Production of the haemopoietic growth factors GM-CSF and interleukin-3 by mast cells in response to IgE receptor-mediated activation

Mast cells have a central role in allergic diseases mediated by specific immunoglobulin E antibody responses to allergens. The binding of IgE to the high-affinity receptor for IgE (Fc epsilon R) on mast cells and basophils enables these cells to react specifically to allergens. Such contact leads to the activation of mast cells and the release of histamine and other pharmacological mediators, causing an immediate hypersensitivity and acute inflammatory reactions, accompanied by the development of allergic symptoms. Here we show that Fc epsilon R-mediated activation of murine mast cells results in the production of the haemopoietic growth factors granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3). IL-3 and GM-CSF, in addition to their role in bone marrow haemopoiesis, also influence inflammation as they have the capacity to recruit, prime and activate inflammatory cells such as neutrophils, macrophages and eosinophils. Secretion of these factors by mast cells in response to allergens may therefore have an important role in local tissue defense.     

Engagement of the high-affinity IgE receptor activates src protein-related tyrosine kinases.

The high-affinity IgE receptor (Fc epsilon RI), which is expressed on the surface of mast cells and basophils, has a central role in immediate allergic responses. In the rat basophilic leukaemia cell line RBL-2H3, which is a model system for the analysis of Fc epsilon RI-mediated signal transduction, surface engagement of Fc epsilon RI induces histamine release and the tyrosine phosphorylation of several distinct proteins. Although the alpha, beta, and gamma subunits of Fc epsilon RI lack intrinsic tyrosine protein kinase (TPK) activity, a kinase that copurifies with Fc epsilon RI phosphorylates the beta and gamma subunits of the receptor on tyrosine residues. We report here that in RBL-2H3 cells, p56lyn and pp60c-src are activated after Fc epsilon RI crosslinking, and p56lyn coimmunoprecipitates with Fc epsilon RI. In the mouse mast-cell line PT-18, another cell type used to study FC epsilon RI-mediated signalling, tyrosine phosphorylation of proteins is also an immediate consequence of receptor crosslinking. Notably, the only detectable src protein-related TPK in PT-18 cells is p62c-yes, and it is this TPK that is activated on Fc epsilon RI engagement and coimmunoprecipitates with the receptor. Therefore, it seems that different src protein-related TPKs can associate with the same receptor and become activated after receptor engagement.     

Phosphorylation and dephosphorylation of the high-affinity receptor for immunoglobulin E immediately after receptor engagement and disengagement.

Triggering of mast cells and basophils by immunoglobulin E (IgE) and antigen induces various biochemical signals, including tyrosine kinase activation, which lead to cell degranulation and the release of mediators of the allergic reaction. The high-affinity receptor for IgE (Fc epsilon RI) responsible for initiating these events is a complex structure composed of an IgE-binding alpha-chain, a beta-chain and a homodimer of gamma-chains. It has been assumed that beta and gamma, which have extensive cytoplasmic domains, play an important but undefined role in coupling Fc epsilon RI to signal transduction mechanisms. Here we show that Fc epsilon RI engagement induces immediate in vivo phosphorylation on beta (tyrosine and serine) and gamma (tyrosine and threonine) by at least two different non-receptor kinases. We take advantage of unique features of this receptor system to demonstrate that the phosphorylation signal is restricted to activated receptors and is immediately reversible upon receptor disengagement by undefined phosphatases. Rapid phosphorylation and dephosphorylation may be a general mechanism to couple and uncouple activated receptors to other effector molecules. This could be particularly relevant to other multimeric receptors containing Fc epsilon RI gamma-chains or the related zeta and eta chains such as the T-cell antigen receptor (TCR) and the low-affinity receptor for immunoglobulin G (Fc gamma RIII, CD16).     

Essential role for Gab2 in the allergic response.

