Regulation of IgE activity in inhalational tolerance via formation of IgG anti-IgE/IgE immune complexes

Background

Allergic asthma is an inflammatory disorder of the airways that results from inappropriate production of IgE against harmless, environmental antigens. Sequestration of free IgE using humanized IgG anti-IgE is an effective therapy for asthma and other atopic disorders. However, the status of free IgE in subjects who have naturally developed immune tolerance to inhaled antigens has not been well studied.

Methods

C57BL/6 mice were sensitized and challenged with ovalbumin (OVA) for 7 days to induce allergic airway disease (AAD) or 6 weeks to induce a state of local inhalational tolerance (LIT). Serum from AAD or LIT mice, diluted to achieve equivalent levels of total OVA-specific IgE, was used to sensitize rat basophil leukemia cells for allergen-mediated degranulation. Levels of degranulation were measured in relation to serum concentrations of free IgE and IgG anti-IgE/IgE immune complexes.

Results

Serum from AAD animals induced a greater degree of basophil degranulation than serum from LIT animals. These results correlated with higher levels of free IgE in AAD animals, whereas LIT mice demonstrated a significant increase in IgG anti-IgE/IgE immune complexes relative to their diseased counterparts.

Conclusions

Sequestration of free IgE by naturally occurring IgG anti-IgE may aid in the development of immune tolerance against inhaled allergens. The decrease in bioavailability of free IgE may, in turn, contribute to the overall reduction of asthma symptoms via a mechanism that mimics the therapeutic effects of humanized IgG anti-IgE.

     

Avian IgY binds to a monocyte receptor with IgG-like kinetics despite an IgE-like structure

An ancestor of avian IgY was the evolutionary precursor of mammalian IgG and IgE, and present day chicken IgY performs the function of human IgG despite having the domain structure of human IgE. The kinetics of IgY binding to its receptor on a chicken monocyte cell line, MQ-NCSU, were measured, the first time that the binding of a non-mammalian antibody to a non-mammalian cell has been investigated (k(+1) = 1.14 +/- 0.46 x 10(5) mol(-1)sec(-1), k(-1) = 2.30 +/- 0.14 x 10(-3) s(-1), and K(a) = 4.95 x 10(7) m(-1)). This is a lower affinity than that recorded for mammalian IgE-high affinity receptor interactions (Ka approximately 10(10) m(-1)) but is within the range of mammalian IgG-high affinity receptor interactions (human: Ka approximately 10(8)-10(9) m(-1) mouse: Ka approximately 10(7)-10(8) m(-1). IgE has an extra pair of immunoglobulin domains when compared with IgG. Their presence reduces the dissociation rate of IgE from its receptor 20-fold, thus contributing to the high affinity of IgE. To assess the effect of the equivalent domains on the kinetics of IgY binding, IgY-Fc fragments with and without this domain were cloned and expressed in mammalian cells. In contrast to IgE, their presence in IgY has little effect on the association rate and no effect on dissociation. Whatever the function of this extra domain pair in avian IgY, it has persisted for at least 310 million years and has been co-opted in mammalian IgE to generate a uniquely slow dissociation rate and high affinity.     

Cytophilic binding of IgE to the macrophage. II. Immunologic release of lysosomal enzyme from macrophages by IgE and anti-IgE in the rat: a new mechanism of macrophage activation.

