Mast cells lacking the high affinity immunoglobulin E receptor are deficient in Fc epsilon RI gamma messenger RNA

A population of cells that express mast cell markers, including the membrane protein p161, but that lack expression of the high affinity IgE receptor, Fc epsilon RI, can be routinely grown from bone marrow. Ionomycin, but not IgE immune complexes, causes these cells to release serotonin and to express IL-3 and IL-13 mRNA, consistent with their being FC epsilon RI-deficient mast cells. These p161+/Fc epsilon RI- mast cells expressed normal amounts of Fc epsilon RI alpha and beta chain mRNA, but extremely low levels of Fc epsilon RI gamma chain mRNA. In addition, this novel mast cell population expressed CD3 zeta chain mRNA, which p161+/Fc epsilon RI+ mast cells did not. CD3 zeta stable transfectants of Abelson-murine leukemia virus-transformed p161+/Fc epsilon RI+ mast cells continued to express Fc epsilon RI. This strongly suggests that the failure of p161+/Fc epsilon RI- mast cells to express IgE receptors was not caused by the presence of CD3 zeta chain. Transfection of human Fc epsilon RI gamma cDNA into p161+/Fc epsilon RI- mast cells rescued IgE binding. These stable transfectants released serotonin in response to cross-linkage of Fc epsilon RI, demonstrating that the molecular defect of p161+/Fc epsilon RI- mast cells is indeed the loss of Fc epsilon RI gamma expression.     

Expression of functional high affinity immunoglobulin E receptors (Fc epsilon RI) on monocytes of atopic individuals

Suggestive evidence indicates that immunoglobulin E (IgE)-dependent activation of mononuclear phagocytes plays an important pathogenic role in allergic tissue inflammation. Prevailing opinion holds that low affinity IgE receptors are the relevant IgE-binding structures on monocytes/macrophages and that functional events occurring after cross- linking of membrane-bound IgE on these cells are mediated by these receptors. Here we demonstrate that peripheral blood monocytes can bind monomeric IgE via the high affinity IgE receptor (Fc epsilon RI) and that Fc epsilon RI expression on these cells is upregulated in atopic persons. Further, we demonstrate that, upon monocyte adherence to substrate, bridging of monocyte Fc epsilon RI is followed by cell activation. We propose that direct interaction of multivalent allergen with Fc epsilon RI(+)-bound IgE on mononuclear phagocytes results in cell signaling via Fc epsilon RI and that the biological consequences of this event may critically influence the outcome of allergic reactions.     

Identification of the low affinity receptor for immunoglobulin E on mouse mast cells and macrophages as Fc gamma RII and Fc gamma RIII

In addition to their well characterized high affinity immunoglobulin E (IgE) receptors (Fc epsilon RI) mast cells have long been suspected to express undefined Fc receptors capable of binding IgE with low affinity. In this paper, we show that Fc gamma RII and Fc gamma RIII, but not Mac-2, on mouse mast cells and macrophages bind IgE-immune complexes. This binding is efficiently competed by 2.4G2, a monoclonal antibody against the extracellular homologous region of both Fc gamma RII and Fc gamma RIII. Furthermore, IgE-immune complexes bind specifically to Fc gamma RII or Fc gamma RIII transfected into COS-7 cells. The association constants of IgE binding estimated from competition experiments are about 3.1 x 10(5) M-1 for Fc gamma RII, and 4.8 x 10(5) M-1 for Fc gamma RIII. Engagement of Fc gamma RII and Fc gamma RIII with IgE-immune complexes (after blocking access to Fc epsilon RI) or with IgG-immune complexes triggers C57.1 mouse mast cells to release serotonin. This release is inhibited by 2.4G2, and at maximum, reaches 30-40% of the intracellular content, about half of the maximal release (60-80%) obtained after Fc epsilon RI engagement. These data demonstrate that mouse Fc gamma RII and Fc gamma RIII are not isotype specific, and that the binding of IgE-immune complexes to these receptors induces cell activation.     

