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.

• 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

Background: Mucosal mast cells (MC) are key players in IgE-mediated food allergy (FA). The evidence on the interaction between gut microbiota, MC and susceptibility to FA is contradictory. Objective: We tested the hypothesis that commensal bacteria are essential for MC migration to the gut and their maturation impacting the susceptibility to FA. Methods: The development and severity of FA symptoms was studied in sensitized germ-free (GF), conventional (CV), and mice mono-colonized with L. plantarum WCFS1 or co-housed with CV mice. MC were phenotypically and functionally characterized. Results: Systemic sensitization and oral challenge of GF mice with ovalbumin led to increased levels of specific IgE in serum compared to CV mice. Remarkably, despite the high levels of sensitization, GF mice did not develop diarrhea or anaphylactic hypothermia, common symptoms of FA. In the gut, GF mice expressed low levels of the MC tissue-homing markers CXCL1 and CXCL2, and harbored fewer MC which exhibited lower levels of MC protease-1 after challenge. Additionally, MC in GF mice were less mature as confirmed by flow-cytometry and their functionality was impaired as shown by reduced edema formation after injection of degranulation-provoking compound 48/80. Co-housing of GF mice with CV mice fully restored their susceptibility to develop FA. However, this did not occur when mice were mono-colonized with L. plantarum. Conclusion: Our results demonstrate that microbiota-induced maturation and gut-homing of MC is a critical step for the development of symptoms of experimental FA. This new mechanistic insight into microbiota-MC-FA axis can be exploited in the prevention and treatment of FA in humans.

• MeSH
• biologické markery MeSH
• buněčná diferenciace genetika   imunologie   MeSH
• cytokiny metabolismus   MeSH
• degranulace buněk genetika   imunologie   MeSH
• gnotobiologické modely MeSH
• mastocyty imunologie   metabolismus   MeSH
• metagenom MeSH
• metagenomika metody   MeSH
• mikrobiota * imunologie   MeSH
• modely nemocí na zvířatech MeSH
• myši MeSH
• náchylnost k nemoci MeSH
• potravinová alergie etiologie   metabolismus   patologie   MeSH
• RNA ribozomální 16S MeSH
• střevní mikroflóra MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Dendritic cells (DCs) and mast cells (MCs) are key players of the immune system, often coming in close proximity in peripheral tissues. The interplay of these cells is, however, still poorly understood, especially with regards to human cells. The reason for that is the absence of a well established in vitro human cell-based study system that would allow a simultaneous preparation of both cell types. In this study, we show a method for simultaneous generation of DCs and MCs from CD34+ stem cell progenitors that were isolated from the non-adherent fraction of non-mobilized peripheral blood mononuclear cells of healthy donors. We observed that combining stem cells factor (SCF), IL-3 and GM-CSF in serum-free StemPro-34 medium allowed CD34+ cells isolated from an equivalent of 450 ml of peripheral blood to expand to 10-92 × 106 cells after 7 weeks of culturing. These cultures comprised of 6-53% of DCs and 1-21% of MCs as determined by the expression of, respectively, CD11c/HLA-DR or CD117/FcεRI. The DCs were CD1a-CD14-, did not express co-stimulatory molecules CD80 and CD83 and chemokine receptor CCR7. However, the DCs expressed co-stimulatory molecule CD86, and had a capacity to uptake dextran, phagocyte latex particles and induce alloreactivity. MCs, on the other hand, degranulated after crosslinking of FcεRI-bound IgE as determined by the externalization of CD107b. Collectively, our data show that CD34+-derived human DCs and MCs can be generated in a single culture using CD34+ cells isolated from non-mobilized human peripheral blood and that this method may allow ex vivo studies on DC-MC interplay in human system.

• MeSH
• antigeny CD34 metabolismus   MeSH
• buněčná diferenciace MeSH
• degranulace buněk imunologie   MeSH
• dendritické buňky imunologie   metabolismus   MeSH
• dextrany imunologie   MeSH
• faktor růstu kmenových buněk metabolismus   MeSH
• faktor stimulující granulocyto-makrofágové kolonie metabolismus   MeSH
• kmenové buňky z periferní krve fyziologie   MeSH
• kultivační média bez séra metabolismus   MeSH
• leukocyty mononukleární MeSH
• lidé MeSH
• mastocyty imunologie   MeSH
• mezibuněčná komunikace imunologie   MeSH
• primární buněčná kultura metody   MeSH
• průtoková cytometrie metody   MeSH
• rekombinantní proteiny metabolismus   MeSH
• separace buněk metody   MeSH
• vrstva buffy coat cytologie   MeSH
• zdraví dobrovolníci pro lékařské studie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Klíčová slova
• ORMDL3 protein, serin-palmitoyl transferáza, techniky in vivo,
• MeSH
• bronchiální astma * etiologie   imunologie   patologie   MeSH
• cytokiny imunologie   MeSH
• degranulace buněk imunologie   MeSH
• down regulace genetika   imunologie   MeSH
• endoplazmatické retikulum imunologie   MeSH
• mastocyty * fyziologie   imunologie   MeSH
• myši MeSH
• receptory IgE imunologie   MeSH
• sfingolipidy biosyntéza   MeSH
• signální transdukce MeSH
• techniky in vitro MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