Rat peritoneal macrophages release lysosome granule-associated β-glucuronidase, but not cytoplasmic leucine aminopeptidase, after successive incubation with purified IgE protein and ?-specific anti-IgE antibody or anti-IgE F(ab′)₂ fragments. The selective release of β-glucuronidase was shown to proceed by a first step of binding of the purified IgE to the cell surface, followed by IgE-anti-IgE reaction on the macrophage, whereas the possibility of cell activation by IgE-anti-IgE complexes in the bulk phase was ruled out. Heating rat IgE destroyed its ability to mediate lysosomal enzyme release. The characteristics of macrophage activation, insofar as the binding of IgE is concerned, were in agreement with those reported for the fixation of IgE to the mononuclear phagocyte, optimal binding of IgE being achieved with 20 min incubation. Preincubation of rat macrophages with rat IgG, either aggregated or not aggregated, did not inhibit the selective release of β-glucuronidase by the successive addition of IgE and anti-IgE antibody. Simultaneous incubation of macrophage monolayers with rat IgE and aggregated rat IgG did not reduce the subsequent activation by addition of anti-IgE. These studies indicated that rat macrophages can bind rat IgE through a specific receptor, with no interference of the classical Fc (γ) receptor, and are triggered to release lysosomal enzymes upon conformational changes of the IgE molecule by anti-IgE antibody. Antibody-dependent macrophage cytotoxicity in rat schistosomiasis is mediated by IgE antibody to the parasite, which may therefore function by activating the macrophage to an efficient effector cell.     

Targeting the extracellular membrane-proximal domain of membrane-bound IgE by passive immunization blocks IgE synthesis in vivo

The classical allergic reaction starts seconds or minutes after Ag contact and is committed by Abs produced by a special subset of B lymphocytes. These Abs belong to the IgE subclass and are responsible for Type I hyperreactivity reactions. Treatment of allergic diseases with humanized anti-IgE Abs leads primarily to a decrease of serum IgE levels. As a consequence, the number of high-affinity IgE receptors on mast cells and basophils decreases, leading to a lower excitability of the effector cells. The biological mechanism behind anti-IgE therapy remains partly speculative; however, it is likely that these Abs also interact with membrane IgE (mIgE) on B cells and possibly interfere with IgE production. In the present work, we raised a mouse mAb directed exclusively against the extracellular membrane-proximal domain of mIgE. The interaction between the monoclonal anti-mIgE Ab and mIgE induces receptor-mediated apoptosis in vitro. Passive immunization experiments lead to a block of newly synthesized specific IgEs during a parallel application of recombinant Bet v1a, the major birch pollen allergen. The decrease of allergen-specific serum IgE might be related to tolerance-inducing mechanisms stopping mIgE-displaying B cells in their proliferation and differentiation.     

Measurement of IgE on human basophils: relation to serum IgE and anti-IgE-induced histamine release.

The number of IgE molecules bound to human basophils was calculated from direct measurements of the IgE dissociated after exposing leukocytes to pH 3.7 acetate buffer in the cold. In 18 donors studied, cell-bound IgE ranged from 4000 to 500,000 molecules/basophil and correlated with the serum IgE concentration (r = 0.89, p less than 0.001) which ranged from 5 to 3,000 ng/ml. Sensitivity of these cells to anti-IgE was tested to explore the relationship between cell-bound IgE and the concentration of anti-IgE required for histamine release. Cells from some nonatopic donors (4000 to 100,000 IgE molecules/basophil) were as sensitive as cells from allergic donors (100,00 to 500,000 IgE molecules/basophil). Moreover, cells from donors having approximately the same cell-bound IgE concentration varied widely in their sensitivity to anti-IgE. We conclude that an intrinsic property of human basophils ("releasability") is an important parameter in determing mediator release.     

