NK cells interact with eosinophils
We next observed that there was a positive relationship between the total number of nasal lavage NK cells and that of eosinophils 1, 6 and 24 hrs after allergen challenge (Figure 3, A-C ). No significant correlation was observed between NK cells and other leukocyte populations (data not shown). In vitro assays previously showed that peripheral blood NK cells are able to trigger apoptosis of autologous blood eosinophils (12). To determine whether soluble mediators were involved in NK cell-induced eosinophil apoptosis, we incubated freshly isolated eosinophils from healthy donors (4 hrs, 37°C) with supernatants from past autologous NK cell-eosinophil co-cultures kept at -80°C (Fig 4, A ). These supernatants did not increase basal levels of eosinophils apoptosis, suggesting the need for a direct contact between eosinophil and NK cells. Based on their CD56 expression, two primary subsets of NK cells are found in human subjects, namely CD56dim and CD56bright (10) . The CD56bright NK cells are commonly described as cytokine-producing cells with poor cytotoxic ability, whereas CD56dim NK cells are best known for their potent cytotoxic activity. To assess the role of one particular subtype is involved in eosinophil apoptosis, NK cells from healthy donors were sorted by FACS, based on their CD56 expression and incubated with autologous eosinophils (4 hrs, 37°C) (Fig 4, B, left panel ). Intriguingly, eosinophil apoptosis was not modified when eosinophils were incubated alone with either the CD56dim or CD56bright NK cell subset, suggesting a combined interaction of both subpopulations while inducing eosinophil apoptosis (Fig 4, B, right panel ).
Upon activation, eosinophils release many mediators, including highly reactive oxygen species that can damage surrounding tissues. Besides the potential pro-resolving effect of NK cells on eosinophils, activator effects by NK cells on eosinophils have also been reported (13). We first observed that isolated eosinophils were by far the most important producers of superoxide anion among different leukocyte populations involved in type 2 allergic inflammation, at rest and when activated (Fig 4, C ). In contrast, NK cells were unable to release superoxide anion (Fig 4, C ). We next wanted to assess superoxide release by eosinophils at different time points after co-culture with NK cells. In the presence of NK cells, superoxide release from eosinophils was significantly reduced after 1 hr of co-incubation. This inhibitor effect was overwhelmed after 4 hrs of co-incubation (Fig 4, D ), confirming the partial activator effect of NK cells on eosinophils (13).