Coevolution of ticks and the vertebrate immune system has led to the development of immunosuppressive molecules that prevent immediate response of skin-resident immune cells to quickly fend off the parasite. In this article, we demonstrate that the tick-derived immunosuppressor sialostatin L restrains IL-9 production by mast cells, whereas degranulation and IL-6 expression are both unaffected. In addition, the expression of IL-1β and IRF4 is strongly reduced in the presence of sialostatin L. Correspondingly, IRF4- or IL-1R-deficient mast cells exhibit a strong impairment in IL-9 production, demonstrating the importance of IRF4 and IL-1 in the regulation of the Il9 locus in mast cells. Furthermore, IRF4 binds to the promoters of Il1b and Il9, suggesting that sialostatin L suppresses mast cell-derived IL-9 preferentially by inhibiting IRF4. In an experimental asthma model, mast cell-specific deficiency in IRF4 or administration of sialostatin L results in a strong reduction in asthma symptoms, demonstrating the immunosuppressive potency of tick-derived molecules.

• MeSH
• bronchiální astma genetika   imunologie   patologie   MeSH
• cystatiny imunologie   farmakologie   MeSH
• degranulace buněk imunologie   MeSH
• genetická transkripce MeSH
• imunosupresiva farmakologie   MeSH
• interakce hostitele a parazita imunologie   MeSH
• interferonové regulační faktory nedostatek   genetika   imunologie   MeSH
• interleukin-1beta genetika   imunologie   MeSH
• interleukin-6 genetika   imunologie   MeSH
• interleukin-9 antagonisté a inhibitory   genetika   imunologie   MeSH
• mastocyty účinky léků   imunologie   patologie   MeSH
• myši inbrední BALB C MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• přirozená imunita účinky léků   MeSH
• promotorové oblasti (genetika) MeSH
• receptory interleukinu-1 genetika   imunologie   MeSH
• regulace genové exprese MeSH
• signální transdukce MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH

Ve své práci prezentujeme srovnání měření aktivace bazofilů pomocí protilátky CD203c neb CD63. Bylo vyšetřeno 18 pacientů, výsledky 15 pacientů byly navzájem srovnány. Dobrou korelaci výsledků vyjadřuje korelační koeficient r = 0,94. Do srovnání nebyli zařazeni 3 pacienti- u 1 pacienta neodpovídaly bazofily na stimulaci anti-IgE ( pozitivní kontrola), 1 pacient měl zvýšenou expresi CD203c bez stimulace, 1 pacientměl spontánně aktivované bazofily. Naše výsledky ukazují, že spolehlivě je možno měřit aktivaci bazofilů pouze pomocí kombinací protilátek CD203c a CD63. Při použití samotné protilátky CD203c nejsme schopni odlišit od sebepacienty : 1) nereagující na stimulaci přes vysokoafinitní receptor pro IgE (non-responders) od těch, kteří reagují, ale nezvyšuje se u nich exprese CD203c; 2) pacienty se spontánně aktivovanými bazofily od těch, kteří mají pouze zvýšenou expresi CD203c i bez stimulace.

Our study was designed to compare basophil activation using CD203c or CD63 antibodies. A total of 18 patients were examined, data of 15 subjects were compared. Good correlation of data is indicated by a correlation coefficient of r = 0.94. The comparison did not include three patients; in 1 patient, basophils did not respond to anti-IgE stimulation (a positive control ), 1 showed increased CD203c expression without stimulation, and 1 patient had spontaneously activated basophils. Our results suggest basophil activation can be reliably determined only using a combination of CD203c and CD63 antibodies. Use of CD203c antibody alone makes it impossible to distinguish: 1) Patients not responding to stimulation via the high-affinity IgE receptor (non-responders) from those who do respond without increased CD203c expression; 2) Patients with spontaneously activated basophils from those with increased CD203c expression only even without stimulation.

• MeSH
• alergeny analýza   imunologie   klasifikace   MeSH
• alergie diagnóza   etiologie   imunologie   MeSH
• bazofily cytologie   imunologie   klasifikace   MeSH
• buňky produkující protilátky cytologie   imunologie   MeSH
• CD antigeny diagnostické užití   imunologie   MeSH
• degranulace buněk imunologie   MeSH
• fluorescence MeSH
• imunologické testy metody   statistika a číselné údaje   využití   MeSH
• lidé MeSH
• průtoková cytometrie metody   přístrojové vybavení   statistika a číselné údaje   MeSH
• receptory IgE analýza   imunologie   klasifikace   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• srovnávací studie MeSH