IgY: a key isotype in antibody evolution

Immunoglobulin Y (IgY) is central to our understanding of immunoglobulin evolution. It has links to antibodies from the ancestral IgM to the mucosal IgX and IgA, as well as to mammalian serum IgG and IgE. IgY is found in amphibians, birds and reptiles, and as their most abundant serum antibody, is orthologous to mammalian IgG. However, IgY has the same domain architecture as IgM and IgE, lacking a hinge region and comprising four heavy‐chain constant domains. The relationship between IgY and the mucosal antibodies IgX and IgA is discussed herein, in particular the question of how IgA could have contributed to the emergence of IgY. Although IgY does not contain a hinge region, amphibian IgF and duck‐billed platypus IgY/O, which are closely related to IgY, do contain this region, as does mammalian IgG, IgA and IgD. A hinge region must therefore have evolved at least three times independently by convergent evolution. In the absence of three‐dimensional structural information for the complete Fc fragment of chicken IgY (IgY‐Fc), it remains to be discovered whether IgY displays the same conformational properties as IgM and IgE, which exhibit substantial flexibility in their Fc regions. IgY has three characterised Fc receptors, chicken Ig‐like receptor AB1 (CHIR‐AB1), the chicken yolk sac IgY receptor (FcRY) and Gallus gallus Fc receptor (ggFcR). These receptors bind to IgY at sites that are structurally homologous to mammalian counterparts; IgA/FcαRI for CHIR‐AB1, IgG/FcRn for FcRY and IgE/Fc?RI and IgG/FcγR for ggFcR. These resemblances reflect the close evolutionary relationships between IgY and IgA, IgG and IgE. However, the evolutionary distance between birds and mammals allows for the ready generation of IgY antibodies to conserved mammalian proteins for medical and biotechnological applications. Furthermore, the lack of reactivity of IgY with mammalian Fc receptors, and the fact that large quantities of IgY can be made quickly and cheaply in chicken eggs, offers important advantages and considerable potential for IgY in research, diagnostics and therapeutics.     

Down-regulation of human basophil IgE and FC epsilon RI alpha surface densities and mediator release by anti-IgE-infusions is reversible in vitro and in vivo

Previously, infusions of an anti-IgE mAb (rhumAb-E25) in subjects decreased serum IgE levels, basophil IgE and FcepsilonRIalpha surface density, and polyclonal anti-IgE and Ag-induced basophil histamine release responses. We hypothesized that these effects would be reversed in vivo by discontinuation of infusions and in vitro by exposing basophils to IgE. Subjects received rhumAb-E25 biweekly for 46 wk. Blood samples taken 0-52 wk after rhumAb-E25 were analyzed for serum IgE and basophil expression of IgE, FcepsilonRIalpha, and CD32. Basophil numbers were unaffected by infusions. Eight weeks after infusions, free IgE levels rose in vivo but did not reach baseline. Basophil IgE and FcepsilonRIalpha rose in parallel with free IgE while CD32 was stable. FcepsilonRI densities, measured by acid elution, returned to 80% of baseline, whereas histamine release responses returned to baseline. Basophils cultured with or without IgE or IgG were analyzed for expression of IgE, FcepsilonRIalpha, and CD32. By 7 days with IgE, expression of IgE and FcepsilonRIalpha rose significantly, whereas cultures without IgE declined. IgE culture did not effect CD32. IgG culture did not effect expression of any marker. The present results strongly suggest that free IgE levels regulate FcepsilonRIalpha expression on basophils.     

Bivalent monoclonal IgY antibody formats by conversion of recombinant antibody fragments

Monoclonal IgY have the potential to become unique tools for diagnostic research and therapeutic purposes since avian antibodies provide several advantages due to their phylogenetic difference when compared to mammalian antibodies. The mechanism of avian immunoglobulin gene diversification renders chicken an excellent source for the generation of recombinant scFv as well as Fab antibody libraries of high diversity. One major limitation of these antibody fragments, however, is their monovalent format, impairing the functional affinity of the molecules and, thereby, their applicability in prevalent laboratory methods. In this study, we generated vectors for conversion of avian recombinant antibody fragments into different types of bivalent IgY antibody formats. To combine the properties of established mammalian monoclonal antibodies with those of IgY constant domains, we additionally generated bivalent murine/avian chimeric antibody constructs. When expressed in HEK-293 cells, all constructs yielded bivalent disulfide-linked antibodies, which exhibit a glycosylation pattern similar to that of native IgY as assessed by lectin blot analysis. After purification by one step procedures, the chimeric and the entire avian bivalent antibody formats were analyzed for antigen binding and interaction with secondary reagents. The data demonstrate that all antibody formats provide comparable antigen binding characteristics and the well established properties of avian constant domains.     

