Regulation of HSP70 on activating macrophages using PDT-induced apoptotic cells

Although anti-tumor immunological responses have been mainly associated with necrosis, apoptosis-associated immune responses have been recently suggested as well. In this study, we investigated anti-tumor immune responses and regulatory mechanisms of HSP70 using apoptotic cells induced by photodynamic therapy (PDT). The relationships between HSP70 release, HSP70 translocation and macrophage responses were studied using confocal fluorescence microscopy, FACS and ELISA. Macrophages incubated with apoptotic cells as well as necrotic tumor cells showed a high level of TNFalpha secretion. Apoptotic cells but not the apoptotic cell supernatants induced TNFalpha secretion. During both necrosis and apoptosis processes, the TNFalpha production was diminished drastically when HSP70 or TLR-2 was inhibited. After the PDT treatment, cytoplasmic HSP70 was released from the necrotic cells, while HSP70 rapidly translocated to the surface of the apoptotic cells. Furthermore, the TNFalpha secretion and the tumor cytotoxicity of splenocytes from mice immunized with apoptotic cells appeared similar to that of splenocytes immunized with necrotic cells. Our in vitro and in vivo results show that apoptosis can potentially have higher impact in inducing immunological responses, hence clarifying the immunological regulatory mechanisms of HSP70 under cell apoptosis and necrosis induced by PDT treatment. These findings could lead to an optimal PDT treatment based on immunological responses.