Differential regulation of phospholipase Cgamma subtypes through FcepsilonRI, high affinity IgE receptor

The high affinity IgE receptor (FcepsilonRI) usually exists as a tetramer composed of alphabetagamma2 subunits. The COOH-tail of beta and gamma subunits contains consensus sequence termed 'immunoreceptor tyrosine-based activation motif' (ITAM). Tyrosine phosphorylated ITAM interacts with signaling proteins that contain the Src homology domain, forming a main amplifying and signaling route for FcepsilonRI. Unlike the COOH-tail, the functional role of NH(2)-tail of beta subunit in the signaling of FcepsilonRI is not clear because it lacks the ITAM sequences. To study the roles of NH(2)-tail of beta subunit, the cDNA library of RBL-2H3 cells was screened by yeast two-hybrid assay, and the NH(2)-tail of the beta subunit was found to interact with phospholipase Cgamma2 (PLCgamma2) but not with PLCgamma1. Since both PLCgamma1 and PLCgamma2 are expressed in RBL-2H3 cells and they possess identical cellular functions, the functional meaning of the protein-protein interaction between PLCgamma2 and NH(2)-tail of beta subunit was studied by comparing the regulatory pathways that control the FcepsilonRI-mediated tyrosine phosphorylation of the two enzymes. Our study shows that PI3-kinase and PMA-sensitive PKCs were required exclusively for the FcepsilonRI-mediated tyrosine phosphorylation of PLCgamma1. Also the FcepsilonRI-mediated tyrosine phosphorylation of PLCgamma1 was more sensitive to the inhibitors of Src and Syk kinases. These results therefore suggest that PLCgamma1 is involved in dynamic regulation of protein kinase C activity and inositol triphosphate levels in response to cellular needs. In contrast, PLCgamma2, through continuous interaction with the NH(2)-tail of beta subunit, co-localizes with FcepsilonRI in the same signaling domain, and maintains the basal cellular PLC activity.     

Disulfide linkage controls the affinity and stoichiometry of IgE Fcepsilon3-4 binding to FcepsilonRI

IgE antibodies cause long-term sensitization of tissue mast cells and blood basophils toward allergen-induced cross-linking and triggering of allergic inflammation. This persistence of IgE binding is due to its uniquely high affinity for the receptor FcepsilonRI and in particular its slow rate of dissociation once bound. The binding interface consists of two subsites, one contributed by each Cepsilon3 domain of IgE Fc in a 1:1 complex. We have investigated the contributions of Cepsilon3 disulfide linkage and glycosylation to the kinetics and affinity of binding of an Fc subfragment (Fcepsilon3-4) to a soluble receptor fragment (sFcepsilonRIalpha). In contrast to IgG Fc where deglycosylation abrogates receptor binding activity, the removal of the N-linked carbohydrate at Asn-394 in Fcepsilon3-4 only reduces binding affinity by a factor of 4, principally because of a faster off-rate. Removal of the inter-heavy chain disulfide bond unexpectedly resulted in a fragment with a much faster off-rate and the potential to form a complex with a 2:1 stoichiometry (sFcepsilonRIalpha:Fcepsilon3-4). This permitted the determination of the affinity of a single, natively folded Cepsilon3 domain for the first time. The low affinity Ka approximately 10(5)-10(6) m-1, similar to that determined previously for an isolated and partially folded Cepsilon3 domain, demonstrates that substantial reduction in affinity can be achieved by preventing the engagement of one of the two Cepsilon3 domains. Recent structural data indicate that conformational change in IgE is required to allow both Cepsilon3 domains to bind, and thus an allosteric inhibitor that prevents access to the second Cepsilon3 has the potential to reduce the ability of IgE to sensitize allergic effector cells.     

Domain one of the high affinity IgE receptor, FcepsilonRI, regulates binding to IgE through its interface with domain two

