Work-related asthma: diagnosis and prognosis of immunological occupational asthma and work-exacerbated asthma

The incidence and prevalence of asthma are increasing. One reason for this trend is the rise in adult-onset asthma, especially occupational asthma, which is 1 of the 2 forms of work-related asthma. Occupational asthma is defined as asthma caused by agents that are present exclusively in the workplace. The presence of pre-existing asthma does not rule out the possibility of developing occupational asthma. A distinction has traditionally been made between immunological occupational asthma (whether IgE-mediated or not) and nonimmunological occupational asthma caused by irritants, the most characteristic example of which is reactive airway dysfunction syndrome. The other form of work-related asthma is known as work-exacerbated asthma, which affects persons with pre-existing or concurrent asthma that is worsened by work-related factors. It is important to differentiate between the 2 entities because their treatment, prognosis, and medical and social repercussions can differ widely. In this review, we discuss diagnostic methods, treatment, and avoidance/nonavoidance of the antigen in immunological occupational asthma and work-exacerbated asthma (AU)

La incidencia y prevalencia del asma van en aumento. El asma de inicio en la edad adulta y especialmente el asma ocupacional (AO) podrían ser una de las causas que influyeran en este incremento. El AO, una de las dos formas de asma relacionada con el trabajo (ART), se define como el asma causada por agentes que están presentes exclusivamente en el lugar de trabajo. Clásicamente, se ha realizado una distinción entre AO inmunológica (mediada o no por un mecanismo IgE) y AO no inmunológica causada por irritantes, cuyo ejemplo más característico es el síndrome reactivo de disfunción de la vía aérea. La presencia de asma previa no descarta la posibilidad de desarrollar AO. El asma exacerbada por el trabajo (AET) es la otra forma de ART y se define como aquel asma pre-existente o concurrente que empeora por factores relacionados con el trabajo. Diferenciar estas dos entidades es importante ya que su tratamiento, pronóstico y repercusiones médica y social, pueden diferir ampliamente. En esta revisión se discuten los diversos métodos diagnósticos, tratamientos y las diferentes estrategias de evitación / no evitación del antígeno tanto en el AO inmunológica como en el AET (AU)

Serum IgE and eosinophil count in allergic rhinitis-Analysis using a modified Bayes’ theorem

Background: To use probability theory to establish threshold values for total serum IgE and eosinophil counts that support a diagnosis of allergic rhinitis and to compare our results with previously published data. Methods: Prospective study of rhinitis patients using a modified version of Bayes’ theorem. Study included 125 patients at the West Los Angeles VA Medical Center diagnosed with rhinitis who completed allergy consultation and immediate hypersensitivity skin testing. Results: Eighty-nine of 125 patients were atopic by prick and/or intradermal skin testing. Using a modified version of Bayes’ theorem and positive and negative probability weights, calculations for different thresholds of serum IgE and eosinophil counts were summated and a posttest probability for atopy was calculated. Calculated posttest probabilities varied according to the threshold used to determine a positive or negative test; however, IgE thresholds greater than 140IU/ml and eosinophil counts greater that 80cells/ml were found to have a high probability of predicting atopy in patients with rhinitis. Moreover, IgE had a greater influence than eosinophil count in determining posttest probability of allergy in this population. Considerable differences were noted in the IgE levels of atopic and non-atopic patients, including those with asthma or a history of smoking. However, these differences were not observed with eosinophil levels. Conclusions: Using a modified version of Bayes’ theorem to determine posttest probability, IgE threshold levels greater than 140IU/ml and eosinophil counts greater than 80cells/ml in an individual with clinical signs and symptoms of rhinitis are likely to correlate with an atopic aetiology. This model of probability may be helpful in evaluating individuals for diagnostic skin testing and certain types of allergy-modifying treatment(AU)

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Five mouse homologues of the human dendritic cell C-type lectin, DC-SIGN.

