Mast cells (MCs) are long-living immune cells highly specialized in the storage and release of different biologically active compounds and are involved in the regulation of innate and adaptive immunity. MC degranulation and replacement of MC granules are accompanied by active membrane remodelling. Tetraspanins represent an evolutionary conserved family of transmembrane proteins. By interacting with lipids and other membrane and intracellular proteins, they are involved in organisation of membrane protein complexes and act as "molecular facilitators" connecting extracellular and cytoplasmic signaling elements. MCs express different tetraspanins and MC degranulation is accompanied by changes in membrane organisation. Therefore, tetraspanins are very likely involved in the regulation of MC exocytosis and membrane reorganisation after degranulation. Antiviral response and production of exosomes are further aspects of MC function characterized by dynamic changes of membrane organization. In this review, we pay a particular attention to tetraspanin gene expression in different human and murine MC populations, discuss tetraspanin involvement in regulation of key MC signaling complexes, and analyze the potential contribution of tetraspanins to MC antiviral response and exosome production. In-depth knowledge of tetraspanin-mediated molecular mechanisms involved in different aspects of the regulation of MC response will be beneficial for patients with allergies, characterized by overwhelming MC reactions.

• Klíčová slova
• Allergy, Antiviral immune response, Exosomes, FcεRI, Mast cell degranulation, Mast cells, Tetraspanins,
• MeSH
• degranulace buněk MeSH
• exozómy metabolismus   MeSH
• lidé MeSH
• mastocyty imunologie   metabolismus   MeSH
• myši MeSH
• signální transdukce MeSH
• tetraspaniny genetika   imunologie   metabolismus   MeSH
• virové nemoci imunologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• tetraspaniny MeSH

A better understanding of the molecular mechanisms leading to mast cell migration and chemotaxis is the long-term goal in mast cell research and is essential for comprehension of mast cell function in health and disease. Various techniques have been developed in recent decades for in vitro and in vivo assessment of mast cell motility and chemotaxis. In this chapter, three microscopy assays facilitating real-time quantification of mast cell chemotaxis and migration are described, focusing on individual cell tracking and data analysis.

• Klíčová slova
• Cell migration, Cell tracking, Chemoattractant, Chemokine, Chemotaxis, Mast cells,
• MeSH
• analýza buněčné migrace metody   MeSH
• biotest metody   MeSH
• buněčný tracking metody   MeSH
• chemotaxe fyziologie   MeSH
• fibronektiny metabolismus   MeSH
• lidé MeSH
• mastocyty cytologie   fyziologie   MeSH
• mikroskopie metody   MeSH
• myši MeSH
• počítačové systémy MeSH
• pohyb buněk fyziologie   MeSH
• prostředí kontrolované MeSH
• sefarosa MeSH
• software MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fibronektiny MeSH
• sefarosa MeSH

Protein 4.1R, a member of the 4.1 family, functions as a bridge between cytoskeletal and plasma membrane proteins. It is expressed in T cells, where it binds to a linker for activation of T cell (LAT) family member 1 and inhibits its phosphorylation and downstream signaling events after T cell receptor triggering. The role of the 4.1R protein in cell activation through other immunoreceptors is not known. In this study, we used 4.1R-deficient (4.1R-KO) and 4.1R wild-type (WT) mice and explored the role of the 4.1R protein in the high-affinity IgE receptor (FcεRI) signaling in mast cells. We found that bone marrow mast cells (BMMCs) derived from 4.1R-KO mice showed normal growth in vitro and expressed FcεRI and c-KIT at levels comparable to WT cells. However, 4.1R-KO cells exhibited reduced antigen-induced degranulation, calcium response, and secretion of tumor necrosis factor-α. Chemotaxis toward antigen and stem cell factor (SCF) and spreading on fibronectin were also reduced in 4.1R-KO BMMCs, whereas prostaglandin E2-mediated chemotaxis was not affected. Antibody-induced aggregation of tetraspanin CD9 inhibited chemotaxis toward antigen in WT but not 4.1R-KO BMMCs, implying a CD9-4.1R protein cross-talk. Further studies documented that in the absence of 4.1R, antigen-mediated phosphorylation of FcεRI β and γ subunits was not affected, but phosphorylation of SYK and subsequent signaling events such as phosphorylation of LAT1, phospholipase Cγ1, phosphatases (SHP1 and SHIP), MAP family kinases (p38, ERK, JNK), STAT5, CBL, and mTOR were reduced. Immunoprecipitation studies showed the presence of both LAT1 and LAT2 (LAT, family member 2) in 4.1R immunocomplexes. The positive regulatory role of 4.1R protein in FcεRI-triggered activation was supported by in vivo experiments in which 4.1R-KO mice showed the normal presence of mast cells in the ears and peritoneum, but exhibited impaired passive cutaneous anaphylaxis. The combined data indicate that the 4.1R protein functions as a positive regulator in the early activation events after FcεRI triggering in mast cells.

