Granulocytes play important roles in various inflammatory responses. The aim of this study was to develop in vitro methods to enable simultaneous analysis of eosinophil and neutrophil adhesion and transmigration in mixed granulocyte preparations. We used fibronectin-coated plates, with or without semipermeable inserts, to measure adhesion and transmigration. Granulocytes, from healthy blood donors, were stimulated with either interleukin (IL)-5 and eotaxin or N-formyl-methionyl-leucyl-phenylalanine (fMLP), during incubation in the wells. Three different detergents: n-octyl-beta-D-glucopyranoside (OG), Triton-X-100 or N-cetyl-N,N, N-trimethylammonium bromide (CTAB), were tested for their ability to cause lysis of granulocytes with minimal effect on eosinophil cationic protein (ECP) and myeloperoxidase (MPO) antigenicity. These two proteins were used for quantitative analysis of eosinophil and neutrophil adhesion/transmigration, and CTAB was the most efficient lysing reagent. Cell-recovery rates, based on ECP/MPO measurements, were > 95% in both assays. The adhesion and transmigration of eosinophils increased in a time-dependent manner upon stimulation with IL-5 and eotaxin. Eosinophil adhesion reached a plateau at 90 min of incubation and transmigration at 240 min. Neutrophils displayed a similar pattern of adhesion and transmigration upon activation with fMLP, reaching respective plateaux at 30 and 90 min. Our study shows that CTAB is an effective detergent for lysing granulocytes, yielding high and reproducible recovery rates of ECP and MPO. Measurement of ECP and MPO, as markers for cell counts, can therefore be used to quantify adhesive and transmigration properties of eosinophils and neutrophils in mixed granulocyte populations.

Background In allergic inflammation eosinophils and TH2‐like lymphocytes are supposed to be the major effector cells and considered to contribute as cellular source of the key cytokine interleukin (IL)‐5. Objective The purpose of this study was to enable detection of IL‐5 containing leucocytes and to investigate whether the number of these cells in the blood circulation differed between healthy and asthmatics before and after allergen provocation. Methods The distribution of intracellular IL‐5 in human peripheral blood eosinophils (PBE) and lymphocytes (PBL) has been investigated using fixation and cell membrane permeabilization with octyl‐glucopyranoside, the FOG‐method, and flow cytometry. The intracellular staining was performed on leucocytes without any prior purification and in vitro stimulation. The specificity of IL‐5 binding to intracellular compartment of both PBE and PBL was confirmed by complete inhibition with human recombinant IL‐5. Results Preformed intracellular IL‐5 was detected in the main population of PBE (> 70%) in both healthy individuals and asymptomatic patients. Moreover, preformed intracellular IL‐5 was also detected in 4.8% and 2.4% of PBL from healthy individuals and asymptomatic patients, respectively. There was a correlation between the absolute number of PBE and IL‐5 positive PBE. In patients with pollen‐related asthma, the number of IL‐5 positive PBE and PBL increased significantly 24 h after an allergen inhalation provocation (P < 0.05). In the healthy control group no differences regarding IL‐5 positive PBE and PBL were obtained pre‐ and post‐allergen challenge. Conclusions In patients with mild allergic asthma, but not in healthy individuals, allergen provocation induces an increased absolute number of IL‐5 positive PBE and PBL. The reason for the relatively high number of IL‐5 positive PBL is unclear, but a plausible explanation might be that other lymphocyte subsets besides CD4+ TH2 can produce IL‐5. However, enumeration of IL‐5 positive leucocytes may be used as an activity marker and also be a useful tool in monitoring the inflammation in asthma.

We have demonstrated a selective recruitment of eosinophils, and higher levels of released ECP and eotaxin, in skin chambers stimulated with allergen, as compared with control chambers. Allergen challenge in skin chambers is a useful tool for studies of eosinophil recruitment, their state of activation, and their involvement in the allergic inflammatory response.