DC-SIGN, a human C-type lectin, is expressed on the surface of dendritic cells (DC), while a closely related human gene, DC-SIGNR or L-SIGN, is found on sinusoidal endothelial cells of liver and lymph node. Both DC-SIGN and DC-SIGNR/L-SIGN can bind ICAM-3 and HIV gp120, and transmit HIV to susceptible cells in trans. Here, we report the cloning of five mouse genes homologous to human DC-SIGN and DC-SIGNR/L-SIGN. Only one gene, named mouse DC-SIGN, is highly expressed in DC, and is not found in a panel of mouse macrophage and lymphocyte cell lines. The other four genes, named mouse SIGNR1 (SIGN-Related gene 1), SIGNR2, SIGNR3 and SIGNR4, are expressed at lower levels in various cells according to RT-PCR and Northern blot analyses on RNA. All the genes of mouse DC-SIGN and SIGNRs map to adjacent regions of chromosome 8 A1.2-1.3. However, like human DC-SIGN, only the mouse DC-SIGN gene is closely juxtaposed to the CD23 gene, while the other four SIGNR genes are located close to each other in a neighboring region. mRNAs of mouse DC-SIGN and three SIGNR genes encode type II transmembrane proteins (DC-SIGN, 238 amino acids; SIGNR1, 325 amino acids; SIGNR3, 237 amino acids; SIGNR4, 208 amino acids), but the SIGNR2 gene only encodes a carbohydrate recognition domain (CRD) without a cytosolic domain and a transmembrane domain (SIGNR2, 178 amino acids). Amino acid sequence similarities between the CRD of human DC-SIGN and the mouse homologues are 67% for DC-SIGN, 69% for SIGNR1, 65% for SIGNR2, 68% for SIGNR3 and 70% for SIGNR4 respectively. However, the membrane proximal neck domains in the mouse genes are much shorter than their counterparts in human DC-SIGN and DC-SIGNR/L-SIGN. This family of mouse C-type lectins is therefore complex, but only one of the new genes, DC-SIGN, is juxtaposed to CD23 and is expressed at high levels in DC.

Expression and purification of stable 33-kDa soluble human CD23 using the Drosophila S2 expression system.

CD23, a 45-kDa type II membrane glycoprotein present on B cells, monocytes, and other human immune cells, is a low-affinity receptor for IgE. The extracellular region of the membrane-bound human CD23 is processed into at least four soluble (s) CD23 forms, with apparent molecular masses of 37, 33, 29, and 25 kDa. High levels of sCD23 are found in patients with allergy, certain autoimmune diseases, or chronic lymphocytic leukemia. Therefore, inhibition of the processing of membrane-bound CD23 to control the cytokine-like effects of sCD23 offers a novel therapeutic opportunity. While the 37-, 29-, and 25-kDa forms of sCD23 have been expressed previously as recombinant proteins, the 33-kDa form has not been purified and characterized. To further investigate the multiple roles of sCD23 fragments and to devise assays to identify potent small-molecule inhibitors of CD23 processing, we have produced the 33-kDa form of sCD23 using Chinese hamster ovary (CHO) and Drosophila S2 cells. The CHO-expressed 33-kDa protein was found to undergo proteolytic degradation during cell growth and during storage of purified protein, resulting in accumulation of a 25-kDa form. The Drosophila system expressed the 33-kDa sCD23 in a stable form that was purified and demonstrated to be more active than the CHO-derived 25-kDa form in a monocyte TNFalpha release assay.

Intracellular expression and release of Fc epsilon RI alpha by human eosinophils.

Although Fc epsilon R have been detected on human eosinophils, levels varied from moderate to extremely low or undetectable depending on the donor and methods used. We have attempted to resolve the conflicting data by measuring levels of IgE, Fc epsilon RI, and Fc epsilon RII in or on human eosinophils from a variety of donors (n = 26) and late-phase bronchoalveolar lavage fluids (n = 5). Our results demonstrated little or no cell surface IgE or IgE receptors as analyzed by immunofluorescence and flow cytometry. Culture of eosinophils for up to 11 days in the presence or absence of IgE and/or IL-4 (conditions that enhance Fc epsilon R on other cells) failed to induce any detectable surface Fc epsilon R. However, immunoprecipitation and Western blot analysis of eosinophil lysates using mAb specific for Fc epsilon RI alpha showed a distinct band of approximately 50 kDa, similar to that found in basophils. Western blotting also showed the presence of FcR gamma-chain, but no Fc epsilon RI beta. Surface biotinylation followed by immunoprecipitation again failed to detect surface Fc epsilon RI alpha, although surface FcR gamma was easily detected. Since we were able to detect intracellular Fc epsilon RI alpha, we examined its release from eosinophils. Immunoprecipitation and Western blotting demonstrated the release of Fc epsilon RI alpha into the supernatant of cultured eosinophils, peaking at approximately 48 h. We conclude that eosinophils possess a sizable intracellular pool of Fc epsilon RI alpha that is available for release, with undetectable surface levels in a variety of subjects, including those with eosinophilia and elevated serum IgE. The biological relevance of this soluble form of Fc epsilon RI alpha remains to be determined.