Promotion of mouse fibroblast collagen gene expression by mast cells stimulated via the Fc epsilon RI. Role for mast cell-derived transforming growth factor beta and tumor necrosis factor alpha

Chronic allergic diseases and other disorders associated with mast cell activation can also be associated with tissue fibrosis, but a direct link between mast cell mediator release and fibroblast collagen gene expression has not been established. Using in situ hybridization, we show that the elicitation of an IgE-dependent passive cutaneous anaphylaxis (PCA) reaction in mice results in a transient, but marked augmentation of steady state levels of type alpha-1 (I) collagen mRNA in the dermis. While peak levels of collagen mRNA expression in the skin are observed 16-24 h after mast cell activation, substantial numbers of dermal cells are strongly positive for collagen mRNA at 1 and 2 h after antigen challenge, before circulating inflammatory cells are recruited into the tissues. Furthermore, experiments in mast cell- reconstituted or genetically mast cell-deficient WBB6F1-W/Wv mice demonstrate that the increased expression of collagen mRNA at sites of PCA reactions is entirely mast cell dependent. In vitro studies show that the supernatants of mouse serosal mast cells activated via the Fc epsilon RI markedly increase type alpha-1 (I) collagen mRNA levels in mouse embryonic skin fibroblasts, and also upregulate collagen secretion by these cells. The ability of mast cell supernatants to induce increased steady state levels of collagen mRNA in mouse skin fibroblasts is markedly diminished by absorption with antibodies specific for either of two mast cell-derived cytokines, transforming growth factor beta (TGF-beta 1) or tumor necrosis factor alpha (TNF- alpha), and is eliminated entirely by absorption with antibodies against both cytokines. Taken together, these findings demonstrate that IgE-dependent mouse mast cell activation can induce a transient and marked increase in steady state levels of type alpha-1 (I) collagen mRNA in dermal fibroblasts and that mast cell-derived TGF-beta 1 and TNF-alpha importantly contribute to this effect.     

The beta subunit of the Fc epsilon RI is associated with the Fc gamma RIII on mast cells

Fc epsilon RI is a tetrameric receptor, composed of a ligand recognition subunit, alpha, a beta chain, and dimeric gamma chains. Previous studies have indicated that the dimeric gamma chain is associated with Fc gamma RIIIA (CD16) on natural killer cells and macrophages as well as the clonotypic T cell receptor. Here we show that in mast cells, in addition to the dimeric gamma chains, the beta subunit is associated not only with Fc epsilon RI, but also with Fc gamma RIIIA. Functional reconstitution studies with a mastocytoma cell line indicate that Fc gamma RIIIA composed of alpha, beta, and gamma subunits has the capacity for signal transduction. These studies suggest that through the association of alternative ligand recognition subunits (alpha epsilon, alpha gamma), a common signal transduction complex (beta gamma 2) mediates similar biochemical and effector functions in response to immunoglobulin G (IgG) and IgE.     

Release of both preformed and newly synthesized tumor necrosis factor alpha (TNF-alpha)/cachectin by mouse mast cells stimulated via the Fc epsilon RI. A mechanism for the sustained action of mast cell-derived TNF-alpha during IgE-dependent biological responses.

Mast cell-associated mediators are generally classified into two groups: the preformed mediators, which are stored in the cells' cytoplasmic granules and are released upon exocytosis, and the newly synthesized mediators, which are not stored but are produced and secreted only after appropriate stimulation of the cell. We now report that tumor necrosis factor alpha (TNF-alpha)/cachectin represents a new type of mast cell-associated mediator, in that IgE-dependent mast cell activation results in the rapid release of preformed stores of the cytokine followed by the synthesis and sustained release of large quantities of newly formed TNF-alpha. We also demonstrate that challenge with specific antigen induces higher levels of TNF-alpha mRNA at skin sites sensitized with IgE in normal mice or mast cell-reconstituted genetically mast cell-deficient WBB6F1-W/W1' mice than at identically treated sites in WBB6F1-W/W1' mice that are devoid of mast cells. These findings identify mast cells as a biologically significant source of TNF-alpha/cachectin during IgE-dependent responses and define a mechanism whereby stimulation of mast cells via the FC epsilon RI can account for both the rapid and sustained release of this cytokine.     

Ontogeny of Fc receptors and complement receptor (CR3) during human myeloid differentiation.