Dos/Gab family scaffolding adapters (Dos, Gab1, Gab2) bind several signal relay molecules, including the protein-tyrosine phosphatase Shp-2 and phosphatidylinositol-3-OH kinase (PI(3)K); they are also implicated in growth factor, cytokine and antigen receptor signal transduction. Mice lacking Gab1 die during embryogenesis and show defective responses to several stimuli. Here we report that Gab2-/- mice are viable and generally healthy; however, the response (for example, degranulation and cytokine gene expression) of Gab2-/- mast cells to stimulation of the high affinity immunoglobulin-epsilon (IgE) receptor Fc(epsilon)RI is defective. Accordingly, allergic reactions such as passive cutaneous and systemic anaphylaxis are markedly impaired in Gab2-/- mice. Biochemical analyses reveal that signalling pathways dependent on PI(3)K, a critical component of Fc(epsilon)RI signalling, are defective in Gab2-/- mast cells. Our data identify Gab2 as the principal activator of PI(3)K in response to Fc(epsilon)RI activation, thereby providing genetic evidence that Dos/Gab family scaffolds regulate the PI(3)K pathway in vivo. Gab2 and/or its associated signalling molecules may be new targets for developing drugs to treat allergy.     

The mast cell binding site on human immunoglobulin E

Antibodies of the immunoglobulin E isotype sensitize mast cells and basophils for antigen-induced mediator release by binding through the Fc portion to a high-affinity receptor (Fc epsilon R1, Ka = 10(9)M-1) on the cell surface causing the clinical manifestations of type I hypersensitivity. As the amino acid sequence of the human epsilon chain is now known, attempts have been made to map the Fc epsilon R1 binding site on IgE to a fragment smaller than Fc epsilon using proteolytic cleavage products, none of which proved to be active. Cleavage between the C epsilon 2 and C epsilon 3 domains released two inactive fragments, suggesting that the junction between these segments could be important in receptor binding. This region is protected against protease digestion in the rat IgE complex with the receptor of rat basophilic leukaemia cells. Here we report the mapping of the mast cell receptor binding site on human IgE to a sequence of 76 amino acids at the C epsilon 2/C epsilon 3 junction. Recombinant peptides containing this sequence inhibit passive sensitization of skin mast cells in vivo and sensitize mast cells to degranulation by anti-IgE in vitro almost as efficiently as a myeloma IgE. Fragments containing the separate domains are inactive. Additional sequences are required for rapid assembly of fragments into disulphide-linked dimers, suggesting that a single chain can form the active site. In a three-dimensional model of the human Fc epsilon, the two identical segments are far apart. Each folds to generate a cleft between the C epsilon 2 and C epsilon 3 domains on the surface of the Fc epsilon. The docking of IgE on to mast cells could take place within this cleft.     

The B-cell binding site on human immunoglobulin E

Immunoglobulin E comprises the main immunoglobulin class associated with allergy. Its multifarious activities are mediated by two types of Fc receptors found on different cell populations, Fc epsilon R1 on mast cells and basophils, and Fc epsilon R2 on inflammatory cells (monocytes, eosinophils and platelets) and B lymphocytes. Recombinant epsilon-chain fragments synthesized in Escherichia coli have provided the means of mapping the receptor-binding sites on human IgE, and blocking IgE-receptor interactions. We have previously shown that the Fc epsilon R1 binding site is contained within a sequence (Gln 301-Arg 376) spanning the C epsilon 2 and C epsilon 3 domains. Here we show that Fc epsilon R2 can recognize a motif in the C epsilon 3 domain that is formed on dimerization of one or both of the flanking (C epsilon 2 and C epsilon 4) domains. Glycosylation of IgE is not required for the activity of either receptor.     