• Klíčová slova
• 4.1R protein, chemotaxis, degranulation, mast cell, passive cutaneous anaphylaxis,
• MeSH
• chemotaxe imunologie   MeSH
• degranulace buněk imunologie   MeSH
• mastocyty imunologie   metabolismus   MeSH
• mikrofilamentové proteiny imunologie   metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• pasivní kožní anafylaxe imunologie   MeSH
• receptory IgE imunologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Epb41 protein, mouse MeSH Prohlížeč
• mikrofilamentové proteiny MeSH
• receptory IgE MeSH

C-terminal Src kinase (CSK) is a major negative regulator of Src family tyrosine kinases (SFKs) that play critical roles in immunoreceptor signaling. CSK is brought in contiguity to the plasma membrane-bound SFKs via binding to transmembrane adaptor PAG, also known as CSK-binding protein. The recent finding that PAG can function as a positive regulator of the high-affinity IgE receptor (FcεRI)-mediated mast cell signaling suggested that PAG and CSK have some non-overlapping regulatory functions in mast cell activation. To determine the regulatory roles of CSK in FcεRI signaling, we derived bone marrow-derived mast cells (BMMCs) with reduced or enhanced expression of CSK from wild-type (WT) or PAG knockout (KO) mice and analyzed their FcεRI-mediated activation events. We found that in contrast to PAG-KO cells, antigen-activated BMMCs with CSK knockdown (KD) exhibited significantly higher degranulation, calcium response, and tyrosine phosphorylation of FcεRI, SYK, and phospholipase C. Interestingly, FcεRI-mediated events in BMMCs with PAG-KO were restored upon CSK silencing. BMMCs with CSK-KD/PAG-KO resembled BMMCs with CSK-KD alone. Unexpectedly, cells with CSK-KD showed reduced kinase activity of LYN and decreased phosphorylation of transcription factor STAT5. This was accompanied by impaired production of proinflammatory cytokines and chemokines in antigen-activated cells. In line with this, BMMCs with CSK-KD exhibited enhanced phosphorylation of protein phosphatase SHP-1, which provides a negative feedback loop for regulating phosphorylation of STAT5 and LYN kinase activity. Furthermore, we found that in WT BMMCs SHP-1 forms complexes containing LYN, CSK, and STAT5. Altogether, our data demonstrate that in FcεRI-activated mast cells CSK is a negative regulator of degranulation and chemotaxis, but a positive regulator of adhesion to fibronectin and production of proinflammatory cytokines. Some of these pathways are not dependent on the presence of PAG.