The high affinity receptor for IgE, FcepsilonRI, binds IgE through the second Ig-like domain of the alpha subunit. The role of the first Ig-like domain is not well understood, but it is required for optimal binding of IgE to FcepsilonRI, either through a minor contact interaction or in a supporting structural capacity. The results reported here demonstrate that domain one of FcepsilonRI plays a major structural role supporting the presentation of the ligand-binding site, by interactions generated within the interdomain interface. Analysis of a series of chimeric receptors and point mutants indicated that specific residues within the A' strand of domain one are crucial to the maintenance of the interdomain interface, and IgE binding. Mutation of the Arg(15) and Phe(17) residues caused loss in ligand binding, and utilizing a homology model of FcepsilonRI-alpha based on the solved structure of FcgammaRIIa, it appears likely that this decrease is brought about by collapse of the interface and consequently the IgE-binding site. In addition discrepancies in results of previous studies using chimeric IgE receptors comprising FcepsilonRIalpha with either FcgammaRIIa or FcgammaRIIIA can be explained by the presence or absence of Arg(15) and its influence on the IgE-binding site. The data presented here suggest that the second domain of FcepsilonRI-alpha is the only domain involved in direct contact with the IgE ligand and that domain one has a structural function of great importance in maintaining the integrity of the interdomain interface and, through it, the ligand-binding site.     

Lipopolysaccharide reduces intercellular coupling in vitro and arteriolar conducted response in vivo

Our recent in vitro study (Lidington et al. J Cell Physiol 185: 117-125, 2000) suggested that lipopolysaccharide (LPS) reduces communication along blood vessels. The present investigation extended this study to determine whether any effect of LPS and/or inflammatory cytokines [tumor necrosis factor-alpha, interleukin (IL)-1beta, and IL-6] on endothelial cell coupling in vitro could also be demonstrated for an arteriolar conducted response in vivo. Using an electrophysiological approach in monolayers of microvascular endothelial cells, we found that LPS (10 microg/ml) but not these cytokines reduced intercellular conductance (c(i)) (an index of cell communication) and that LPS together with these cytokines did not further reduce c(i). Also, c(i) was restored after LPS washout, and the LPS-induced reduction was prevented by protein tyrosine kinase (PTK) inhibitors (1.5 microM Tyr A9 and 10 nM PP-2). In our in vivo experiments in arterioles of the mouse cremaster muscle, local electrical stimulation evoked vasoconstriction that conducted along arterioles. LPS in the muscle superfusate did not alter local vasoconstriction but reduced the conducted response. Washout of LPS restored the conducted response, whereas PTK inhibitors prevented the effect of LPS. On the basis of a newly developed mathematical model, the LPS-induced reduction in conducted response was predicted to reduce the arteriolar ability to increase resistance to blood flow. We conclude that LPS can reduce communication in in vitro and in vivo systems comparably in a reversible and tyrosine kinase-dependent manner. Based on literature and present results, we suggest that LPS may compromise microvascular hemodynamics at both the arteriolar responsiveness and the conduction levels.     

Inhibition of allergic reactions with monoclonal antibody to the high affinity IgE receptor

A mAb that reacts with the high affinity IgE-R on the rat basophilic leukemia cells (RBL-2H3) was used to inhibit allergic reactions. In vitro, the intact mAb BA3 and its Fab fragment inhibited radiolabeled IgE binding to the RBL-2H3 cells. The mAb binds to the IgE-R with a higher affinity than does IgE. Whereas the intact mAb released histamine from the RBL-2H3 cells, the Fab was inactive. The addition of the Fab fragments to RBL-2H3 inhibited the IgE-mediated histamine release reaction. The Fab fragments also inhibited in vivo passive cutaneous reactions in rats when injected intradermally either before or after IgE. The injection of the mAb Fab i.v. before the injection of the IgE into the skin sites also inhibited reactions, although it was less effective. The results demonstrate that anti-R antibodies can be used as a model for inhibiting immediate hypersensitivity reactions.     

Inhibitory effect of polyphenol-enriched apple extracts on mast cell degranulation in vitro targeting the binding between IgE and FcepsilonRI

Extracts from immature fruit of the apple (Rosaceae, Malus sp.), which contain procyanidins (polymers of catechins) as the major ingredients, are known to inhibit histamine release from mast cells. We analyzed in this study the mechanism for the anti-allergic activity of two polyphenol-enriched apple extracts. These extracts, termed "crude apple polyphenol (CAP)" and "apple condensed tannin (ACT)", reduced the degranulation of mast cells caused by cross-linking of the high-affinity receptor for IgE (FcepsilonRI) with IgE and the antigen in a dose-dependent manner. Furthermore, western blotting revealed that phosphorylation of the intracellular signal-transduction molecules caused by cross-linking of FcepsilonRI was markedly decreased by the addition of CAP or ACT. We then analyzed the effects of CAP and ACT on the binding of the IgE antibody to FcepsilonRI on mast cells, which is the first key step in the allergic reaction mediated by mast cells, and found that this binding was markedly inhibited by both CAP and ACT. These results indicate that the inhibition of binding between FcepsilonRI and IgE by either CAP or ACT was the probable cause of the suppression of mast cell activation. This is the first report demonstrating the molecular mechanism for the anti-allergic effect of procyanidin-enriched extracts from apples.     