Complete structure and expression in transfected cells of high affinity IgE receptor

The high-affinity receptor for immunoglobulin E, Fc epsilon RI, is found exclusively on mast cells and basophils. When multivalent allergens bind to the receptor-bound IgE, the consequent aggregation of the receptors leads to the release of mediators responsible for allergic symptoms. In rodents Fc epsilon RI is a tetrameric complex of non-covalently attached subunits: one IgE-binding alpha subunit, one beta subunit and a dimer of disulphide-linked gamma subunits. Complementary DNA encoding the alpha and the beta subunits has recently been isolated, but expression of IgE-binding by transfected cells has not yet been achieved. Here we report the cloning of cDNA for the gamma subunit, and propose a model for the alpha beta gamma 2 tetramer which accounts for many of the structural features of the receptor. The rodent receptor on the surface of COS 7 cells was expressed only when the cDNAs for all three subunits were cotransfected. Successful expression of human IgE receptors should now be possible, eventually to permit the detailed analysis of the human IgE-receptor interaction and assist the search for therapeutically effective inhibitors.     

Cell-to-cell spread of calcium signals mediated by ATP receptors in mast cells.

Rat basophilic leukaemia cells, like mast cells from which they are derived, have surface Fc epsilon receptors that trigger secretion of inflammatory mediators when crosslinked. Both GTP-binding proteins and a rise in cytosolic calcium concentration ([Ca2+]i) are implicated in the secretory mechanism. Here we use a video-imaging technique to report that transient rises in [Ca2+]i initiated in an individual cell can spread from cell to cell in a wave-like pattern by means of a secreted intermediate, in the absence of gap-junctional communication. We find that the leukaemia cells, peritoneal mast cells and mucosal mast cells have cell-surface P2-type purinergic receptors that can trigger similar [Ca2+]i transients. We provide evidence that ATP is rapidly released, and that it can amplify [Ca2+]i signals and initial secretory responses during antigen-stimulation of rat basophilic leukaemia cells.     

A macrophage Fc gamma receptor and the mast cell receptor for IgE share an identical subunit

Fc receptors for immunoglobulins are found on many immune cells and trigger essential functions of the immune defence system. With the exception of the high-affinity receptor for immunoglobulin E (Fc epsilon RI), these receptors were thought to consist of single polypeptides. Fc epsilon RI is a tetrameric complex of one alpha-subunit, one beta-subunit and two gamma-subunits. Here we report the cloning of a polypeptide identical to the gamma-chains of Fc epsilon RI, from mouse macrophages that do not express this receptor. Biosynthetic labelling and gene transfer together show that these gamma-chains associate with one of the macrophage receptors (Fc gamma RIIa). The human homologue, Fc gamma RIII (CD16), from natural killer cells is also expected to associate with gamma-chains. It is possible that these gamma-chains and the homologous zeta-chains of the T-cell antigen receptor belong to a new family of related proteins which share a common role in the signal transducing pathway.     

Lymphokine-induced IgM secretion by clones of neoplastic B cells

The induction of antibody secretion by B cells requires T-cell-derived factors1-5. Such factors have been described1,2,6-12 but the precise relationship among these various factors is not clear, and it has been difficult to demonstrate that these factors act directly on the B cell and do not exert their effect via T cells or macrophages. In this report we describe the direct induction of IgM synthesis and secretion in cloned lines of long-term tissue culture adapted neoplastic B cells (BCL1) by T-cell supernatants from phorbol-12-myristate 13-acetate (PMA)-induced EL-4 cells or concanavalin A (Con A)-induced 7.1.1a cells5,9. We have termed this activity BCDFmu (B-cell differentiation factor for IgM). The supernatants containing BCDFmu induce activated and neoplastic B cells to secrete IgM5 and the factor responsible is distinct from BCGF13, interleukin-2 (IL-2)5, the classical T-cell replacing factor (TRF) described by Schimpl and Wecker5, and immune interferon (IFN gamma)5.     