• Klíčová slova
• C-terminal Src kinase, LYN, SHP-1, STAT5, cytokines, degranulation, mast cell, phosphoprotein associated with glycosphingolipid-enriched microdomains,
• MeSH
• analýza rozptylu MeSH
• buňky kostní dřeně fyziologie   MeSH
• C-terminální Src kinasa MeSH
• cytokiny metabolismus   MeSH
• degranulace buněk MeSH
• fibronektiny metabolismus   MeSH
• fosfoproteiny metabolismus   MeSH
• fosforylace MeSH
• genetické vektory MeSH
• HEK293 buňky MeSH
• lidé MeSH
• mastocyty fyziologie   MeSH
• membránové proteiny metabolismus   MeSH
• mezibuněčné signální peptidy a proteiny MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• receptory IgE metabolismus   MeSH
• signální transdukce imunologie   MeSH
• skupina kinas odvozených od src-genu metabolismus   fyziologie   MeSH
• transkripční faktor STAT5 metabolismus   MeSH
• tyrosin metabolismus   MeSH
• tyrosinfosfatasa nereceptorového typu 6 metabolismus   MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• C-terminální Src kinasa MeSH
• CSK protein, human MeSH Prohlížeč
• cytokiny MeSH
• fibronektiny MeSH
• fosfoproteiny MeSH
• lyn protein-tyrosine kinase MeSH Prohlížeč
• membránové proteiny MeSH
• mezibuněčné signální peptidy a proteiny MeSH
• Pag protein, mouse MeSH Prohlížeč
• Pag1 protein, mouse MeSH Prohlížeč
• Ptpn6 protein, mouse MeSH Prohlížeč
• receptory IgE MeSH
• skupina kinas odvozených od src-genu MeSH
• transkripční faktor STAT5 MeSH
• tyrosin MeSH
• tyrosinfosfatasa nereceptorového typu 6 MeSH
• vápník MeSH

The plasma membrane contains diverse and specialized membrane domains, which include tetraspanin-enriched domains (TEMs) and transmembrane adaptor protein (TRAP)-enriched domains. Recent biophysical, microscopic, and functional studies indicated that TEMs and TRAP-enriched domains are involved in compartmentalization of physicochemical events of such important processes as immunoreceptor signal transduction and chemotaxis. Moreover, there is evidence of a cross-talk between TEMs and TRAP-enriched domains. In this review we discuss the presence and function of such domains and their crosstalk using mast cells as a model. The combined data based on analysis of selected mast cell-expressed tetraspanins [cluster of differentiation (CD)9, CD53, CD63, CD81, CD151)] or TRAPs [linker for activation of T cells (LAT), non-T cell activation linker (NTAL), and phosphoprotein associated with glycosphingolipid-enriched membrane microdomains (PAG)] using knockout mice or specific antibodies point to a diversity within these two families and bring evidence of the important roles of these molecules in signaling events. An example of this diversity is physical separation of two TRAPs, LAT and NTAL, which are in many aspects similar but show plasma membrane location in different microdomains in both non-activated and activated cells. Although our understanding of TEMs and TRAP-enriched domains is far from complete, pharmaceutical applications of the knowledge about these domains are under way.

• Klíčová slova
• CD9, IgE receptor, LAT, NTAL, membrane microdomains, plasma membrane, signal transduction,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Mast cells play crucial roles in both innate and adaptive arms of the immune system. Along with basophils, mast cells are essential effector cells for allergic inflammation that causes asthma, allergic rhinitis, food allergy and atopic dermatitis. Mast cells are usually increased in inflammatory sites of allergy and, upon activation, release various chemical, lipid, peptide and protein mediators of allergic reactions. Since antigen/immunoglobulin E (IgE)-mediated activation of these cells is a central event to trigger allergic reactions, innumerable studies have been conducted on how these cells are activated through cross-linking of the high-affinity IgE receptor (FcεRI). Development of mature mast cells from their progenitor cells is under the influence of several growth factors, of which the stem cell factor (SCF) seems to be the most important. Therefore, how SCF induces mast cell development and activation via its receptor, KIT, has been studied extensively, including a cross-talk between KIT and FcεRI signaling pathways. Although our understanding of the signaling mechanisms of the FcεRI and KIT pathways is far from complete, pharmaceutical applications of the knowledge about these pathways are underway. This review will focus on recent progresses in FcεRI and KIT signaling and chemotaxis.

