Improved recovery of functionally active eosinophils and neutrophils using novel immunomagnetic technology.

Clinically relevant and reliable reports derived from in vitro research are dependent on the choice of cell isolation protocols adopted between different laboratories. Peripheral blood eosinophils are conventionally isolated using density-gradient centrifugation followed by immunomagnetic selection (positive/negative) while neutrophils follow a more simplified dextran-sedimentation methodology. With the increasing sophistication of molecular techniques, methods are now available that promise protocols with reduced user-manipulations, improved efficiency, and better yield without compromising the purity of enriched cell populations. These recent techniques utilize immunomagnetic particles with multiple specificities against differential cell surface markers to negatively select non-target cells from whole blood, greatly reducing the cost/time taken to isolate granulocytes. Herein, we compare the yield efficiencies, purity and baseline activation states of eosinophils/neutrophils isolated using one of these newer protocols that use immunomagnetic beads (MACSxpress isolation) vs. the standard isolation procedures. The study shows that the MACSxpress method consistently allowed higher yields per mL of peripheral blood compared to conventional methods (P<0.001, n=8, Wilcoxon paired test), with high isolation purities for both eosinophils (95.0±1.7%) and neutrophils (94.2±10.1%) assessed by two methods: Wright's staining and flow cytometry. In addition, enumeration of CD63+ (marker for eosinophil activation) and CD66b+ (marker for neutrophil activation) cells within freshly isolated granulocytes, respectively, confirmed that conventional protocols using density-gradient centrifugation caused cellular activation of the granulocytes at baseline compared to the MACSxpress method. In conclusion, MACSxpress isolation kits were found to be superior to conventional techniques for consistent purifications of eosinophils and neutrophils that were suitable for activation assays involving degranulation markers.

Calcitriol Reduces Eosinophil Necrosis Which Leads to the Diminished Release of Cytotoxic Granules.

BACKGROUND: Asthma severity and eosinophilia correlate with a deficiency in vitamin D and its active metabolite calcitriol. Calcitriol modulates numerous leukocyte functions, but its effect on eosinophils is not fully understood. We postulated that calcitriol exerts a direct effect on eosinophil biology by modulating cell survival. METHODS: Purified peripheral blood eosinophils from atopic donors were incubated in the presence of calcitriol for up to 14 days with or without IL-5. The effect of calcitriol on eosinophil viability was measured using the annexin-V/propidium iodide flow cytometry assay. We also examined the release of eosinophil peroxidase (EPX) in media using a flow cytometry assay with anti-EPX antibodies, and the enzymatic activity of EPX was measured by an OPD-based colorimetric assay. RESULTS: We observed that calcitriol sustained cell viability in eosinophils with a concurrent reduction of necrotic cells. This effect was amplified by the addition of IL-5. In parallel, we observed that a physiological dose of calcitriol (10 nM) significantly reduced eosinophil necrosis and cytolytic release of EPX in media when coincubated with IL-5. CONCLUSION: These results suggest that calcitriol may exert a direct effect on eosinophils by reducing necrosis and the cytolytic release of inflammatory mediators like EPX.

Identification of human eosinophils in whole blood by flow cytometry.

Identification of eosinophils in whole blood samples by flow cytometry is often problematic. There are usually only a low number and percentage of cells that may be detected, and it may be difficult to discriminate eosinophils from other granulocytes. Here, we propose a simple approach using the eosinophil's intrinsic autofluorescence properties, combined with detection of CCR3 expression, to reliably identify eosinophils in a mixed leukocyte population.

Eosinophils in human oral squamous carcinoma; role of prostaglandin D2.

Eosinophils are often predominant inflammatory leukocytes infiltrating oral squamous carcinoma (OSC) sites. Prostaglandins are secreted by oral carcinomas and may be involved in eosinophil infiltration. The objective of this study was to determine the factors contributing to eosinophil migration and potential anti-neoplastic effects on OSC. Eosinophil degranulation was evaluated by measuring release of eosinophil peroxidase (EPO). Eosinophil chemotaxis towards OSC cells was assessed using artificial basement membrane. Eosinophil infiltration was prominent within the tissue surrounding the OSC tumor mass. We observed growth inhibition of the OSC cell line, SCC-9, during co-culture with human eosinophils, in vitro, which correlated with EPO activity that possesses growth inhibitory activity. The PGD2 synthase inhibitor, HQL-79, abrogated migration towards SCC-9. Our data suggest that OSC-derived PGD2 may play an important role via CRTH2 (the PGD2 receptor on eosinophils) in eosinophil recruitment and subsequent anti-tumor activity through the action of eosinophil cationic proteins.