Cbl family proteins: balancing FcεRI-mediated mast cell and basophil activation

The binding of IgE to high-affinity IgE receptors (FcεRI) expressed on the surface of mast cells and basophils initiates a cascade of signaling events that results in the release of a wide array of proinflammatory mediators. In order to limit the intensity and duration of cell activation, FcεRI aggregation has been understood to additionally generate negative signals through the coordinated action of adapters, phosphatases, and ubiquitin ligases. Among them, Cbl family proteins negatively regulate FcεRI-mediated signals mainly by promoting ubiquitination of the activated receptor subunits and associated protein tyrosine kinases. Notably, FcεRI ubiquitination has become recognized as an important signal for the internalization and delivery of engaged receptor complexes to lysosomes for degradation. The surface expression of activated FcεRI complexes is further downregulated through a pathway that is functionally separable from Cbl ligase activity and is dependent on the interaction of Cbl proteins with adapters involved in clathrin-dependent endocytosis. In this article, we review recent advances in our understanding of the molecular mechanisms through which Cbl proteins negatively regulate FcεRI-mediated mast cell and basophil functions.     

Transcriptional response of human mast cells stimulated via the FcεRI and identification of mast cells as a source of IL-11

Background  

In asthma and other allergic disorders, the activation of mast cells by IgE and antigen induces the cells to release histamine and other mediators of inflammation, as well as to produce certain cytokines and chemokines. To search for new mast cell products, we used complementary DNA microarrays to analyze gene expression in human umbilical cord blood-derived mast cells stimulated via the high-affinity IgE receptor (FcεRI).     

Phospholipase C-β3 regulates FcɛRI-mediated mast cell activation by recruiting the protein phosphatase SHP-1

Mast cells are major effectors in high-affinity IgE receptor (Fc?RI)-dependent allergic reactions. Here we show that phospholipase C (PLC)-β3 is crucial for Fc?RI-mediated mast cell activation. Plcb3(-/-) mice showed blunted Fc?RI-dependent late-phase, but not acute, anaphylactic responses and airway inflammation. Accordingly, Fc?RI stimulation of Plcb3(-/-) mast cells exhibited reduced cytokine production but normal degranulation. Reduced cytokine production in Plcb3(-/-) cells could be accounted for by increased activity of the negative regulatory Src family kinase Lyn and reduced activities of the positive regulatory protein kinases MAPKs. Mechanistically, PLC-β3 constitutively interacts with Fc?RI, Lyn, and SHP-1 (protein phosphatase). SHP-1 probably recognizes its substrates Lyn and MAPKs via the recently described kinase tyrosine-based inhibitory motif, KTIM. Consistent with PLC-β3- and SHP-1-mediated repression of Lyn activity by dephosphorylation at Tyr396, Fc?RI-mediated phenotypes were similar in Plcb3(-/-) and SHP-1 mutant mast cells. Thus, we have defined a PLC-β3- and SHP-1-mediated signaling pathway for Fc?RI-mediated cytokine production.     

An involvement of neurokinin-1 receptor in FcεRΙ-mediated RBL-2H3 mast cell activation

Objective and design

To determine whether the neurokinin-1 receptor (NK1R) plays a role in the activation of RBL-2H3 mast cells after FcεRΙ aggregation.

Materials and methods

NK1R expression in RBL-2H3 cells was inhibited by small hairpin RNA (shRNA) against NK1R, and determined by western blotting. For activation, both NK1R knockdown and control RBL-2H3 cells were sensitized by dinitrophenol (DNP)-specific IgE and stimulated with the antigen DNP-bovine serum albumin (BSA). Following the activation of RBL-2H3 cells, monocyte chemoattractant protein (MCP-1) production and intracellular calcium flux were monitored by ELISA and confocal microscopy assay, respectively. For investigation of the signaling mechanism, phosphorylation of mitogen-activated protein kinases (MAPKs) after RBL-2H3 cell activation was assessed by western blotting.

Results

shRNA-NK1R mediated an effective inhibition of NK1R expression in RBL-2H3 cells. Protein production of MCP-1 was reduced by more than 55?% in NK1R knockdown RBL-2H3 cells compared with control RBL-2H3 cells. In addition, both calcium mobilization and phosphorylation levels of MAPKs (Erk1/2, JNK, and p38) after DNP-BSA stimulation (via FcεRΙ) were decreased due to the inhibition of NK1R expression.

Conclusion

NK1R is required for the activation of RBL-2H3 cells following FcεRΙ engagement and involved in the regulation of MAPK signaling pathways.     

Negative signals from FcεRI engagement attenuate mast cell functions

Mast cells have long been recognized as the critical tissue-based effector cells in IgE-mediated allergic diseases. Ligation of the high-affinity receptor for IgE (FcεRI), constitutively expressed on mast cells, promotes cell activation and immediate release and production of pro-inflammatory mediators. Besides these positive signals, FcεRI aggregation has recently been understood to generate negative intracellular signals capable of limiting mast cell functional responses. This review is aimed at providing a summary of the mechanisms through which FcεRI engagement can generate negative signals and regulate mast-cell function. Similar mechanisms are employed by other receptors expressed by immune cells, such as T cell and B cell receptors, pointing to a general concept in negative immunoreceptor signaling.     

