Supplementary Material for: Human Eosinophils Show Chemotaxis to Lymphoid Chemokines and Exhibit Antigen-Presenting-Cell-Like Properties upon Stimulation with IFN-γ, IL-3 and GM-CSF

<i>Background:</i> Eosinophils are multifunctional leukocytes. Under physiological conditions, they circulate in the blood and through the tissues to serve their functions. In certain inflammatory states, they enter the T-cell areas of lymph nodes (LNs) that drain the inflamed tissue and communicate with T cells in LNs, but the underlying mechanism that regulates their trafficking to LNs is not yet fully explored. Here, we report that a human eosinophilic leukemia cell line, EoL-1, and human peripheral blood (PB) eosinophils become reactive to the lymphoid chemokines CCL21 and CCL25 upon stimulation. <i>Methods:</i> EoL-1 cells were differentiated with dibutyryl cyclic AMP (dEoL-1) and subsequently pulsed with IFN-γ, IL-3 and GM-CSF. The eosinophil fraction was purified from normal human adult PB and incubated for 1 day with the same cytokine combination. <i>Results:</i> Upon cytokine stimulation, dEoL-1 cells expressed chemokine receptors CCR7, CCR9 and CCR3 and developed chemotactic responsiveness to CCL21, CCL25 and CCL11, which bind to the respective receptors. Human PB eosinophils also showed chemotactic responsiveness to CCL21 and CCL25 upon stimulation with IFN-γ, IL-3 and GM-CSF. In addition, the cytokine-stimulated dEoL-1 cells expressed costimulatory molecules, including CD40, CD80, CD86 and HLA-DR, and also expressed a tolerogenic and Th2-polarizing enzyme, indoleamine 2,3-dioxygenase. <i>Conclusions:</i> These in vitro observations raise the possibility that eosinophils may utilize lymphoid chemokines to enter LNs and serve antigen-presenting functions in the LN under certain inflammatory conditions.