Two different Fc receptors for IgG (Fc gamma R) have been identified on human leukocytes: a high avidity receptor (Fc gamma Rhi) present on monocytes but not on neutrophils, and a low avidity receptor (Fc gamma Rlo) present on neutrophils but not on monocytes. Fc gamma Rlo can be inhibited and the receptor precipitated by monoclonal antibody 3G8. We have used this monoclonal antibody to study the course of Fc gamma Rlo appearance on bone marrow cells, leukocytes of patients with chronic myelogenous leukemia (CML), and HL-60 and U937 cells induced to differentiate with agents such as dimethyl sulfoxide (DMSO), retinoic acid, phorbol myristate acetate, and lymphokine. We report that Fc gamma Rlo is a late differentiation antigen, first expressed at the metamyelocyte stage. Since precursors to metamyelocytes bear Fc gamma R, and the promyelocyte line HL-60 bears Fc gamma Rhi, there must be a progressive loss of Fc gamma Rhi during myeloid differentiation and the reciprocal expression of Fc gamma Rlo. Results of immunoprecipitation and polyacrylamide gel analysis of the proteins are consistent with these results. We have also studied the receptor for the C3bi complement component (CR3), which is blocked and immunoprecipitated by monoclonal antibody OKM10. During DMSO-driven differentiation of HL-60 cells, we find that CR3 is induced on all cells, whereas Fc gamma Rlo is induced on only 24% of cells, suggesting that CR3 appears earlier during differentiation than Fc gamma Rlo does.     

Immunopharmacology of human mast cells and basophils

Human mast cells and basophils play a key role in the pathogenesis of several immunological and inflammatory disorders, not only by producing inflammatory and fibrogenic mediators, but also by directly (CD40 ligand) and indirectly secreting various cytokines and chemokines. Studies carried out to evaluate the effects of drugs that modulate the release of mediators and cytokines from these cells have contributed to clarifying the biochemical mechanism by which immunological and non-immunological stimuli activate these cells. Significant differences have been documented between human mast cells and basophils as regard the pharmacological agents that modulate the release of mediators, between mast cells isolated from different anatomical sites, and between compounds of the same class of drugs. Efforts to gain insight into the biochemical events occurring during immunological activation of mast cells and basophils could lead to the identification of new biochemical targets for therapeutic interventions in several immunological disorders.     

Different roles for the Fc epsilon RI gamma chain as a function of the receptor context

The high affinity immunoglobulin E receptor (Fc epsilon RI) and the B and T cell antigen receptors (TCR) are multimeric complexes containing subunits with cytoplasmic antigen recognition activation motifs (ARAMs). The presence of multiple motifs may be a way to amplify a single signal or provide independent activation modules. Here we have compared the signaling capacity of the same Fc epsilon RI gamma motif in the context of two different receptors, Fc epsilon RI and TCR/CD3, simultaneously reconstituted on the surface of the same zeta-deficient T cell line. Both reconstituted receptors mediate early (phosphorylation) and late (interleukin [IL]-2 release) signals. Mutation of the two tyrosine residues of ARAM gamma alters early signaling by both receptors, but the set of substrates phosphorylated via ARAM gamma is different for each receptor and is thus dependent on the receptor context. Furthermore, the mutations prevent Fc epsilon RI- but not TCR/CD3-mediated IL-2 release. These data demonstrate that ARAM gamma is necessary for allowing both receptors to phosphorylate the complete set of substrates, and that the CD3 complex, unlike the Fc epsilon RI beta chain, contains activation modules capable of compensating for the absence of a functional ARAM gamma in generating late signals such as IL-2 release.     