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.     

Modeling the response time of an in vivo glucose affinity sensor

A mathematical model was developed to describe the dose-response relationship of an optical glucose sensor. The basis for glucose detection is the reversible competitive displacement of a ligand from a receptor protein with specific binding sites for certain carbohydrates. Detection of glucose is based on measurements of the change in fluorescent lifetime of the donor-labeled protein, as it binds to the acceptor-labeled ligand. The sensor was modeled as a hollow fiber membrane, permeable to glucose, which encapsulates a solution of the receptor protein and competing ligand. Model equations that describe the diffusion of glucose through the fiber membrane and the subsequent displacement reactions within the fiber lumen were solved numerically to predict the response time of the sensor following a step change in bulk glucose concentration. The incorporation of an external mass transfer boundary layer was found to increase the response time by a factor of 3.7 over the well-stirred case. On the basis of the results of a parametric study, the response time of the sensor was found to be most sensitive to the diffusion coefficient of glucose in the membrane. When compared to experimental response times for an intensity-based affinity sensor using Concanavalin A as the receptor protein and dextran as the competing ligand, the model predictions were found to be significantly shorter than those observed. The effect of the in vivo environment on the performance of the sensor was also investigated through the incorporation of a fibrotic capsule layer. The additional diffusional resistance offered by the capsular tissue resulted in a 5-fold increase in the response time of the sensor.     

Dietary pulverized konjac glucomannan prevents the development of allergic rhinitis-like symptoms and IgE response in mice

Konjac is a traditional Japanese food with a peculiar texture, and it has been suggested that its main ingredient, konjac glucomannan (KGM), ameliorates metabolic disorders such as diabetes and hypercholesteremia. We have found that feeding with pulverized KGM (PKGM) prevents skin inflammation in a murine model of atopic dermatitis. Here, we show that dietary PKGM suppresses allergic rhinitis-like symptoms in mice upon immunization and nasal sensitization with ovalbumin (OVA). The PKGM-fed mice showed a much lower frequency of sneezing than in control animals. We also found that PKGM supplementation exclusively suppressed OVA-specific IgE response without affecting IgG1/IgG2a responses as well as systemic Th1/Th2 cytokine production. These results suggest that PKGM can be a beneficial foodstuff in preventing nasal allergy such as seasonal pollinosis.     

IgE and mast cells in allergic disease

Immunoglobulin E (IgE) antibodies and mast cells have been so convincingly linked to the pathophysiology of anaphylaxis and other acute allergic reactions that it can be difficult to think of them in other contexts. However, a large body of evidence now suggests that both IgE and mast cells are also key drivers of the long-term pathophysiological changes and tissue remodeling associated with chronic allergic inflammation in asthma and other settings. Such potential roles include IgE-dependent regulation of mast-cell functions, actions of IgE that are largely independent of mast cells and roles of mast cells that do not directly involve IgE. In this review, we discuss findings supporting the conclusion that IgE and mast cells can have both interdependent and independent roles in the complex immune responses that manifest clinically as asthma and other allergic disorders.     

A minimal receptor-Ig chimera of human FcepsilonRI alpha-chain efficiently binds secretory and membrane IgE

We constructed a soluble minimal receptor-Ig chimera in which the two extracellular domains of human Fcepsilonhain (D1 and D2) were fused to the dimerizing C-terminal domain of human IgG1 heavy chain (gamma1-CH3). The protein was expressed and actively secreted by Chinese hamster ovary (CHO) cells as a fully glycosylated soluble dimeric protein. It showed efficient binding both to human membrane-bound IgE isoforms and to the two secretory IgE isoforms. Moreover, the dimeric receptor binds IgE with the expected 1:2 stoichiometry. The receptor-Ig chimera, in 2-fold molar excess, inhibited engagement of secretory IgE to rat basophilic leukemia cells expressing the human alphabetagamma receptor. Full self-nature and inability to bind Fcgamma receptors make this protein an attractive candidate for clinical applications and a novel biotechnological tool for atopic allergy research.     