IgE generation and mast cell effector function in mice deficient in IL-4 and IL-13

IL-4 and IL-13 are potent cytokines that drive production of IgE, which is critical to the development of atopic disease. In this study, we directly compared IgE generation and IgE-dependent mast cell effector function in mouse strains lacking IL-4, IL-13, IL-4 + IL-13, or their common receptor component, IL-4Ralpha. Although serum IgE was undetectable under resting conditions in most animals deficient in one or both cytokines, peritoneal mast cells from mice lacking IL-4 or IL-13 had only partial reductions in surface IgE level. In contrast, peritoneal mast cells from IL-4/13(-/-) and IL-4Ralpha(-/-) animals were severely deficient in surface IgE, and showed no detectable degranulation following treatment with anti-IgE in vitro. Surprisingly, however, intradermal challenge with high concentrations of anti-IgE Ab induced an ear-swelling response in these strains, implying some capacity for IgE-mediated effector function in tissue mast cells. Furthermore, upon specific immunization with OVA, both IL-4/IL-13(-/-) and IL-4Ralpha(-/-) mice produced detectable levels of serum IgE and Ag-specific IgG1, and generated strong ear-swelling responses to intradermal administration of anti-IgE. These findings suggest that a mechanism for IgE production exists in vivo that is independent of IL-4 or IL-13.     

The pharmacological basis of anti-IgE therapy

The treatment of asthma and allergic rhinitis using unique, humanized anti-IgE monoclonal antibodies with very particular binding specificities is now supported by the results of multiple phase II and III human clinical studies. The therapeutic efficacy of this approach is attributable to several pharmacological mechanisms. In addition to the expected effects of these monoclonal antibodies in neutralizing free IgE and inhibiting IgE production by B cells, several indirect biochemical and cellular effects have been uncovered during the course of the clinical trials. These include the accumulation of potentially beneficial IgE-anti-IgE immune complexes and the downregulation of the high-affinity IgE Fc receptors (FcvarepsilonRI) on basophils and mast cells. This article analyzes the structural basis of the specificity of the anti-IgE antibodies and pertinent results from in vitro experiments, animal model studies, and human clinical trials in an attempt to provide a cogent pharmacological interpretation of the therapeutic effects of anti-IgE therapy in both the near- and long term. The development of anti-IgE therapy over the past 10 years provides an interesting example of the emergence of a conceptually new, biotechnology-produced pharmaceutical.     

Relationships between epitopes on IgE recognized by defined monoclonal antibodies and by the FC epsilon receptor on basophils

The present study investigated whether the sites on the FC region of the IgE molecule, recognized by different anti-IgE monoclonal antibodies (mAb), are identical to those recognized by the Fc receptor (Fc epsilon R). The anti-IgE mAb recognize different clusters of epitopes on the Fc region of IgE and could interfere to different degrees with the binding of IgE to mast cells and basophils, but still recognized cell-bound IgE. Analysis of the stoichiometry and affinity binding of 125I anti-IgE mAb Fab' to free IgE have revealed that anti-IgE mAb of one group (51.3) recognized three repetitive determinants on the IgE Fc portion, and another group (95.3) recognized only one determinant. When these stoichiometric studies were performed with cell-bound IgE, it was found that only one of the sites recognized by 51.3 mAb was involved in the Fc epsilon R binding site. On the other hand, the site recognized by 95.3 mAb was not the Fc epsilon R binding site. Such findings establish mAb 51.3 as a useful tool for isolating the IgE peptides involved in the binding site to the receptor.     

Fluorometric enzyme-linked immunosorbent assay for the measurement of IgE antibody to mite Dermatophagoides farinae

We have developed a fluorometric enzyme-linked immunosorbent assay for measuring IgE antibody to Dermatophagoides farinae. Polystyrene microplates were coated with proteins extracted from the mites. The IgE antibody which attached to the solid-phase antigen was detected by anti-IgE antibody conjugated with beta-galactosidase. Four-methylumbelliferyl-beta-D-galactoside was used as the enzyme substrate and the fluorescence intensity of the reaction product was measured. The antibody levels determined by this method well correlated with those determined by the radioallergosorbent test (RAST). This method is simpler and less expensive to carry out than the RAST when dealing with a large number of serum specimens for seroepidemiological studies of asthma and nasal allergy.     