• Klíčová slova
• Chemotaxis, IgE receptor, KIT receptor, Mast cell, Plasma membrane, Signal transduction,
• MeSH
• chemotaxe * účinky léků   MeSH
• lidé MeSH
• mastocyty cytologie   účinky léků   MeSH
• signální transdukce * účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH

Aggregation of the high-affinity receptor for IgE (FcεRI) in mast cells initiates activation events that lead to degranulation and release of inflammatory mediators. To better understand the signaling pathways and genes involved in mast cell activation, we developed a high-throughput mast cell degranulation assay suitable for RNA interference experiments using lentivirus-based short hairpin RNA (shRNA) delivery. We tested 432 shRNAs specific for 144 selected genes for effects on FcεRI-mediated mast cell degranulation and identified 15 potential regulators. In further studies, we focused on galectin-3 (Gal3), identified in this study as a negative regulator of mast cell degranulation. FcεRI-activated cells with Gal3 knockdown exhibited upregulated tyrosine phosphorylation of spleen tyrosine kinase and several other signal transduction molecules and enhanced calcium response. We show that Gal3 promotes internalization of IgE-FcεRI complexes; this may be related to our finding that Gal3 is a positive regulator of FcεRI ubiquitination. Furthermore, we found that Gal3 facilitates mast cell adhesion and motility on fibronectin but negatively regulates antigen-induced chemotaxis. The combined data indicate that Gal3 is involved in both positive and negative regulation of FcεRI-mediated signaling events in mast cells.

• MeSH
• aktiny metabolismus   MeSH
• buněčná adheze MeSH
• chemotaxe MeSH
• cytokiny genetika   metabolismus   MeSH
• fosforylace MeSH
• galektin 3 genetika   metabolismus   MeSH
• lyzozomy metabolismus   MeSH
• malá interferující RNA MeSH
• mastocyty cytologie   fyziologie   MeSH
• myši inbrední BALB C MeSH
• prostaglandin D2 metabolismus   MeSH
• receptory IgE genetika   metabolismus   MeSH
• signální transdukce MeSH
• ubikvitinace MeSH
• vápník metabolismus   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• aktiny MeSH
• cytokiny MeSH
• galektin 3 MeSH
• Lgals3 protein, mouse MeSH Prohlížeč
• malá interferující RNA MeSH
• prostaglandin D2 MeSH
• receptory IgE MeSH
• vápník MeSH

Single-nucleotide polymorphism studies have linked the chromosome 17q12-q21 region, where the human orosomucoid-like (ORMDL)3 gene is localized, to the risk of asthma and several other inflammatory diseases. Although mast cells are involved in the development of these diseases, the contribution of ORMDL3 to the mast cell physiology is unknown. In this study, we examined the role of ORMDL3 in antigen-induced activation of murine mast cells with reduced or enhanced ORMDL3 expression. Our data show that in antigen-activated mast cells, reduced expression of the ORMDL3 protein had no effect on degranulation and calcium response, but significantly enhanced phosphorylation of AKT kinase at Ser 473 followed by enhanced phosphorylation and degradation of IκBα and translocation of the NF-κB p65 subunit into the nucleus. These events were associated with an increased expression of proinflammatory cytokines (TNF-α, IL-6, and IL-13), chemokines (CCL3 and CCL4), and cyclooxygenase-2 dependent synthesis of prostaglandin D2. Antigen-mediated chemotaxis was also enhanced in ORMDL3-deficient cells, whereas spreading on fibronectin was decreased. On the other hand, increased expression of ORMDL3 had no significant effect on the studied signaling events, except for reduced antigen-mediated chemotaxis. These data were corroborated by increased IgE-antigen-dependent passive cutaneous anaphylaxis in mice with locally silenced ORMDL3 using short interfering RNAs. Our data also show that antigen triggers suppression of ORMDL3 expression in the mast cells. In summary, we provide evidence that downregulation of ORMDL3 expression in mast cells enhances AKT and NF-κB-directed signaling pathways and chemotaxis and contributes to the development of mast cell-mediated local inflammation in vivo.