Cloning of complementary DNA encoding T-cell replacing factor and identity with B-cell growth factor II

Proliferation and maturation of antigen-stimulated B cells are regulated by several soluble factors derived from macrophages and T cells. These soluble factors are functionally divided into two groups: B-cell growth factor (BCGF), thought to be involved in B-cell proliferation; and B-cell differentiation factor (BCDF), responsible for maturation of activated B cells into immunoglobulin-secreting cells. This classification needs to be re-examined in the light of the recent cloning of complementary DNA encoding IgG1 induction factor (interleukin-4, IL-4) from the 2.19 mouse T-cell line. Recombinant IL-4 has BCGF and BCDF activities and affects B cells, T cells and mast cells (refs 7, 8; our unpublished data). Another well-characterized B-cell factor is T-cell replacing factor (TRF), which, when secreted by the murine T-cell hybridoma B151K12, is defined by two activities: induction of IgM secretion by BCL1 leukaemic B-cell line; and induction of secondary anti-dinitrophenol (DNP) immunoglobulin G (IgG) synthesis in vitro by DNP-prime B cells. Although TRF from B151K12 was classified as BCDF, purified TRF has BCGF-II activity. To elucidate the molecular properties of TRF we isolated cDNA encoding TRF from the 2.19 T-cell line and report here the structure and multiple activities of this lymphokine.     

A hapten-specific chimaeric IgE antibody with human physiological effector function

Immunoglobulin E (IgE) has a central role in allergic reactions although it rarely exceeds 5 micrograms ml-1 even in the serum of severely allergic individuals. Both mast cells and basophils possess receptors which bind the Fc portion of IgE with high affinity; crosslinking of membrane-bound IgE by allergen results in degranulation of the cell and release of a variety of pharmacologically active mediator including histamine. Myeloma IgE has been successfully used to block the skin sensitizing activity of allergic sera; however, human myeloma IgE is clearly in limited supply. The emergence of techniques allowing the stable introduction of immunoglobulin gene DNA into myeloma cells has allowed us to construct a mouse cell line that secretes a chimaeric IgE, lambda 1 antibody whose heavy chain is composed of a human C epsilon constant region fused to a mouse variable (VH) region. This chimaeric IgE is specific for the hapten 4-hydroxy-3-nitro-phenacetyl (NP) and can, when crosslinked by antigen, trigger the degranulation of human basophils. When not crosslinked, however, the chimaeric IgE can prevent the passive sensitization of these cells by sera from allergic subjects.     

Receptors for B-cell stimulatory factor-1 expressed on cells of haematopoietic lineage

B-cell stimulatory factor-1 (BSF-1) is a T-cell product of relative molecular mass 20,000 (Mr, 20K) initially described as a cofactor required for DNA synthesis by resting mouse B cells stimulated with low concentrations of anti-IgM antibodies. It acts on resting B cells to enhance the expression of class II major histocompatibility complex (MHC) molecules, to prepare these cells to respond more promptly to subsequent stimuli, such as anti-IgM antibodies, and causes the secretion of IgG1 and IgE by B cells stimulated with lipopolysaccharide (LPS). BSF-1 has been shown to stimulate T cell lines, resting T cells and some mast cell lines. Recently, the designation interleukin-4 (IL-4) has been suggested for BSF-1. We report here the existence of high-affinity cell-surface receptors specific for BSF-1 on both B and T lymphocytes, and on cells of several other haematopoietic lineages, including mast cell, macrophage and undifferentiated haematopoietic cell lines. Resting B and T lymphocytes express receptors, which increase in number upon activation of B cells with LPS or anti-IgM, and of T cells with concanavalin A. Cross-linking of 125I-labelled-BSF-1 to its receptors creates a complex of Mr approximately 80,000.     

Tyrosine-containing motif that transduces cell activation signals also determines internalization and antigen presentation via type III receptors for IgG.

