgE and ricin A-chain were derivatized with SPDP. RBL cells were killed in a dose-dependent manner by this IgE-ricin A-chain conjugate (2.5 × 10−6−2.5 × 10−9 M) without requiring the addition of monensin or ricin B-chain. These data indicate that the intracellular route and processing of internalized immunotoxin is critical to eliciting toxicity.