Site-specific N-glycosylation of chicken serum IgG

Avian serum immunoglobulin (IgG or IgY) is functionally equivalent to mammalian IgG but has one additional constant region domain (CH2) in its heavy (H) chain. In chicken IgG, each H-chain contains two potential N-glycosylation sites located on CH2 and CH3 domains. To clarify characteristics of N-glycosylation on avian IgG, we analyze N-glycans from chicken serum IgG by derivatization with 2-aminopyridine (PA) and identified by HPLC and MALDI-TOF-MS. There were two types of N-glycans: (1) high-mannose-type oligosaccharides (monoglucosylated 26.8%, others 10.5%) and (2) biantennary complex-type oligosaccharides (neutral, 29.9%; monosialyl, 29.3%; disialyl, 3.7%) on molar basis of total N-glycans. To investigate the site-specific localization of different N-glycans, chicken serum IgG was digested with papain and separated into Fab [containing variable regions (VH + VL) + CH1 + CL] and Fc (containing CH3 + CH4) fragments. Con A stained only Fc (CH3 + CH4) and RCA-I stained only Fab fractions, suggesting that high-mannose-type oligosaccharides were located on Fc (CH3 + CH4) fragments, and variable regions of Fab contains complex-type N-glycans. MS analysis of chicken IgG-glycopeptides revealed that chicken CH3 domain (structurally equivalent to mammalian CH2 domain) contained only high-mannose-type oligosaccharides, whereas chicken CH2 domain contained only complex-type N-glycans. The N-glycosylation pattern on avian IgG is more analogous to that in mammalian IgE than IgG, presumably reflecting the structural similarity to mammalian IgE.     

