Excretory-secretory products secreted by Paragonimus westermani delay the spontaneous cell death of human eosinophils through autocrine production of GM-CSF

Background: Eosinophils play important roles in tissue inflammatory responses associated with helminth infections. Excretory-secretory products (ESP) produced by tissue-invasive helminths contain a large quantity of proteolytic enzymes that can modulate the host's immune responses. However, little is known regarding the roles of worm-derived products that are responsible for eosinophilic inflammatory responses in helminth infections. Objective: In the present study, we investigated whether ESP produced by Paragonimus westermani, which cause pulmonary or extrapulmonary paragonimiasis in human beings, regulates both cell survival and death of human eosinophils. Methods: The ESP was obtained from P. westermani newly excysted metacercariae (PwNEM). Eosinophils were purified from peripheral blood of healthy donors, and the purified eosinophils were incubated with or without the ESP secreted by PwNEM. The viability of eosinophils was assessed by staining with propidium iodide using the flow cytometer. Results: When eosinophils were incubated with a low concentration of the ESP produced by PwNEM, which totally consists of proteolytic enzymes, eosinophil cell death was delayed compared with results for cells incubated with medium alone. In fact, the ESP at a low concentration stimulated eosinophils to produce detectable levels of GM-CSF that can delay eosinophil cell death. In contrast, eosinophil cell death was dose-dependently accelerated when cells were incubated with high concentrations of the ESP. To see whether the dose-dependent biphasic survival effect of the ESP on eosinophils is primarily due to the protease activity contained in the ESP, a high dose of the ESP was treated with heat at 56°C for 30 min before being added to eosinophils. Attenuating protease activity in a high dose of the ESP by heat treatment reversed the ESP-afforded eosinophil cell death. This prolonged survival of eosinophils induced by the heated ESP was remarkably inhibited by anti-GM-CSF-neutralizing mAb and Jak2 kinase inhibitor AG-490. Conclusion: These results suggest that the proteases in the ESP secreted by PwNEM are able to regulate eosinophil survival through the autocrine production of GM-CSF. Thus, the enhanced eosinophil survival induced by Paragonimus-secreted products may contribute to the elicitation of eosinophilic inflammatory responses at the worm-infected lesion in human paragonimiasis.