Phosphorylation reduces the affinity of protein 4.1 for spectrin

The phosphorylation of protein 4.1 by the membrane kinase and casein kinase A has been investigated. Each of these kinases catalyzed the incorporation of 2 mol of phosphate per mole of protein 4.1. The presence of both kinases in the reaction mixture did not lead to an increase in the incorporation of phosphates into the protein. An analysis of the acid hydrolysis products of the 32P-labeled protein 4.1 indicated that the radioactivities were distributed between phosphothreonine and phosphoserine in a ratio of about 2 to 1. The effects of phosphorylation on the binding of protein 4.1 to spectrin were investigated by using sucrose density gradient centrifugation. The affinity of protein 4.1 for spectrin was reduced about 5-fold, from a KD of 2 X 10(-6) M to a KD of 9.4 X 10(-6) M, by phosphorylation. The phosphorylation of spectrin, on the other hand, appeared to increase slightly its affinity for protein 4.1. The results suggest that phosphorylation may lead to a relaxation of the cytoskeletal network and the formation of a more flexible membrane structure that is important to red cell function.     

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.     

Analysis of proteins binding to the ITAM motif of the beta-subunit of the high-affinity receptor for IgE (FcepsilonRI)

Aggregation of the multichain (alphabetagamma2) high-affinity IgE receptor (Fcepsilon RI) initiates a signaling cascade that results in the release of allergic mediators. The cytoplasmic tails of the FcepsilonRI-beta and -gamma subunits contain immunoreceptor tyrosine-based activation motifs (ITAMs). Phosphorylation of the gammaITAM mediates activation of Syk kinase and is sufficient for triggering the responses induced by Fcepsilon RI crosslinking. Phosphorylation of the betaITAM is insufficient to mediate cell activation. The rat betaITAM contains three tyrosines (Tyr218, Tyr224, and Tyr228) with an intermediate noncanonical tyrosine. Synthetic peptides based on the ITAM of the FcepsilonRI-beta subunit were used to investigate the role of each phosphotyrosine in the binding of signaling proteins to this motif. Among the proteins that bind to phosphorylated beta ITAM are Syk, Grb2, Shc, SHIP, and SHP-1, and binding does not depend on previous cell activation. Nonphosphorylated peptides do not bind these proteins. Syk binding to beta-peptides is dependent on the number and position of phosphotyrosines in the ITAM. Phosphorylation of Tyr218 seems to be most important for Syk binding. Recruitment of Syk and other signaling proteins to the beta-subunit might be important for its amplifier role.     

Indinavir reduces Cryptosporidium parvum infection in both in vitro and in vivo models

The use of highly active antiretroviral therapy in persons with acquired immunodeficiency syndrome has reduced the prevalence of infection with Cryptosporidium parvum and the length and severity of its clinical course. This effect has in most cases been attributed to the recovery of the host immunity; however, some works suggest that human immunodeficiency virus protease inhibitors, indinavir in particular, which is one of the human immunodeficiency virus protease inhibitors used in highly active antiretroviral therapy, may be capable of controlling Microsporidia and Cryptosporidium infections, which are refractory to other treatments. The objective of the present study was to investigate the effect of human immunodeficiency virus protease inhibitors on C. parvum infections. Since preliminary experiments using ritonavir, saquinavir, and indinavir showed a drastic reduction of C. parvum infection both in vivo (neonatal Balb/c mice) and in vitro (human ileocecal adenocarcinoma tumour cell line) models, indinavir alone was tested in successive experiments. In vitro, the treatment of the sporulated oocysts with different concentrations of indinavir reduced the percentage of human ileocecal adenocarcinoma tumour cell line infected cells in a dose-dependent manner. For established infection, the treatment with 50 microM of indinavir decreased the percentage of infected cells in a time-dependent manner. In vivo, mice treated with indinavir at the same time they were infected with the oocysts showed a 93% reduction in the number of oocysts present in the entire intestinal contents and a 91% reduction in the number of intracellular parasites in the ileum. For established infection, indinavir treatment reduced the number of oocysts in the entire intestinal content by about 50% and the number of intracellular parasites in the ileum by about 70%. These data show that indinavir directly interferes with the cycle of C. parvum, resulting in a marked reduction in oocyst shedding and in the number of intracellular parasites. Protease inhibitors could be considered as good candidates for the treatment of cyptosporidiosis in immunosuppressed persons.