A Monomeric Chicken IgY Receptor Binds IgY with 2:1 Stoichiometry

IgY is the principal serum antibody in birds and reptiles, and an IgY-like molecule was the evolutionary precursor of both mammalian IgG and IgE. A receptor for IgY on chicken monocytes, chicken leukocyte receptor AB1 (CHIR-AB1), lies in the avian leukocyte receptor cluster rather than the classical Fc receptor cluster where the genes for mammalian IgE and IgG receptors are found. IgG and IgE receptors bind to the lower hinge region of their respective antibodies with 1:1 stoichiometry, whereas the myeloid receptor for IgA, FcαRI, and the IgG homeostasis receptor, FcRn, which are found in the mammalian leukocyte receptor cluster, bind with 2:1 stoichiometry between the heavy chain constant domains 2 and 3 of each heavy chain. In this paper, the extracellular domain of CHIR-AB1 was expressed in a soluble form and shown to be a monomer that binds to IgY-Fc with 2:1 stoichiometry. The two binding sites have similar affinities: K_a1 = 7.22 ± 0.22 × 10⁵ m⁻¹ and K_a2 = 3.63 ± 1.03 × 10⁶ m⁻¹ (comparable with the values reported for IgA binding to its receptor). The affinity constants for IgY and IgY-Fc binding to immobilized CHIR-AB1 are 9.07 ± 0.07 × 10⁷ and 6.11 ± 0.02 × 10⁸ m⁻¹, respectively, in agreement with values obtained for IgY binding to chicken monocyte cells and comparable with reported values for human IgA binding to neutrophils. Although the binding site for CHIR-AB1 on IgY is not known, the data reported here with a monomeric receptor binding to IgY at two sites with low affinity suggest an IgA-like interaction.Fc receptors link the specificity of the adaptive immune system with the effector mechanisms of innate immune cells. In birds and reptiles, IgY is the principal serum antibody, and both mammalian IgG and IgE have evolved from an IgY-like ancestor, so studies of IgY offer insights into their origins (1). The historical contribution of chicken immunology to a wider understanding of the subject has been considerable (2), and recently several chicken IgY-Fc receptors have been identified. In this paper, the chicken antibody, IgY, is shown to bind to a chicken leukocyte receptor, CHIR-AB1,⁴ in a different manner from that of its mammalian orthologues, IgG and IgE, to their respective Fc receptors.Phagocytosis, mediated in mammals by IgG, and passive cutaneous anaphylaxis, mediated by both IgG and IgE in mammals, have been observed in chickens (3, 4), presumably both effected by IgY. In vitro, IgY binds to monocyte cell lines (5, 6), and an IgY receptor (CHIR-AB1) has been identified that is able to mediate the influx of calcium into cells (5).The genes for the mammalian high affinity IgE receptor, and several IgG receptors, are located in the classical Fc receptor cluster, whereas in chickens, this cluster is represented by a single gene, the product of which has been expressed and found not to bind IgY (7). Intriguingly, the first IgY leukocyte receptor, CHIR-AB1, was found to be a member of the chicken leukocyte receptor cluster (LRC) (5), adjacent to over 100 genes with high intersequence homology (8). This finding, together with phylogenetic analysis of the orthologous Fc receptor gene clusters (7, 9), implies that during the evolution of the IgY-like ancestor of both IgG and IgE, antibody-Fc binding function migrated from proteins expressed in the LRC to those in the classical Fc receptor cluster. The human LRC is the site of FcαRI, the leukocyte receptor for IgA (an antibody involved in mucosal immunity), the fetal IgG receptor (FcRn, involved in adult IgG homeostasis), and also a number of natural killer cell receptors including the HLA-G ligand, KIR2DL4 (10). A further leukocyte receptor for chicken IgY, also related to LRC receptors, was identified recently, on chromosome 20 (11), and remains to be characterized.Typically, the stoichiometry of the receptor-antibody complex differs for receptors located in the classical Fc receptor cluster and the LRC. Crystal structures of IgG complexes with FcγRIII and of IgE with FcϵRI show 1:1 receptor:antibody stoichiometry, with the receptor binding across both heavy chains in the lower hinge (12). In contrast, the crystal structure of FcαRI complexed with IgA shows 2:1 stoichiometry (13) as does that of FcRn with IgG (14), with the two receptors binding between the heavy chain constant domains 2 and 3 on each heavy chain. The IgY/receptor interaction could have either stoichiometry; on the one hand, IgY is an orthologue of IgG and IgE, which can both show 1:1 stoichiometry, but on the other hand, the location of the IgY receptor, CHIR-AB1, in the same gene cluster as the IgA and FcRn receptors suggests the possibility of a 2:1 stoichiometry. Consistent with either of these binding modes, the crystal structure of IgY-Fc reveals that many of the residues located in the receptor-binding sites in human IgE, IgG, and IgA are present and accessible in IgY (15).The single extracellular domain of the chicken leukocyte IgY receptor, CHIR-AB1, has been expressed in insect cells by Arnon et al. (16), who showed that this preparation consists of a mixture of soluble monomer and dimer. Because of the heterogeneity of the protein, it was not possible to ascertain whether the observed 2:1 stoichiometry of receptor binding to antibody involved two monomers or a single dimer binding to IgY. Thus, it was not possible to answer the question of whether the antibody-receptor complex most resembles that of human IgA or of IgG and IgE. We have expressed the extracellular domain of CHIR-AB1 in human HEK cells. It is a monomer, and we report here that it binds to IgY and IgY-Fc with 2:1 stoichiometry.     