WIP regulates signaling via the high affinity receptor for immunoglobulin E in mast cells

Wiskott-Aldrich syndrome protein-interacting protein (WIP) stabilizes actin filaments and is important for immunoreceptor-mediated signal transduction leading to actin cytoskeleton rearrangement in T and B cells. Here we report a role for WIP in signaling pathways downstream of the high affinity receptor for immunoglobulin (Ig)E (FcepsilonRI) in mast cells. WIP-deficient bone marrow-derived mast cells (BMMCs) were impaired in their capacity to degranulate and secrete interleukin 6 after FcepsilonRI ligation. Calcium mobilization, phosphorylation of Syk, phospholipase C-g2, and c-Jun NH2-terminal kinase were markedly decreased in WIP-deficient BMMCs. WIP was found to associate with Syk after FcepsilonRI ligation and to inhibit Syk degradation as evidenced by markedly diminished Syk levels in WIP-deficient BMMCs. WIP-deficient BMMCs exhibited no apparent defect in their subcortical actin network and were normal in their ability to form protrusions when exposed to an IgE-coated surface. However, the kinetics of actin changes and the cell shape changes that follow FcepsilonRI signaling were altered in WIP-deficient BMMCs. These results suggest that WIP regulates FcepsilonRI-mediated mast cell activation by regulating Syk levels and actin cytoskeleton rearrangement.     

Identification of Fc epsilon RIneg mast cells in mouse bone marrow cell cultures. Use of a monoclonal anti-p161 antibody

A monoclonal hamster antibody (K-1) specific for a 161-kD mast cell surface glycoprotein was derived. p161 is expressed on normal and cultured mast cells and on some macrophages, but not on basophils or other hematopoietic cells. A population of Fc epsilon Rneg cells expressing p161 was found in short term cultures of bone marrow cells grown in interleukin (IL)-3. These cells were purified and propagated for extended periods in IL-3. They express c-kit and Fc gamma RII/III, contain alcian blue-positive granules and histamine, and secrete IL-3 in response to ionomycin treatment. Their morphology is consistent with that of mast cells. We propose that they represent Fc epsilon RIneg mast cells that can be detected and purified because of their p161 expression.     

Physical and functional association of the high affinity immunoglobulin G receptor (Fc gamma RI) with the kinases Hck and Lyn

The high affinity immunoglobulin G (IgG) receptor Fc gamma RI (CD64) is expressed constitutively on monocytes and macrophages, and is inducible on neutrophils. Fc gamma RI has recently been shown to be associated with the signal transducing gamma subunit of the high-affinity IgE receptor (Fc epsilon RI gamma). Induction of cytoplasmic protein tyrosine phosphorylation by Fc gamma RI cross-linking is known to be important in mediating Fc gamma RI-coupled effector functions. Recently, syk has been implicated in this role. We now report that the src-type kinases hck and lyn are physically and functionally associated with Fc gamma RI. Hck and lyn coimmunoprecipitated with Fc gamma RI from detergent lysates of normal human monocytes and of the monocytic line THP-1. Hck and lyn showed rapidly increased phosphorylation and increased exogenous substrate kinase activity after cross-linking of Fc gamma RI. These results demonstrate both physical and functional association of the Fc gamma RI/Fc epsilon RI gamma receptor complex with hck and lyn, and suggest a potential signal transducing role for these kinases in monocyte/macrophage activation.     

Antigen-independent induction of histamine synthesis by immunoglobulin E in mouse bone marrow-derived mast cells

Immunoglobulin (Ig)E-mediated activation of mast cells has long been thought to occur only when Fc(epsilon)RI receptor-bound IgE is cross-linked via multivalent antigens. However, recent studies have raised the possibility that mast cells may be activated by the binding of IgE to the Fc(epsilon)RI receptor in the absence of antigen. Here we demonstrate that IgE binding without antigen induces the expression of histidine decarboxylase (HDC) in mouse interleukin (IL)-3-dependent bone marrow-derived mast cells (BMMCs). The induction of HDC by the binding of IgE was found to require an influx of extracellular calcium ions, which was attenuated by pretreatment with U73122, a phospholipase C inhibitor. Furthermore, the increase in HDC activity upon sensitization with IgE was completely suppressed by pretreatment of BMMCs with protein kinase C inhibitors, such as H7, staurosporine, and G?6976. In addition, immediate activation of the tyrosine kinase Lyn was not detectable upon treatment with IgE. These results suggest that the binding of IgE to its receptor in the absence of antigen results in de novo synthesis of HDC in BMMCs through a signaling pathway distinct to that operating during antigen-stimulated Fc(epsilon)RI activation.     

The balance between sphingosine and sphingosine-1-phosphate is decisive for mast cell activation after Fc epsilon receptor I triggering.