Crystal structure of the human high-affinity IgE receptor.

Allergic responses result from the activation of mast cells by the human high-affinity IgE receptor. IgE-mediated allergic reactions may develop to a variety of environmental compounds, but the initiation of a response requires the binding of IgE to its high-affinity receptor. We have solved the X-ray crystal structure of the antibody-binding domains of the human IgE receptor at 2.4 A resolution. The structure reveals a highly bent arrangement of immunoglobulin domains that form an extended convex surface of interaction with IgE. A prominent loop that confers specificity for IgE molecules extends from the receptor surface near an unusual arrangement of four exposed tryptophans. The crystal structure of the IgE receptor provides a foundation for the development of new therapeutic approaches to allergy treatment.

A conformational rearrangement upon binding of IgE to its high affinity receptor.

One of the critical steps in the allergic reaction is the binding of immunoglobulin E (IgE) to its high affinity receptor (FcepsilonRI). FcepsilonRI is a tetrameric complex composed of an alpha-chain, a beta-chain, and a dimeric gamma-chain. The extracellular portion of the alpha-chain (alpha-t) is sufficient for the binding of IgE. The Fc portion of IgE contains two copies of the FcepsilonRI binding sites. In contrast, the binding stoichiometry is 1:1. Previously, it was hypothesized that the binding of FcepsilonRI to IgE results in a conformational change in IgE that precludes the binding of a second molecule (Presta, L., Shields, R., O'Connel, L., Lahr, S., Porter, J. , Gorman, C., and Jardieu, P.(1994) J. Biol. Chem. 269, 26368-26373). Here we characterize the secondary structure of IgE and alpha-t and analyze their interaction by circular dichroism spectroscopy. Binding experiments show that when IgE interacts with alpha-t there is a 15-26% decrease of the negative ellipticity at 217 nm. Together, the absence of an alpha-helix element in alpha-t and the small contribution of alpha-t to the spectra of the complex indicate that upon binding, a major conformational rearrangement must occur on IgE. In addition, we analyze the thermal unfolding of alpha-t, IgE, and their complex. Despite the several domains that constitute IgE and alpha-t, these molecules unfold cooperatively with two-state kinetics.

Persistence of tyrosine-phosphorylated FcepsilonRI in deactivated cells.

Engagement of the high affinity IgE receptor (FcepsilonRI) with a multimeric antigen leads to immediate tyrosine phosphorylation of its beta and gamma subunits, recruitment, and activation of the tyrosine kinase Syk, and later to cell degranulation. Monovalent hapten treatment reverses these events, resulting in receptor dephosphorylation and an abrupt arrest of cell degranulation. Thus far, it has been assumed that there is a direct linkage between receptor tyrosine phosphorylation, Syk activation and phosphorylation, and cell degranulation. However, we show here that when FcepsilonRI receptors are cross-linked for extended periods of time, hapten-mediated receptor dephosphorylation is delayed. These receptors, which remain tyrosine-phosphorylated despite the addition of hapten, are progressively targeted to a Triton X-100-insoluble fraction, suggesting their progressive association with the membrane skeleton. In contrast to FcepsilonRI receptors, hapten-induced Syk dephosphorylation and the consequent arrest of degranulation are not affected by prolonged cross-linking. Thus, some tyrosine-phosphorylated receptors persist in deactivated cells. We propose that, with time, some tyrosine-phosphorylated receptors become unaccessible to phosphatases and, in addition, unable to activate Syk. This inactive status of tyrosine-phosphorylated FcepsilonRI may be the result of membrane skeleton compartmentalization. However, another population of clustered receptors that includes the ones most recently formed is still immediately sensitive to hapten deactivation. This latter population is critical in maintaining Syk activity and cell degranulation. The shift from a transiently active state of phosphorylated receptors toward an inactive state could be a general mechanism of desensitization also utilized by other antigen receptors.