Type III receptors for IgG (Fc gamma RII; ref. 1), high-affinity IgE receptors (Fc epsilon RI; ref. 2), as well as the T- and B-cell antigen receptors, consist of multiple components with specialized ligand-binding and signal transduction functions. Fc gamma RII alpha (ligand-binding) and gamma (signal-transducing) subunits are expressed in macrophages, a cell type involved in the uptake of antigen, its processing and the presentation of the resulting peptides to major histocompatibility complex class II-restricted T lymphocytes. Here we show that murine Fc gamma RIII, transfected into Fc gamma R-negative antigen-presenting B-lymphoma cells, mediate rapid ligand internalization and strongly increase the efficiency of antigen presentation when antigen is complexed to IgG. Efficient internalization and antigen presentation via Fc gamma RIII did not require the cytoplasmic domain of the ligand-binding alpha-chain, but did require the gamma-subunit. Using chimaeric molecules, we show that gamma-chain contains a signal for receptor internalization and that the mutation of either of the two tyrosine residues present in its cytoplasmic domain prevents efficient internalization and antigen presentation of immune complexes. Thus, associated chains and their tyrosine-containing motif are not exclusively involved in cell activation, but also determine multimeric receptor internalization.     

A single amino acid in the glycosyl phosphatidylinositol attachment domain determines the membrane topology of Fc gamma RIII

Cell-surface proteins are associated with the lipid bilayer either as membrane-spanning molecules or as glycosyl phosphatidylinositol (GPtdIns)-linked proteins. Proteins destined for GPtdIns anchoring are synthesized as precursors with a hydrophobic C-terminal transmembrane domain, which is removed during the processing of these proteins in the endoplasmic reticulum (ref. 1). We have investigated the structural requirements for GPtdIns anchoring through the study of two closely related proteins which exhibit alternative membrane attachment. The IgG Fc receptor, Fc gamma RIII, is GPtdIns-linked on neurophils (III-1) whereas on natural killer (NK) cells and macrophages it is found as a transmembrane-anchored molecule (III-2), able to mediate antibody-dependent cellular cytotoxicity and phagocytosis. At the primary structural level, the III-1 gene differs from that encoding III-2 by only nine nucleotide substitutions, which result in six amino-acid differences, and the absence of 21 amino acids at the C terminus. We have analysed a series of III-1 and III-2 mutants in transient expression assays, and show that Ser 203 in the GPtdIns attachment domain is the dominant residue in determining whether the molecule can be GPtdIns-anchored. As in the case of its murine homologue, Fc gamma RII alpha, surface expression of the III-2 molecule is dependent on co-expression of a second subunit, the gamma chain of F epsilon RI. Our data also suggest that gamma chain can associate with the III-1 precursor, preventing GPtdIns attachment, favouring instead a transmembrane form.     

Extensive proliferation of mature connective-tissue type mast cells in vitro

There are two phenotypically distinct subpopulations of mast cells in rodents: connective tissue-type mast cells (CTMC) and mucosal mast cells (MMC). These populations differ in their location, cell size, staining characteristics, ultrastructure, mediator content and T-cell dependency. Several investigators recently reported a further subclass of mast cells which arise when normal mouse haematopoietic cells are cultured with interleukin-3 (IL-3); IL-3 is an activity similar or identical to mast-cell growth factor, histamine-producing factor, or P-cell stimulating factor. These cultured mast cells are in many ways similar to MMC; they stain with Alcian blue but not safranin, contain chondroitin sulphate E proteoglycan rather than heparin proteoglycan and have relatively low histamine content, as do MMC. Although proliferation of MMC is known to be T-cell dependent in vivo and thought to be IL-3-dependent in vitro, the factors on which CTMC proliferation depends remain elusive. Here we show that mature CTMC purified from mouse peritoneal cells can proliferate in vitro in methylcellulose culture and maintain the appearance and function of CTMC. We also present evidence that mature CTMC cannot proliferate in the presence of pure IL-3 alone.     