Over the last few years, sphingolipids have been identified as potent second messenger molecules modulating cell growth and activation. A newly emerging facet to this class of lipids suggests a picture where the balance between two counterregulatory lipids (as shown in the particular example of ceramide and sphingosine-1-phosphate in T lymphocyte apoptosis) determines the cell fate by setting the stage for various protein signaling cascades. Here, we provide a further example of such a decisive balance composed of the two lipids sphingosine and sphingosine-1-phosphate that determines the allergic responsiveness of mast cells. High intracellular concentrations of sphingosine act as a potent inhibitor of the immunoglobulin (Ig)E plus antigen-mediated leukotriene synthesis and cytokine production by preventing activation of the mitogen-activated protein kinase pathway. In contrast, high intracellular levels of sphingosine-1-phosphate, also secreted by allergically stimulated mast cells, activate the mitogen-activated protein kinase pathway, resulting in hexosaminidase and leukotriene release, or in combination with ionomycin, give cytokine production. Equivalent high concentrations of sphingosine-1-phosphate are dominant over sphingosine as they counteract its inhibitory potential. Therefore, it might be inferred that sphingosine-kinase is pivotal to the activation of signaling cascades initiated at the Fc epsilon receptor I by modulating the balance of the counterregulatory lipids.     

Mechanism of formation of human IgE-binding factors (soluble CD23): III. Evidence for a receptor (Fc epsilon RII)-associated proteolytic activity

There is mounting evidence that Fc epsilon RII (CD23) and its soluble fragments (IgE-binding factors [BFs] or soluble CD23) have pleiotropic activities. IgE-BFs are formed mainly by the proteolytic cleavage of surface Fc epsilon RII; they are first released as 37- and 33-kD unstable molecules that are subsequently transformed into 25-kD IgE-BFs. In this study, purified and radioiodinated 37-kD IgE-BFs as well as 45-kD Fc epsilon RII were used as substrates to identify the proteases leading to the formation of 25-kD IgE-BFs. These substrates generate 25-kD IgE-BFs when incubated with several Fc epsilon RII-bearing cells, including CHO1-7 cells (transfected with Fc epsilon RII cDNA); by contrast Fc epsilon RII- cells, including CHO control cells, have no effect. Highly purified unlabeled native 37-kD and recombinant 29-kD IgE-BFs also cleave labeled 45-kD Fc epsilon RII into 25-kD IgE-BFs. The proteolytic activity of these purified IgE-BFs is specifically removed by immunoprecipitation with an antibody against IgE-BFs. These data strongly suggest that Fc epsilon RII and some of its soluble fragments play an active role in the proteolytic mechanism generating IgE-BFs. They are supported by the observation that IgE-BFs released by CHO1-7 cells are cleaved exactly at the same sites as B cell-derived IgE-BFs. Taken collectively, the results are compatible with an autoproteolytic process.     

Isolation and expression of cDNA clones encoding a human receptor for IgG (Fc gamma RII)

We have cloned and expressed a cDNA encoding a human receptor for IgG (Fc gamma R) from the monocyte cell line U937. The deduced structure is a 35-kD transmembrane protein with homology to the mouse Fc[gamma 2b/gamma 1] receptor amino acid sequence of approximately 60% in the extracellular domain. The signal sequence is homologous to the mouse Fc gamma R alpha cDNA clone, while the transmembrane domain shares homology with mouse Fc gamma R beta cDNAs. The cytoplasmic domain is apparently unique. The extracellular domain shows significant homology to proteins of the Ig gene superfamily, including the human c-fms protooncogene/CSF-1 receptor. Mouse Ltk- cells transfected with the human Fc gamma R cDNA express a cell-surface receptor that selectively binds human IgG and is recognized by the anti-Fc gamma RII mAb IV.3. Antibodies against peptides derived from the human Fc gamma R sequence specifically stain U937 cells, but not an Fc gamma RII-bearing B-lymphoblastoid cell line (Daudi). These results identify the human Fc gamma RII as the homologue of mouse Fc[gamma 2b/gamma 1] R, and provide evidence for heterogeneity of Fc gamma RII expressed on monocytes and B cells.