SUMMARYInterleukin-5 (IL-5) and eotaxin are the most important cytokines/chemokines responsible for regulating eosinophil locomotion and are known to play a co-operative role in the selective recruitment of eosinophils to inflamed tissues. Following exposure to chemoattractants, eosinophils undergo a series of events, including reorganization of actin filaments and subsequent rapid shape changes, culminating in chemotaxis. In this study we examined the signalling pathways for eosinophil shape change regulated by eotaxin and IL-5, primarily using a gated autofluorescence/forward-scatter assay. Eotaxin and IL-5 were able to elicit shape change with peaks at 10 and 60 min, respectively, and IL-5 triggered the shape change more efficiently than eotaxin. The pharmacological inhibitors of mitogen-activated protein kinase (MAP kinase) and p38 blocked both eotaxin-and IL-5-induced eosinophil shape change in a dose-dependent manner. In addition, depletion of intracellular Ca 2þ and inhibition of protein kinase A (PKA) strongly reduced eosinophil shape change. In contrast, even when used at high concentrations, protein tyrosine kinase (PTK) inhibitors caused only a slight reduction in the ability to change shape. However, treatment with protein kinase C (PKC) inhibitors, such as GF109203X and staurosporine, resulted in a striking inhibition of eosinophil shape change by IL-5, but not eotaxin. Data from the inhibition of activation and chemotaxis of the extracellular signal-regulated kinases (ERK1/2) by the PKC inhibitors were also consistent with findings from the experiments on shape change. Collectively, two eosinophilselective cytokines/chemokines probably regulate eosinophil shape change via a largely overlapping signalling pathway, with involvement of PKC restricted to the IL-5 signal alone.

Background: Th2 cytokines play pivotal roles in allergic inflammation, including eosinophilia, and their actions are antagonized by Th1 cytokines, conferring them therapeutic potential. Methods: In this study, we examined the ability of a number of cytokines to suppress the activation of eosinophils that function as effector cells for allergic airway diseases. Results: Interleukin (IL)-5, IL-6, and tumor necrosis factor (TNF) induced an eosinophil shape change, whereas interferon (IFN)-γ significantly inhibited the shape change. Other cytokines, including IL-1β, IL-4, IL-10 and IL-13, had little or only slightly enhancing or reducing effects on the shape change. We further analyzed the IFN-γ effect, showing that pretreatment with IFN-γ strongly suppressed IL-5-induced eosinophil shape change, and cycloheximide (CHX) abrogated the suppression by IFN-γ, suggesting that new protein synthesis is required for the inhibitory effect by this cytokine. In agreement with these results, IFN-γ blocked the eosinophil migration and ERK phophorylation induced by IL-5, and the addition of CHX restored eosinophil chemotaxis. Conclusions: Collectively, IFN-γ may attenuate eosinophilic inflammation by directly negating eosinophil mobilization.