Activation of the classical pathway of human complement by a human monoclonal IgE, IgE(DES)

Activation of the classical pathway of human complement by monoclonal IgE from patient DES was demonstrated by using IgE(DES) coupled to latex particles. This material depleted human serum of C1 and C4 hemolytic activities. In addition, C3bi was deposited in a calcium-dependent way onto the insolubilized IgE as shown by the agglutination of latex by conglutinin. The alternative pathway was also activated. These anticomplementary activities were dose and time dependent. Moreover, we confirmed that another monoclonal IgE, IgE(PS), activated the alternative pathway exclusively. Particular attention was paid to exclude contamination by other immunoglobulins or C-reactive protein, generation of artifacts due to the chemical coupling, and the presence of proteolytic enzymes in the IgE(DES) preparation. Moreover, evidence is also presented against the involvement of IgG or IgM anti-IgE autoantibodies that could activate the classical pathway after their binding to insolubilized IgE(DES). Although one cannot exclude the possibility that IgE(DES) or IgE(PS) are abnormal proteins, these findings suggest the existence of an isotypic or allotypic variation of IgE.     

A novel family of hairpin peptides that inhibit IgE activity by binding to the high-affinity IgE receptor

A family of structured peptides that bind to FcepsilonRIalpha, the alpha-chain of the high-affinity receptor for IgE, has been identified. Binding selections using FcepsilonRIalpha and polyvalent peptide-phage libraries yielded a dominant 18-residue peptide-phage clone, as well as related sequences that did not resemble fragments of IgE. Synthetic peptides based on these sequences inhibited IgE binding to its receptor, and were found by NMR analysis to adopt a stable beta-hairpin structure in solution. Optimized peptides with micromolar receptor affinity exhibited high stability in biological fluids and inhibited cellular histamine release in an in vitro bioassay of IgE activity. The structure-activity relationships of these peptides, which are less than 1% of the size of IgE, suggest an overlap between their binding site and that of IgE on FcepsilonRI. Thus, the peptides demonstrate that blocking a small epitope on this receptor chain is sufficient to block IgE activity. Such structured peptides represent a possible starting point for the design of novel antagonists, and offer the potential for testing in vivo a new approach for treating allergic disease.     

Studies of IgE-dependent histamine releasing factors: heterogeneity of IgE

Nasal lavage fluids from unstimulated individuals contain a histamine-releasing factor (HRF) similar to those which we have previously described from macrophages, platelets, and from blister fluids obtained during the late cutaneous reaction. The nasal HRF was partially purified by ion-exchange chromatography and gel filtration. Although some m.w. heterogeneity was observed, the majority of the HRF eluted at an apparent m.w. range of 15,000 to 30,000. This partially purified HRF induced histamine release from basophils of certain individuals. Histamine release occurred via a mechanism which is IgE-dependent in that: basophils desensitized by exposure to anti-IgE in the absence of calcium no longer respond to HRF, and desensitization with HRF reduces responsiveness to anti-IgE; and removal of IgE from the basophil surface by using lactic acid renders cells unresponsive to HRF. We have further defined this IgE dependence and have shown that the reason that only selected basophil donors respond to HRF is due to a previously unrecognized, functional heterogeneity of IgE. Thus, passive sensitization using sera from responders restored the responsiveness of acid-stripped basophils and conferred responsiveness to basophils of a nonresponder with naturally unoccupied IgE receptors. Sera from nonresponders failed to do this even though similar numbers of IgE molecules were put onto the basophil surface in each case. This property of responder sera was due to IgE because both heating sera at 56 degrees C for 2 hr and passage of sera over anti-IgE-Sepharose (which removes greater than 90% of the IgE) markedly reduced the ability of sera to induce responsiveness, and because an excess of either purified IgE myeloma or purified penicillin-specific IgE antibody from a nonresponder competitively inhibited the ability of IgE from responder sera to induce responsiveness to HRF. We conclude that nasal lavage fluids contain an HRF which induces basophil histamine release in a specific, IgE-dependent fashion but only from individuals with the appropriate type of IgE. Because we have shown that basophils are recruited into the nose during the late-phase reaction, we suggest that nasal HRF may induce these cells to release histamine and other mediators which could contribute to the symptomatology of the late-phase reaction.     

