Mast cell activation: A complex interplay of positive and negative signaling pathways

Mast cells regulate the immunological responses causing allergy and autoimmunity, and contribute to the tumor microenvironment through generation and secretion of a broad array of preformed, granule‐stored and de novo synthesized bioactive compounds. The release and production of mast cell mediators is the result of a coordinated signaling machinery, followed by the FcεRI and FcγR antigen ligation. In this review, we present the latest understanding of FcεRI and FcγR signaling, required for the canonical mast cell activation during allergic responses and anaphylaxis. We then describe the cooperation between the signaling of FcR and other recently characterized membrane‐bound receptors (i.e., IL‐33R and thymic stromal lymphopoietin receptor) and their role in the chronic settings, where mast cell activation is crucial for the development and the sustainment of chronic diseases, such as asthma or airway inflammation. Finally, we report how the FcR activation could be used as a therapeutic approach to treat allergic and atopic diseases by mast cell inactivation. Understanding the magnitude and the complexity of mast cell signaling is necessary to identify the mechanisms underlying the potential effector and regulatory roles of mast cells in the biology and pathology of those disease settings in which mast cells are activated.     

Anti-IgE as a mast cell–stabilizing therapeutic agent

The mast cell–stabilizing effects of current drugs for treating allergic diseases are dissected into 2 parts: (1) the reduction of potency or readiness to release and synthesize mediators on intracellular activation signaling triggered by the aggregation of FcεRI and (2) the reduction of sensitivity to extracellular allergen stimulation caused by the loss of IgE-FcεRI on the mast cell surface. The long-used cromones represent the first class of therapeutics, and the new-concept drug anti-IgE represents the second class. The kinetics of FcεRI reduction on the mast cell surface suggests that such effects should be significant after a few weeks of anti-IgE treatment. In the initial few weeks of anti-IgE treatment, additional pharmacologic mechanisms contribute to anti-IgE's therapeutic effects.     

Human dendritic cell 1 and dendritic cell 2 subsets express FcepsilonRI: correlation with serum IgE and allergic asthma

BACKGROUND: Type 1 dendritic cells (DC1) express the high-affinity IgE receptor (FcepsilonRI); however, the regulation of FcepsilonRI expression by DCs is not well understood. Type 2 DC (DC2) expression of FcepsilonRI has not been demonstrated. OBJECTIVE: We hypothesized that DC2 cellsalso express FcepsilonRI and that expression of FcepsilonRI by the DC1 and DC2 subsets correlates with serum IgE and allergic asthma disease status. METHODS: To test these hypotheses, we quantitated FcepsilonRI alpha chain expression by the peripheral blood precursor DC1 (pDC1) and pDC2 subsets by using flow cytometry. RESULTS: FcepsilonRI was expressed by the pDC1 and pDC2 subsets, as well as tissue DCs from tonsils. Relative FcepsilonRI expression by basophil, pDC1, and pDC2 subsets was 12:6.5:1, respectively. In both pDC subsets, FcepsilonRI expression was significantly greater in allergic asthmatic subjects than in nonatopic control subjects. pDC1 and pDC2 expression of FcepsilonRI was highly correlated to serum IgE concentration. The pDC1, pDC2, and basophil subsets demonstrated a similar magnitude of increase in FcepsilonRI expression relative to changes in serum IgE. CONCLUSIONS: FcepsilonRI expression is characteristic of both the DC1 and DC2 subsets. Furthermore, FcepsilonRI expression by these cells is highly correlated to serum IgE and to basophil FcepsilonRI expression and is greater in subjects with allergic asthma. These data support the concept that novel therapeutic approaches directly targeted at FcepsilonRI expression would affect both the sensitization and the effector phases of the allergen-specific immune response.     