Recombinant soluble form of the human high-affinity receptor for IgE prevents anaphylactic shock in mice.

The ectodomain of the alpha subunit of the human high-affinity receptor for IgE (Fc epsilon RI) has been engineered as a recombinant soluble protein (sFc epsilon RI alpha) and purified in large quantity from the supernatant of transfected mammalian cells. The sFc epsilon RI alpha was apparently heterogeneous in terms of molecular weight (40 to 60 kd), and this heterogeneity was largely due to N-linked carbohydrates on the molecule. The amino terminal sequence was homogeneous and identical to the sequence predicted from the complementary DNA sequence. The amino acid composition of the sFc epsilon RI alpha was in agreement with the values expected from the cDNA sequence. The sFc epsilon RI alpha formed a complex with IgE, and gel filtration analyses of the complex of the sFc epsilon RI alpha and human IgE supported the idea that the stoichiometry of the IgE binding was 1:1. Passive anaphylactic shock in mice was suppressed when the sFc epsilon RI alpha was intravenously injected after sensitization with anti-trinitrophenyl IgE and challenged with the appropriate antigen. In this model the sFc epsilon RI alpha was effective when administered 48 hours before the antigen challenge, but not 24 hours before the challenge. These results suggest that the sFc epsilon RI alpha facilitated the dissociation of IgE from Fc epsilon RI on mast cells in vivo and suppressed type I allergic reactions.

Tyrosine phosphorylation of protein kinase C-delta in response to the activation of the high-affinity receptor for immunoglobulin E modifies its substrate recognition.

The delta isoform of protein kinase C is phosphorylated on tyrosine in response to antigen activation of the high-affinity receptor for immunoglobulin E. While protein kinase C-delta associates with and phosphorylates this receptor, immunoprecipitation of the receptor revealed that little, if any, tyrosine-phosphorylated protein kinase C-delta is receptor associated. In vitro kinase assays with immunoprecipitated tyrosine-phosphorylated protein kinase C-delta showed that the modified enzyme had diminished activity toward the receptor gamma-chain peptide as a substrate but not toward histones or myelin basic protein peptide. We propose a model in which the tyrosine phosphorylation of protein kinase C-delta regulates the kinase specificity toward a given substrate. This may represent a general mechanism by which in vivo protein kinase activities are regulated in response to external stimuli.

Fc epsilon RI-mediated recruitment of p53/56lyn to detergent-resistant membrane domains accompanies cellular signaling.

Detergent-resistant plasma membrane structures, such as caveolae, have been implicated in signalling, transport, and vesicle trafficking functions. Using sucrose gradient ultracentrifugation, we have isolated low-density, Triton X-100-insoluble membrane domains from RBL-2H3 mucosal mast cells that contain several markers common to caveolae, including a src-family tyrosine kinase, p53/56lyn. Aggregation of Fc epsilon RI, the high-affinity IgE receptor, causes a significant increase in the amount of p53/56lyn associated with these low-density membrane domains. Under our standard conditions for lysis, IgE-Fc epsilon RI fractionates with the majority of the solubilized proteins, whereas aggregated receptor complexes are found at a higher density in the gradient. Stimulated translocation of p53/56lyn is accompanied by increased tyrosine phosphorylation of several proteins in the low-density membrane domains as well as enhanced in vitro tyrosine kinase activity toward these proteins and an exogenous substrate. With a lower detergent-to-cell ratio during lysis, significant Fc epsilon RI remains associated with these membrane domains, consistent with the ability to coimmunoprecipitate tyrosine kinase activity with Fc epsilon RI under similar lysis conditions [Pribluda, V. S., Pribluda, C. & Metzger, H. (1994) Proc. Natl. Acad. Sci. USA 91, 11246-11250]. These results indicate that specialized membrane domains may be directly involved in the coupling of receptor aggregation to the activation of signaling events.

