ABSTRACT
BACKGROUND: Mast cell (MC) - eosinophil (Eos) activating cross-talk might be critical for the severity and chronicity of allergy. Among soluble mediators, eosinophil major basic protein (MBP), a hallmark of allergy, is particularly important because it was shown to activate specific MC subtypes. We previously demonstrated that MBP activates IgE-desensitized rat MC and human lung and cord blood-derived MC (CBMC) after priming with fibroblast membranal stem cell factor. However, a distinct mechanism for this activation was missing. Therefore, we aimed to investigate it. METHODS: Major basic protein-1 activation of CBMC primed with fibroblast-derived membranes (FBM) was measured by ß-hexosaminidase and tryptase release. Chemical cross-linking followed by micrometric flow cytometry probed direct interactions. Antibodies neutralized integrin-ß1 and recognized its active form. Pertussis toxin (Ptx) was used to decrease integrin-ß1 active form expression. Hematopoietic cell kinase (Hck) was identified by immunoprecipitation (IP) and silenced by siRNA. RESULTS: Major basic protein-1-induced CBMC activation is mediated partly by MBP1-integrin-ß1 interaction on the MC surface. FBM prime CBMC via a G protein, as confirmed by Ptx, to shift integrin-ß1 to its active form. Following MBP1 binding, integrin-ß1 binds Hck that further transduces the activation signal. MC priming with FBM leads to up-regulation in Hck protein level. MC integrin-ß1 neutralization inhibits MBP1-induced activation and uptake. Hck silencing results with reduced MBP1-induced activation. CONCLUSIONS: Fibroblast-derived membranes, integrin-ß1, and Hck are involved in MBP1-induced activation of CBMC and therefore represent a distinct mechanism for this activation. This finding might implicate integrin-ß1 and Hck as targets for decreasing MC - Eos activating cross-talk in allergy.
Subject(s)
Cell Membrane/immunology , Eosinophil Major Basic Protein/immunology , Eosinophils/immunology , Eosinophils/metabolism , Fibroblasts/immunology , Integrin beta1/immunology , Mast Cells/immunology , Animals , Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/pharmacology , Cell Communication/immunology , Cell Membrane/metabolism , Eosinophil Major Basic Protein/metabolism , Fibroblasts/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Gene Silencing , Humans , Integrin beta1/metabolism , Mice , Protein Binding , Proto-Oncogene Proteins c-hck/genetics , Proto-Oncogene Proteins c-hck/metabolismABSTRACT
BACKGROUND: Eosinophils are involved in several inflammatory processes including allergic inflammation. It has been shown that eosinophil functions may be regulated by activating or inhibitory receptors. Hypoxia is a feature of inflamed tissues and has recently been shown to regulate eosinophil viability and pro-angiogenic potential. In this study, the effect of hypoxia and GM-CSF on the inhibitory receptor CD300a in human peripheral blood eosinophils was investigated. METHODS: CD300a expression on eosinophils was analyzed by flow cytometry and evaluated by immuno-fluorescence; mRNA levels were evaluated by RT-PCR. RESULTS: An increase in the expression of CD300a was observed in hypoxic eosinophils compared to the normoxic ones. GM-CSF strongly induced CD300a increase also after 3 h in culture. In addition, hypoxia augmented mRNA levels of CD300a. Inhibition of hypoxia-inducible factor (HIF)-1 abolished the hypoxia-/GM-CSF-induced CD300a increase. CONCLUSION: CD300a expression is up-regulated by hypoxia, and GM-CSF where HIF-1 might play an important role. These results are important for the understanding of eosinophils behavior in inflamed tissue and suggest a new effect on their function in allergic inflammation. Taken together our data point out CD300a as a novel target for the treatment of allergy.