The inhibitory receptor CD300a is up-regulated by hypoxia and GM-CSF in human peripheral blood eosinophils.

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.

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.

The role of eosinophil major basic protein in angiogenesis.

BACKGROUND: Eosinophil-derived major basic protein (MBP) plays an active role in allergic inflammation and tissue remodelling. However, its role in angiogenesis has not been established as yet. Therefore our objective was to investigate whether MBP exhibits any direct pro-angiogenic effects. METHODS: Rat aortic endothelial cells and human umbilical vascular endothelial cells were cultured with different concentrations of MBP and their viability (Trypan blue exclusion test), proliferation (thymidine incorporation) and capillary-like structure formation (matrigel assay) were investigated in vitro. The angiogenic activity of MBP was then tested in vivo using the chick chorio allantoic membrane (CAM) assay. RESULTS: Subcytotoxic concentrations of MBP induce endothelial cell proliferation and enhance the pro-mitogenic effect of vascular endothelial growth factor (VEGF), but do not affect their VEGF release. MBP promotes capillarogenesis by endothelial cells seeded on matrigel and sprouting formation in the CAM assay. Furthermore, we have shown that the pro-angiogenic effect of MBP is not due to its cationic charge since stimulation of the CAMs with the synthetic polycation, poly-L-arginine does not induce any angiogenic effects. CONCLUSIONS: These data demonstrate that MBP has pro-angiogenic effects in vitro and in vivo, providing a novel mechanism whereby MBP can participate in tissue inflammation and remodelling in atopic diseases.

Efficient purification of eosinophils from human tissues: a comparative study.

BACKGROUND: Eosinophils are key effector cells in allergy and in other inflammatory diseases. Although they carry out their function in the tissues, no efficient method exists allowing for consistent purification of tissue eosinophils for culture. Rather, studies rely mainly on peripheral blood eosinophils. This study aimed to determine the most efficient protocol for purifying eosinophils from nasal polyp tissue. METHODS: Nasal polyps were obtained from patients undergoing surgical polypectomy. The polyps were minced and enzymatically digested. Surface receptor analysis was performed by flow cytometry. In order to obtain optimal purification, the nasal polyp cell suspension was subjected to two methods of purification: 1) positive magnetic selection of CCR3+cells, or 2) negative selection using CD3/CD14/CD16 magnetic beads. Enriched tissue eosinophils were cultured with or without IL-3, IL-5 or GM-CSF, and their survival was evaluated by flow cytometry. RESULTS: Tissue-derived eosinophils exhibited surface expression of NEC2, DNAM-1, NTBa, 2B4, and CD300a comparable to similarly prepared eosinophils obtained from the peripheral blood of the same patients. Positive selection consistently yielded eosinophils of high purity (>90%) with 63% viability. In contrast, negative selection yielded better viability (88%), reduced purity (66%), and could be utilized for in vitro activation experiments. CONCLUSION: Eosinophils can be purified from nasal polyps. Negative selection appears to be advantageous due to improved viability of the eosinophils, which may be cultured and activated in vitro. This methodology is an important advance in studying tissue eosinophils for further investigations on inflammatory tissue responses.

Tissue remodeling and angiogenesis in asthma: the role of the eosinophil.

This review covers the role of eosinophils in asthma-associated tissue remodeling and angiogenesis focusing on angiogenesis which is a recently discovered feature of asthma. In addition, novel directions for eosinophil-targeted/angiogenesis-targeted pharmacological intervention are discussed as new approaches in the treatment of asthma.