Soluble form of the human high-affinity receptor for IgE inhibits recurrent allergic reaction in a novel mouse model of type I allergy.

The recombinant soluble form of the human high-affinity receptor for IgE alpha subunit (sFc epsilon RI alpha) is able to block the binding of IgE to the cell surface Fc epsilon RI and prevent the activation of mast cells and basophils. To evaluate its anti-allergic effects in vivo, we established a novel model for type I allergy by transplanting antigen-specific IgE-secreting hybridomas into syngeneic mice. The hybridomas continuously produced anti-2,4,6-trinitrophenyl IgE in vivo, and ear swelling responses could be elicited upon applying picryl chloride to the ears of these mice, which reached their maximum 1-2 h after the antigen challenge. The second swelling response, in extent comparable to the first response, was induced by the second antigen challenge several days later. When the sFC epsilon RI alpha was intravenously administered before the first challenge and periodically between the first and the second challenges, the ear swelling response to the second challenge, but not to the first challenge, was suppressed almost completely. Immunohistochemical examination revealed that treatment with the sFc epsilon RI alpha blocked the binding of IgE to the cell surface IgE receptors after the first challenge. Thus, our results indicate that the sFc epsilon RI alpha suppresses recurrent allergic reactions by preventing IgE binding to the cell surface Fc epsilon RI.

Hydrodynamic studies of a complex between the Fc fragment of human IgE and a soluble fragment of the Fc epsilon RI alpha chain.

The interaction between immunoglobulin E (IgE) and its high-affinity receptor Fc epsilon RI is central to allergic disease. The binding site for Fc epsilon RI lies in the third constant region domain of the epsilon heavy chain of IgE (C epsilon 3). Identical epitopes on the two C epsilon 3 domains in the IgE-Fc are predicted to be on opposite sides of the structure, and therefore each could bind independently to a receptor molecule. Titrations, however, reveal that the IgE-Fc forms an equimolar complex with a soluble fragment of the Fc epsilon RI alpha chain (sFc epsilon RI alpha), and the molecular weight of the complex, as determined by sedimentation equilibrium, confirms this stoichiometry. The measured sedimentation coefficients of the two ligands are in good agreement with computed values for a compact IgE-Fc and an elongated sFc epsilon RI alpha structure. The calculated sedimentation coefficients for possible models of a 1:1 complex lead to exclusion of all highly extended geometries for the complex. Possible explanations for the paradoxical stoichiometry of the IgE-Fc/sFc epsilon RI alpha complex, in terms of the curved shape of IgE, a conformational change in IgE when the receptor binds, and steric interference between two molecules of Fc epsilon RI binding to identical sites, are discussed.

Activation of protein tyrosine kinase p72syk by Fc epsilon RI aggregation in rat basophilic leukemia cells. p72syk is a minor component but the major protein tyrosine kinase of pp72.

Aggregation of the high affinity IgE receptors (Fc epsilon RI) on rat basophilic leukemia RBL-2H3 cells results in protein tyrosine phosphorylations. Previously we reported that there is prominent tyrosine phosphorylation of approximately 72-kDa proteins (pp72) and that the tyrosine kinase p72syk is one component of pp72. Here we studied further the relationship of p72syk to pp72. The aggregation of Fc epsilon RI induced the activation of p72syk which was parallel to its tyrosine phosphorylation. By in vitro kinase assay of immune complexes purified with anti-phosphotyrosine antibodies, p72syk was the major pp72 tyrosine kinase. However, by immunoblotting with anti-phosphotyrosine antibodies, p72syk was a minor component of pp72. The heterogeneous nature of pp72 was indicated by different studies. Under optimum conditions of one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, pp72 consisted of a heterogeneous group of 69-, 71-, and 72-kDa tyrosine-phosphorylated proteins. There were differences in the tyrosine phosphorylation of these proteins in cells activated in the absence of extracellular calcium or when stimulation was with the calcium ionophore A23187 or with phorbol myristate acetate. One of the proteins migrating at 69 kDa was p72syk. By two-dimensional gel electrophoresis pp72 was found to consist of multiple tyrosine-phosphorylated protens including 71-80-kDa proteins that associate with p53/56lyn. A 75-kDa tyrosine-phosphorylated protein, different from pp72, was identified as p75HS1 (SPY75). These results demonstrate the heterogeneous nature of the pp72 and that p72syk is activated after Fc epsilon RI aggregation.

