Eosinophil major basic protein activates human cord blood mast cells primed with fibroblast membranes by integrin-ß1.

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.

Interacting mast cells and eosinophils acquire an enhanced activation state in vitro.

BACKGROUND: Mast cells (MCs) and eosinophils (Eos), the key effector cells in allergy, are abundantly co-localized particularly in the late and chronic stages of allergic inflammation. Recent evidence has outlined a specialized 'allergic effector unit' in which MCs and Eos communicate via both soluble mediators and physical contact. However, the functional impact of this bi-directional crosstalk on the cells' effector activities has not yet been revealed. We aimed to investigate whether MC/eosinophil interactions can influence the immediate and late activation phenotypes of these cells. METHODS: Human and murine MCs and Eos were co-cultured under various conditions for 1-2 h or 1-3 days, and in selected experiments cell-cell contact was blocked. Cell migration and mediator release were examined, and flow cytometry was applied to stain intracellular signaling molecules and surface receptors. RESULTS: Eosinophils enhanced basal MCs mediator release and co-stimulated IgE-activated MCs through physical contact involving CD48-2B4 interactions. Reciprocally, resting and IgE-stimulated MCs led to eosinophil migration and activation through a paracrine-dependent mechanism. Increased phosphorylation of activation-associated signaling molecules, and enhanced release of tumor necrosis factor α, was observed in long-term co-cultures. Eosinophils also showed enhanced expression of intercellular adhesion molecule 1, which depended on direct contact with MCs. CONCLUSIONS: Our findings reveal a new role for MC/eosinophil interplay in augmenting short- and long-term activation in both cells, in a combined physical/paracrine manner. This enhanced functional activity may thus critically contribute to the perpetuation of the inflammatory response in allergic conditions.

Mast cells in allergy and beyond.

Mast cells (MC) are highly granulated tissue dwelling cells, widely distributed throughout the body in connective tissues and on mucosal surfaces. They are derived from bone marrow progenitors that migrate into the blood and subsequently into the tissues, where they undergo final maturation. Mast cell proliferation, differentiation, survival and activation are regulated by stem cell factor, the ligand for the c-kit tyrosine kinase receptor, expressed on the mast cell surface. They release a large number of pro-inflammatory and immunoregulatory mediators after activation induced by either immunoglobulin E-dependent or immunoglobulin E-independent mechanisms. Mast cells have been most widely studied in the context of allergic reactions and parasite infections, but there is now compelling evidences that they are important players in innate and acquired immunity, wound healing, fibrosis, tumors and autoimmune diseases. This review will discuss current advances in these fields.

The third wave of myotome colonization by mitotically competent progenitors: regulating the balance between differentiation and proliferation during muscle development.

The myotome is formed by a first wave of pioneer cells originating from the entire dorsomedial region of epithelial somites and a second wave that derives from all four lips of the dermomyotome but generates myofibers from only the rostral and caudal edges. Because the precedent progenitors exit the cell cycle upon myotome colonization, subsequent waves must account for consecutive growth. In this study, double labeling with CM-DiI and BrdU revealed the appearance of a third wave of progenitors that enter the myotome as mitotically active cells from both rostral and caudal dermomyotome edges. These cells express the fibroblast growth factor (FGF) receptor FREK and treatment with FGF4 promotes their proliferation and redistribution towards the center of the myotome. Yet, they are negative for MyoD, Myf5 and FGF4, which are, however, expressed in myofibers. The proliferating progenitors first appear around the 30-somite stage in cervical-level myotomes and their number continuously increases, making up 85% of total muscle nuclei by embryonic day (E)4. By this stage, generation of second-wave myofibers, which also enter from the extreme lips is still under way. Formation of the latter fibers peaks at 30 somites and progressively decreases with age until E4. Thus, cells in these dermomyotome lips generate simultaneously distinct types of muscle progenitors in changing proportions as a function of age. Consistent with a heterogeneity in the cellular composition of the extreme lips, MyoD is normally expressed in only a subset of these epithelial cells. Treatment with Sonic hedgehog drives most of them to become MyoD positive and then to become myofibers, with a concurrent reduction in the proportion of proliferating muscle precursors.

The sub-lip domain--a distinct pathway for myotome precursors that demonstrate rostral-caudal migration.

We have previously reported that the myotome is formed by a first wave of pioneer cells generated from all along the dorsomedial portion of the epithelial somite and a second wave of cells issued from all four edges of the dermomyotome. Cells from the extreme rostral and caudal edges directly generate myofibers that elongate towards the opposite pole of each segment and along the pre-existing myotomal scaffold. In contrast, cells from the dorsomedial and ventrolateral lips first reach the extreme edges and then contribute to myofiber formation. The mechanism by which these epithelial cells translocate remained unknown and was the goal of the present study. We have found that epithelial cells along the dorsomedial and ventrolateral lips of the dermomyotome first delaminate into the immediate underlayer of the corresponding lips, the sub-lip domain, then migrate longitudinally along this pathway until reaching the extreme edges from which they differentiate into myofibers. Cells of the sub-lip domain are negative for Pax3 and desmin but express MyoD, Myf5 and FREK, suggesting that they are specific myogenic progenitors.