• Klíčová slova
• Chemotaxis, Degranulation, Mast cell, ORMDL3 knockdown, Proinflammatory cytokines, Prostaglandin D2, RNA interference,
• MeSH
• chemotaxe * MeSH
• cytokiny genetika   imunologie   MeSH
• degranulace buněk * MeSH
• down regulace MeSH
• kultivované buňky MeSH
• mastocyty cytologie   imunologie   metabolismus   MeSH
• membránové proteiny genetika   imunologie   MeSH
• messenger RNA genetika   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• receptory IgE imunologie   MeSH
• upregulace MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokiny MeSH
• membránové proteiny MeSH
• messenger RNA MeSH
• ORMDL3 protein, mouse MeSH Prohlížeč
• receptory IgE MeSH

The transmembrane adaptor protein PAG/CBP (here, PAG) is expressed in multiple cell types. Tyrosine-phosphorylated PAG serves as an anchor for C-terminal SRC kinase, an inhibitor of SRC-family kinases. The role of PAG as a negative regulator of immunoreceptor signaling has been examined in several model systems, but no functions in vivo have been determined. Here, we examined the activation of bone marrow-derived mast cells (BMMCs) with PAG knockout and PAG knockdown and the corresponding controls. Our data show that PAG-deficient BMMCs exhibit impaired antigen-induced degranulation, extracellular calcium uptake, tyrosine phosphorylation of several key signaling proteins (including the high-affinity IgE receptor subunits, spleen tyrosine kinase, and phospholipase C), production of several cytokines and chemokines, and chemotaxis. The enzymatic activities of the LYN and FYN kinases were increased in nonactivated cells, suggesting the involvement of a LYN- and/or a FYN-dependent negative regulatory loop. When BMMCs from PAG-knockout mice were activated via the KIT receptor, enhanced degranulation and tyrosine phosphorylation of the receptor were observed. In vivo experiments showed that PAG is a positive regulator of passive systemic anaphylaxis. The combined data indicate that PAG can function as both a positive and a negative regulator of mast cell signaling, depending upon the signaling pathway involved.

• MeSH
• anafylaxe genetika   MeSH
• buňky kostní dřeně metabolismus   fyziologie   MeSH
• C-terminální Src kinasa MeSH
• degranulace buněk MeSH
• fosfolipasy typu C metabolismus   MeSH
• fosfoproteiny genetika   MeSH
• fosforylace MeSH
• intracelulární signální peptidy a proteiny metabolismus   MeSH
• kinasa Syk MeSH
• malá interferující RNA MeSH
• mastocyty metabolismus   fyziologie   MeSH
• membránové proteiny genetika   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• protoonkogenní proteiny c-fyn biosyntéza   MeSH
• protoonkogenní proteiny c-kit metabolismus   MeSH
• receptory IgE metabolismus   MeSH
• RNA interference MeSH
• signální transdukce MeSH
• skupina kinas odvozených od src-genu biosyntéza   metabolismus   MeSH
• tyrosinkinasy metabolismus   MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• C-terminální Src kinasa MeSH
• fosfolipasy typu C MeSH
• fosfoproteiny MeSH
• Fyn protein, mouse MeSH Prohlížeč
• intracelulární signální peptidy a proteiny MeSH
• kinasa Syk MeSH
• lyn protein-tyrosine kinase MeSH Prohlížeč
• malá interferující RNA MeSH
• membránové proteiny MeSH
• Pag1 protein, mouse MeSH Prohlížeč
• protoonkogenní proteiny c-fyn MeSH
• protoonkogenní proteiny c-kit MeSH
• receptory IgE MeSH
• skupina kinas odvozených od src-genu MeSH
• Syk protein, mouse MeSH Prohlížeč
• tyrosinkinasy MeSH
• vápník MeSH