High-level expression of the truncated alpha chain of human high-affinity receptor for IgE as a soluble form by baculovirus-infected insect cells. Biochemical characterization of the recombinant product.

The binding subunit of human high-affinity receptor for IgE (Fc epsilon RI alpha) was efficiently expressed as a truncated form in insect cells. The soluble (s)Fc epsilon RI alpha purified from culture medium by affinity chromatography with an anti-(alpha chain) mAb was nearly homogeneous and had an IgE-binding activity. The amino acid composition and the revealed N-terminal amino acid sequence of sFc epsilon RI alpha suggested that it was properly processed in insect cells. The apparent molecular mass (35 kDa) of purified sFc epsilon RI alpha was smaller than that of sFc epsilon RI alpha produced by CHO transfectants. The reduction of the apparent molecular mass after N-glycanase treatment showed the recombinant product was N-glycosylated. Peptide mapping of native and deglycosylated sFc epsilon RI alpha indicated that three Asn residues (Asn21, Asn42 and Asn166) should be almost fully glycosylated, and that two Asn residues (Asn74 and Asn135) were partially glycosylated.

Src family tyrosine kinase p53/56lyn, a serine kinase and Fc epsilon RI associate with alpha-galactosyl derivatives of ganglioside GD1b in rat basophilic leukemia RBL-2H3 cells.

The monoclonal antibody (mAb) AA4 recognizes two alpha-galactosyl derivatives of the GD1b ganglioside on rat mast cells and on the rat basophilic leukemia RBL-2H3 cultured cell line. Here we demonstrate that mAb AA4 coprecipitated both protein tyrosine and serine kinases. In contrast, a monoclonal antibody to the GD3 ganglioside did not coprecipitate any kinase activity. In kinase assays of mAb AA4 immunoprecipitates there were phosphorylated proteins of 71-80, 53/56, and 41/42 kDa. All proteins were phosphorylated on tyrosine, whereas the 71-80- and 41/42-kDa proteins were also phosphorylated on serine residues. The precipitation of these proteins by mAb AA4 correlated with the presence of the alpha-galactosyl derivatives of GD1b. The 53/56-kDa proteins were identified as the Src-related tyrosine kinase p53/56lyn. The presence of p53/56lyn in the mAb AA4 immunoprecipitates was specific and was observed when several different detergents were used. The same 71-80-kDa tyrosine-phosphorylated proteins were immunoprecipitated by mAb AA4 and anti-Lyn antibodies and may play a role in the interaction of p53/56lyn with the gangliosides. Although there is a weak association of the high affinity IgE receptor with these gangliosides, the coprecipitation of p53/56lyn with mAb AA4 was not secondary to the association of this kinase with receptor. These complexes of gangliosides and several proteins that include p53/56lyn, a serine kinase, and the high affinity IgE receptor could play an important role in receptor-mediated signal transduction.

The gamma chain of the high-affinity receptor for IgE is a major functional subunit of the T-cell antigen receptor complex in gamma delta T lymphocytes.

T-cell activation is a consequence of the clonotypic T-cell antigen receptor (TCR) binding to an antigen followed by signal transduction via the invariant subunits of the TCR/CD3 complex. gamma delta TCR cells are a small subset of T cells that populate both the epithelial and lymphoid tissues and have unique antigen specificity and function. However, the composition of invariant chains within the gamma delta TCR/CD3 complex has not been well characterized. Here we report that, unlike the majority of alpha beta T cell, gamma delta T cells isolated from spleen and intestinal epithelial tissue express high levels of the gamma chain of the high-affinity receptor for IgE (Fc epsilon RI gamma) as one invariant subunit of their TCR/CD3 complex. Fc epsilon RI gamma exists as both a homodimer and a heterodimer associated with the TCR zeta chain. Moreover, stimulation of the gamma delta TCR results in rapid tyrosine phosphorylation of Fc epsilon RI gamma. Our results suggest that utilization of distinct receptor signaling components may enable the coupling of antigen stimulation to the activation of different signal transduction pathways in alpha beta and gamma delta T cells.