High‐affinity immunoglobulin E receptor expression is increased in large and small airways in fatal asthma

Background IgE and its high‐affinity receptor Fc?RI play an important role in allergy and asthma. The distribution of Fc?RI expression in the airways and within the airway wall, however, is largely unknown. Objective In this study, we aimed to map the distribution of Fc?RI in different layers of large airways (LA) and small airways (SA) in lung tissue from non‐smoking and smoking patients who died of asthma [fatal asthma (FA)] and non‐smoking controls (CTR). Methods Postmortem lung tissue from 24 cases of non‐smoking FA, 13 smoking FA patients and from 19 subjects who died of non‐pulmonary causes (CTR) was immunohistochemically stained for Fc?RI and AA1 (mast cell tryptase marker). The expression of these markers was analysed in inner, muscle, and outer layers of both LA and SA by image analysis. Results Fc?RI expression was higher in non‐smoking and smoking FA compared with CTR in the inner and outer layer of SA. In the outer layer of LA, Fc?RI expression was higher in non‐smoking FA compared with CTR. AA1 was higher in non‐smoking FA compared with smoking FA and CTR in the outer layer of the SA, which was correlated with Fc?RI in this layer. Conclusion Our results show that the expression of Fc?RI is higher in both LA and SA in FA compared with CTR. These differences are predominantly found in the outer layer where they can be attributed in part to the increased mast cell numbers. These results indicate an increased capacity to mount IgE‐mediated reactions in FA, both in LA and SA. Cite this as: I. den Otter, L. F. F. Silva, A. L. N. Carvalho, R. C. Pires‐Neto, R. Annoni, D. S. Ferreira, I. Bajema, A. van Schadewijk, K. F. Rabe, M. Dolhnikoff, P. J. Sterk, P. S. Hiemstra and T. Mauad, Clinical & Experimental Allergy, 2010 (40) 1473–1481.     

Kinetic Analysis of the Interaction between Recombinant Human FcRIα and Serum IgEs from Allergic Patients

Binding of IgE to the high-affinity IgE receptor (FcεRI) is the essential event for allergic reaction. Although there are many reports on binding kinetics between myeloma IgE and FcεRI, little is known about the kinetics between heterogeneous polyclonal IgE in the serum and FcεRIα. To elucidate the binding characteristics of heterogeneous serum IgE, we measured kinetic parameters of binding between IgE from allergic patients and a recombinant ectodomain of the human FcεRIα subunit by real-time interaction analysis based on surface plasmon resonance. Purified IgE monomer from the plasma of allergic patients displayed kinetics for the interaction with FcεRIα similar to those of myeloma IgE. In the case of crude IgE samples from allergic patients, one of seven specimens showed significantly higher affinity than highly purified IgE, suggesting that it is possible for IgEs in this specimen to form complexes of higher molecular weight.     

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

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

Human IgE mediates stimulus secretion coupling in rat basophilic leukemia cells transfected with the α chain of the human high-affinity receptor

Cell surface expression of the a chain of the human (h) Fee receptor type 1 (FceR1) can be observed following stable transfection of RBL-2H3 cells with the ha chain of the FceR1 complex. Association and dissociation constants for hIgE are similar to those previously reported for the ligand and its receptor. The demonstration of mediator release following the cross-linking of hFc?R1α by hIgE and antigen or anti-hFc?R1α antibody suggests that this system will assist the identification of structural motifs and specific amino acids that are involved in the generation of the signal(s) that will initiate and/or propagate the secretion of mediators from mast cells and basophils.     

Disease activity in systemic lupus erythematosus is associated with an altered expression of low-affinity Fcγ receptors and costimulatory molecules on dendritic cells

Dendritic cells (DCs) play a pivotal role in the interface between immunity and maintenance of peripheral tolerance. The capture of immunoglobulin G (IgG)-containing immune complexes (ICs) by low-affinity Fcγ receptors (FcγRs) expressed on DCs may influence the immunogenicity/tolerogenicity of these cells, depending on the activating/inhibitory potential of FcγRs. Because of the key role that low-affinity FcγRs play in determining the magnitude of the response in IC-driven inflammation, these receptors are likely to play a role in autoimmune diseases, such as systemic lupus erythematosus (SLE). To evaluate if an altered expression of costimulatory molecules and/or FcγRs could account for disease severity, we evaluated the expression of these molecules on immature and mature DCs derived from peripheral blood monocytes of SLE patients and healthy donors. Our results show an increased expression of the costimulatory molecules CD40 and CD86. Furthermore, the ratio of CD86/CD80 is higher in SLE patients compared with healthy donors. Conversely, while the expression of activating FcγRs was higher on DCs from SLE patients, expression of inhibitory FcγRs was lower, compared with DCs obtained from healthy donors. As a result, the activating to inhibitory FcγR ratio was significantly higher in DCs from SLE patients. The altered ratio of activating/inhibitory FcγRs on mature DCs showed a significant correlation with the activity of SLE, as determined by the SLE Disease Activity Index (SLEDAI) score. We postulate that the increased ratio of activating/inhibitory FcγRs expressed on DCs from SLE patients can contribute to the failure of peripheral tolerance in the IC-mediated phase of autoimmune pathogenesis.     