Avian IgY antibodies and their recombinant equivalents in research, diagnostics and therapy

The generation and use of avian antibodies is of increasing interest in a wide variety of applications within the life sciences. Due to their phylogenetic distance, mechanisms of immune diversification and the way in which they deposit IgY immunoglobulin in the egg yolk, chickens provide a number of advantages compared to mammals as hosts for immunization. These advantages include: the one-step purification of antibodies from egg yolk in large amounts facilitates having a virtually continuous supply; the epitope spectrum of avian antibodies potentially grants access to novel specificities; the broad absence of cross-reactivity with mammalian epitopes avoids assay interference and improves the performance of immunological techniques. The polyclonal nature of IgY antibodies has limited their use since avian hybridoma techniques are not well established. Recombinant IgY, however, can be generated from mammalian monoclonal antibodies which makes it possible to further exploit the advantageous properties of the IgY scaffold. Moreover, cloning and selecting the immune repertoire from avian organisms is highly efficient, yielding antigen-specific antibody fragments. The recombinant approach is well suited to circumvent any limitations of polyclonal antibodies. This review presents comprehensive information on the generation, purification, modification and applications of polyclonal and monoclonal IgY antibodies.     

IL-4 requirements for the generation of secondary in vivo IgE responses.

IL-4 has been shown to induce B lymphocytes to switch from the expression of membrane IgM to the expression of membrane IgE and to be required for the generation of primary polyclonal and secondary Ag-specific IgE responses in mice. To further define the role of IL-4 in the generation of memory IgE responses, we investigated the ability of a combination of anti-IL-4 and anti-IL-4R mAb to block the generation of secondary IgE responses induced by: 1) a second infection with the nematode parasites Nippostrongylus brasiliensis or Heligmosomoides polygyrus; or 2) injection of anti-IgD antibody-primed mice with anti-IgE antibody. The latter stimulus was designed to induce intrinsic membrane IgE-expressing B cells to differentiate into IgE-secreting cells. Although the IgE responses induced by a second nematode infection were completely inhibited by the combination of anti-IL-4 and anti-IL-4R mAb, anti-IgE antibody-induced IgE responses in anti-IgD primed mice were not inhibited by these antibodies to a large degree. Additional experiments demonstrated that the anti-IgE antibody-induced memory IgE response was dependent on CD4+ T cells but did not involve the low affinity B cell Fc epsilon RII. Taken together, these observations provide evidence that IL-4 is required for virgin B lymphocytes to develop into IgE-expressing cells, but is not required for B cells that express intrinsic membrane IgE to differentiate into IgE-secreting cells in a T-dependent response. Furthermore, these data suggest that secondary IgE responses in the parasite models that we have studied develop from B cells that had not previously switched to the expression of IgE.     

The crystal structure of CHIR-AB1: a primordial avian classical Fc receptor

CHIR-AB1 is a newly identified avian immunoglobulin (Ig) receptor that includes both activating and inhibitory motifs and was therefore classified as a potentially bifunctional receptor. Recently, CHIR-AB1 was shown to bind the Fc region of chicken IgY and to induce calcium mobilization via association with the common γ-chain, a subunit that transmits signals upon ligation of many different immunoreceptors. Here we describe the 1.8-Å-resolution crystal structure of the CHIR-AB1 ectodomain. The receptor ectodomain consists of a single C2-type Ig domain resembling the Ig-like domains found in mammalian Fc receptors such as FcγRs and FcαRI. Unlike these receptors and other monomeric Ig superfamily members, CHIR-AB1 crystallized as a 2-fold symmetrical homodimer that bears no resemblance to variable or constant region dimers in an antibody. Analytical ultracentrifugation demonstrated that CHIR-AB1 exists as a mixture of monomers and dimers in solution, and equilibrium gel filtration revealed a 2:1 receptor/ligand binding stoichiometry. Measurement of the 1:1 CHIR-AB1/IgY interaction affinity indicates a relatively low affinity complex, but a 2:1 CHIR-AB1/IgY interaction allows an increase in apparent affinity due to avidity effects when the receptor is tethered to a surface. Taken together, these results add to the structural understanding of Fc receptors and their functional mechanisms.