Physical association between the high-affinity IgG receptor (Fc gamma RI) and the gamma subunit of the high-affinity IgE receptor (Fc epsilon RI gamma).

To investigate the structural basis of transmembrane signaling via the high-affinity IgG receptor (Fc gamma RI), the identity of Fc gamma RI-associated proteins in THP-1 human monocytic cells was examined. Anti-Fc gamma RI monoclonal antibody (mAb) 197 immunoprecipitates from 125I-labeled THP-1 cells solubilized in 1% digitonin buffer were found to contain a protein migrating at 12 kDa on reduction. This protein comigrated with the 12-kDa protein precipitated by the anti-high-affinity IgE receptor gamma chain (Fc epsilon RI gamma) mAb 4D8. Similarly, a 70-kDa band immunoprecipitated by mAb 4D8 comigrated with the 70-kDa protein band corresponding to Fc gamma RI in mAb 197 immunoprecipitates. On two-dimensional nonreducing-reducing gel analysis, the 12-kDa protein present in both mAb 197 and mAb 4D8 immunoprecipitates migrated as a disulfide-linked homodimer. Analysis of 1% Nonidet P-40 eluates of the digitonin immunoprecipitates under reducing conditions demonstrated the presence of a 12-kDa band in the mAb 197 immunoprecipitate that could be reprecipitated with mAb 4D8. Conversely, a 70-kDa band in the mAb 4D8 immunoprecipitate could be reprecipitated with mAb 197. Similar to these findings, both mAb 197 and mAb 4D8 precipitated a 12-kDa disulfide-linked homodimeric protein from digitonin lysates of 125I-labeled human neutrophils after induction of Fc gamma RI expression with interferon gamma but not from unstimulated neutrophils. Northern blot analysis confirmed the presence of Fc epsilon RI gamma mRNA in interferon gamma-induced human neutrophils. We conclude that Fc epsilon RI gamma, a member of a family of proteins implicated in transmembrane signaling via immune recognition receptors, associates with Fc gamma RI in human cells.

Purification and characterization of human recombinant IgE-Fc fragments that bind to the human high affinity IgE receptor.

The Fc-region of immunoglobulin E (IgE) comprising C epsilon 2, C epsilon 3, and C epsilon 4 domains is sufficient for binding to the alpha chain of the high affinity IgE-Fc receptor (Fc epsilon RI alpha). In order to identify the smallest Fc fragment capable of binding to the Fc epsilon RI alpha with high affinity, various regions of the IgE-Fc molecule were expressed in COS cells and investigated for their ability to bind Fc epsilon RI alpha. The smallest fragment that showed Fc epsilon RI alpha binding activity spans amino acids 329-547 and lacks the entire C epsilon 2 domain. Two active fragments, viz. Fc epsilon(315-547) (containing Cys328 which is responsible for interchain S-S bonding) and Fc epsilon(329-547), have been overexpressed in CHO cells and purified to homogeneity. The purified proteins bind to the Fc epsilon RI alpha with high affinity, similar to native IgE. SDS-polyacrylamide gel electrophoresis analyses indicate that Fc epsilon(315-547) is an S-S-linked dimer of apparent molecular mass of 68 kDa. Fc epsilon(329-547) appears on SDS-gel as three distinct bands at approximately 32 kDa, both under reducing and nonreducing conditions. However, size exclusion chromatography and analytical ultracentrifugation studies suggest that Fc epsilon(329-547) also remains associated as a dimer. The presence of N-linked glycosylation was detected in both proteins. The deglycosylated form of Fc epsilon(315-547) was isolated after Endo F/N-glycosidase F digestion and demonstrated to have binding activity comparable to that of the mock-digested protein. These results suggest that the presence of N-linked sugars is not necessary for Fc epsilon RI alpha binding. Both proteins blocked the release of histamine from RBL cells expressing human Fc epsilon RI alpha in a dose-dependent manner. The availability of these recombinant IgE-Fc proteins will be helpful in elucidating the key epitopes essential for the binding of IgE to its high affinity receptor.