Nitric oxide pathway is induced by FcεRI and up-regulated by stem cell factor in mouse mast cells

Murine stem cell factor (SCF) induces the differentiation of mucosal mast cells (MMC) into connective tissue mast cells (CTMC) and potentiates mediator release induced by aggregation of high-affinity IgE receptors (FcεRI). In the present work, we investigated the effect of FcεRI aggregation on nitric oxide (NO) pathway induction in the different subsets of mast cells, as well as the contribution of SCF in this induction. Inducible NO synthase (iNOs) expression was not evidenced in non-stimulated MMC obtained by culture of hematopoietic progenitors in the presence of interleukin-3, whereas IgE-antigen-stimulated MMC expressed iNOs mRNA and protein and synthesized nitrites. Long-term treatment of MMC with SCF, allowing them to differentiate into CTMC, induced iNOs expression in non-stimulated cells and up-regulated iNOs expression and generation of NO derivatives induced by IgE-antigen stimulation. Thus, NO derivatives generated by mast cells could participate in inflammatory reactions during allergic stimulation.     

Targeted elimination of cells expressing the high-affinity receptor for IgE (FcϵRI) by a Pseudomonas exotoxin-based chimeric protein

The interaction between IgE and its high-affinity receptor Fc?RI found on mast cells and basophils is the primary effector pathway in allergic response. To achieve a targeted elimination of cells expressing Fc?RI receptors, we constructed a chimeric protein in which a Fc fragment of mouse IgE is attached to a truncated form of Pseudomonas exotoxin (PE). To prepare the targeting moiety, we used a DNA sequence corresponding to amino acids 301–437, representing 30 residues of domain 2 and domain 3 of the mouse IgE constant region. This sequence was fused at the 5′ of a cDNA encoding PE₄₀, a truncated form of PE lacking the cell binding domain. The chimeric protein, termed Fc_2′?3?PE₄₀, was expressed in Escherichia coli and partially purified. The protein is highly cytotoxic to mouse mast cell lines and bone marrow-derived primary mast cells. This cytotoxicity is specific, as it could be blocked upon addition of whole IgE. Moreover, the protein had no effect on other cell lines of hemopoietic origin. The Fc_2′?3?PE₄₀ chimeric protein offers a new approach to the treatment of allergic disorders.     

Syk‐dependent regulation of Hrs phosphorylation and ubiquitination upon FcεRI engagement: Impact on Hrs membrane/cytosol localization

Several lines of evidence suggest that Syk controls immune receptor endocytic trafficking. However, the Syk substrates that regulate this process are not currently known. Here, we demonstrate that Syk knockdown prevents the trafficking of engaged high affinity IgE receptor (FcεRI) to a degradative compartment in mast cells. We then concentrate our attention on hepatocyte growth factor‐regulated tyrosine kinase substrate (Hrs) as potential Syk substrate, since it serves as critical regulator for FcεRI entry into lysosomes. We show that Hrs undergoes antigen‐dependent tyrosine phosphorylation and ubiquitination, and identify Syk as the kinase responsible for Hrs phosphorylation. Syk was also required for Hrs ubiquitination catalyzed by c‐Cbl E3 ligase. Syk‐dependent regulation of Hrs covalent modifications, without affecting protein stability, controlled Hrs localization. The majority of phosphorylated Hrs forms were observed only in membrane compartments, whereas ubiquitinated Hrs was predominantly cytosolic, suggesting that both modifications might impact on Hrs function. Together, these findings provide a major step forward in understanding how Syk orchestrates endocytosis of engaged immune receptors.