Leukaemia inhibitory factor is identical to the myeloid growth factor human interleukin for DA cells

Leukaemia inhibitory factor (LIF) is a cytokine that induces macrophage differentiation of the murine M1 myeloid leukaemia cell line. We have isolated a cDNA clone encoding a novel human haemopoietic growth factor, human interleukin for DA cells (HILDA) that supports the proliferation of the murine interleukin-3-dependent leukaemic cell line, DA-la (refs 3-5). HILDA proved to be identical to LIF. The demonstration that the differentiation factor LIF will also serve as a growth factor for at least one myeloid leukaemic cell line provides further evidence that the distinction between growth-promoting and differentiation-inducing activities are largely determined by the target cell type.     

A cloned cell with NK function resembles basophils by ultrastructure and expresses IgE receptors

Natural killer (NK) cells are defined by their ability to lyse certain tumour cells in vitro without previous exposure to them, and have been postulated as effectors of immune surveillance against spontaneous neoplasms. Because they kill some non-neoplastic lymphoid cells, they may also have a role in immunoregulation. NK cell activity resides in a small proportion of normal mouse spleen cells (less than 5%) that have been difficult to characterize completely. They may represent a heterogeneous group of effector cells whose precise relationship to other myelopoietic or immunological cells has remained obscure. We have previously described a cloned mouse cell line (Cl. Ly 1-2-NK-1+/11) with the functional characteristics of natural killer cells activated by interferon or other factors. We now find that this cloned line, like basophils and mast cells, expresses high-affinity plasma membrane receptors (Fc epsilon R) specific for IgE antibody. In addition, the clone contains cytoplasmic granules similar by ultrastructure to those of basophils of the mouse and other species. Our findings indicate that cells sharing morphological and biochemical features of basophilic granulocytes can mediate NK lysis.     

Structure of the Fc fragment of human IgE bound to its high-affinity receptor Fc epsilonRI alpha

The initiation of immunoglobulin-E (IgE)-mediated allergic responses requires the binding of IgE antibody to its high-affinity receptor, Fc epsilonRI. Crosslinking of Fc epsilonRI initiates an intracellular signal transduction cascade that triggers the release of mediators of the allergic response. The interaction of the crystallizable fragment (Fc) of IgE (IgE-Fc) with Fc epsilonRI is a key recognition event of this process and involves the extracellular domains of the Fc epsilonRI alpha-chain. To understand the structural basis for this interaction, we have solved the crystal structure of the human IgE-Fc-Fc epsilonRI alpha complex to 3.5-A resolution. The crystal structure reveals that one receptor binds one dimeric IgE-Fc molecule asymmetrically through interactions at two sites, each involving one C epsilon3 domain of the IgE-Fc. The interaction of one receptor with the IgE-Fc blocks the binding of a second receptor, and features of this interaction are conserved in other members of the Fc receptor family. The structure suggests new approaches to inhibiting the binding of IgE to Fc epsilonRI for the treatment of allergy and asthma.     

Insights into IgA-mediated immune responses from the crystal structures of human FcalphaRI and its complex with IgA1-Fc

Immunoglobulin-alpha (IgA)-bound antigens induce immune effector responses by activating the IgA-specific receptor FcalphaRI (CD89) on immune cells. Here we present crystal structures of human FcalphaRI alone and in a complex with the Fc region of IgA1 (Fcalpha). FcalphaRI has two immunoglobulin-like domains that are oriented at approximately right angles to each other. Fcalpha resembles the Fcs of immunoglobulins IgG and IgE, but has differently located interchain disulphide bonds and external rather than interdomain N-linked carbohydrates. Unlike 1:1 FcgammaRIII:IgG and Fc epsilon RI:IgE complexes, two FcalphaRI molecules bind each Fcalpha dimer, one at each Calpha2-Calpha3 junction. The FcalphaRI-binding site on IgA1 overlaps the reported polymeric immunoglobulin receptor (pIgR)-binding site, which might explain why secretory IgA cannot initiate phagocytosis or bind to FcalphaRI-expressing cells in the absence of an integrin co-receptor.