Disparity in FcεRI-induced degranulation of primary human lung and skin mast cells exposed to adenosine

Inhaled and intravenously administered adenosine induces mast cell-mediated (histamine-dependent) bronchospasm in asthmatics without causing urticaria. A differential response to adenosine by human lung and skin mast cells is shown: low concentrations potentiate FcεRI-induced degranulation of human lung mast cells but not that of skin mast cells. Human lung mast cells were found to express ∼3-fold more A3AR messenger RNA (mRNA) than skin mast cells, suggesting the involvement of the G_i-linked A3AR. Indeed, the adenosine-induced potentiation was sensitive to inhibition by pertussis toxin and, furthermore, could be induced with an A3AR-specific agonist. This study reveals a previously unrecognized disparity in the response to adenosine by primary human mast cells from lung and skin that might explain why adenosine induces a pulmonary but not dermatologic allergy-like response in vivo. In addition, we identify the A3AR as a potentiating receptor of FcεRI-induced degranulation, thereby implicating it in the in vivo bronchoconstrictive response to adenosine in asthmatics.     

Protocatechuic acid inhibits human dendritic cell functional activation: Role of PPARγ up-modulation

Polyphenols have been shown to exhibit anti-inflammatory, anti-oxidant and immunomodulatory activities. However, the effects of anthocyanins, flavonoids of great nutritional interest, in particular of their metabolite protocatechuic acid (PCA) on the phenotypic and functional maturation of human dendritic cells (DCs) are still largely unknown. In this study, we report that PCA is efficiently taken up and accumulated in human monocyte-derived DCs (MD-DCs). PCA exposure of MD-DCs markedly impaired the production of proinflammatory cytokines and chemokines (i.e. IL-6, IL-8 and CCL2) in response to bacterial endotoxin and leptin, and down-regulated the lipopolysaccharide (LPS)-induced migratory response of MD-DCs to CCL19. Conversely, the phenotypic profile induced by LPS-mediated activation as well as IL-12 production was not affected. Interestingly, we found that PPARγ is a main factor in the PCA-induced effects as blocking its activity abolish PCA capacity to down-regulate IL-6 and IL-8, but not CCL2, secretion and to inhibit MD-DC migration. In keeping with this observation, cytosol to nucleus translocation and PPARγ activity were found to be directly stimulated by PCA exposure of MD-DCs.     

Maspin inhibits macrophage phagocytosis and enhances inflammatory cytokine production via activation of NF-κB signaling

Maspin (mammary serine protease inhibitor) is a non-inhibitory member of the serine protease inhibitor superfamily and a tumor suppressor in several cancers due to its ability to inhibit cell invasion, angiogenesis, and promote apoptosis. However, its immunomodulatory function remains largely unexplored. Thus, we explored the potential link between Maspin and macrophage function, first evaluating the regulatory effects of conditioned medium (CM) of a Maspin-overexpressing CHO cell strain on mouse peritoneal macrophage phagocytosis and cytokine secretion. Next, we used a transwell co-culture system and recombinant Maspin (rMaspin) to confirm the effects of Maspin on macrophages, and attempted to clarify the underlying mechanisms. We found that irrespective of CM, rMaspin or co-culture of Maspin-overexpressing cells with macrophages impaired macrophages phagocytosing Saccharomyces cerevisiae. Furthermore, q-RT-PCR or ELISA confirmed increased IL-1β, TNF-α, IFN-γ, IL-6, IL-12, IL-10, and M1 marker iNOS production in macrophages after Maspin stimulation, but TGF-β and M2 marker Arg-1 production were suppressed. Western blot showed activated NF-κB signaling in Maspin-stimulated macrophages; upregulated cytokines were lowered, and impaired phagocytosis recovered after blocking NF-κB signaling with PDTC. Thus, Maspin mildly inhibited phagocytic activity, but markedly enhanced inflammatory cytokine production and likely skewed macrophages towards M1 polarization, partially due to activation of NF-κB signaling. These results reveal a novel biological function of Maspin in modulating macrophage activity and may open a new avenue for Maspin-based tumor therapy.     

Negative regulation of dendritic cell activation in psoriasis mediated via CD100–plexin-B2

Psoriasis is a chronic inflammatory skin disease in which dendritic cells (DCs) play a pivotal role by inducing Th1/Th17 immune responses; however, the regulation of DC activation in psoriasis remains largely unknown. Previously we found that the level of soluble CD100 was increased in sera of psoriasis patients, and CD100 promoted the activation of inflammasome in keratinocytes. In the present study, CD100 knockout mice were utilized for generation of imiquimod (IMQ)-induced psoriatic dermatitis, with the result that skin inflammation in the early, but not late, phase of the psoriatic dermatitis was significantly exacerbated compared to that in wild-type controls. This was attributed mainly to the deficiency of CD100 in hematopoietic cells. Bone marrow-derived DCs, but not T cells or keratinocytes, from CD100 knockout mice produced significantly increased levels of IL-1β, IL-36, and IL-23 upon stimulation with IMQ in a plexin-B2-dependent manner. Moreover, the surface level of plexin-B2 on DCs of psoriasis patients was lower than that of healthy individuals, and CD100 attenuated IMQ-induced production of IL-1β and IL-36 from monocyte-derived DCs of psoriasis patients. Our results uncovered a negative regulatory mechanism for DCs activation in psoriasis, which was mediated via CD100–plexin-B2 in a cell type- and receptor-specific manner. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.