Eosinophils oxidize damage-associated molecular pattern molecules derived from stressed cells.

Eosinophils (Eos) are found at increased numbers within necrotic areas of tumors. We show that necrotic material from cell lysates containing damage-associated molecular pattern molecules induce eosinophil degranulation (release of major basic protein and eosinophil peroxidase) and enhance their oxidative burst while the stimulatory capacity of cell lysates is significantly diminished following oxidation. High mobility group box 1 (HMGB1), a prototypic damage-associated molecular pattern molecule, released following necrosis but not apoptosis, induced a similar effect on Eos. Additionally, we demonstrate that HMGB1 enhances eosinophil survival and acts as a chemoattractant. Consistently, we show that Eos express an HMGB1 receptor, the receptor for advanced glycation end product, and that anti-receptor for advanced glycation end product could diminish the HMGB1-mediated effects. Of all tested biologic activities, Eos respond most sensitively to the presence of necrotic material including HMGB1 with generation of peroxide. We postulate that Eos "sense" necrotic cell death, migrating to and responding to areas of tissue injury/necrosis. Oxidation of cell lysates reduces their biologic activity when compared with native lysates. We postulate that eosinophil-associated modulation of immunity within tumor and other damaged tissues may be primarily by promoting oxidative degradation of necrotic material. Novel therapeutic strategies may be considered by advancing oxidative denaturation of released necrotic material using Eos or other aerobic strategies.

Quality of tumor lysates used for pulsing dendritic cells is influenced by the method used to harvest adherent tumor cells.

BACKGROUND: Lysates from tumor cells are reported to induce maturation of dendritic cells (DCs) and are used in clinical settings for DC-based vaccination against solid tumors. Nevertheless, the maturation inducing effect of tumor lysates on DCs is discussed controversially and the efficacy of tumor vaccines varies significantly. FINDINGS: Using three individual adherent colorectal tumor cell lines we also faced the difficulty to obtain consistent results regarding maturation inducing effect of tumor lysates on DCs. Therefore, we compared different methods to prepare tumor cell lysate and could demonstrate that trypsinizing as a method to harvest adherent tumor cells has a significant negative impact on biologic activity of tumor lysates. Specifically, we assessed induction of maturation markers CD40, CD80, and CD86 on DCs which were treated with differently prepared lysates. CONCLUSIONS: Trypsinizing is a very common way of harvesting adherent cells from culture flasks. Our results shall call investigators' attention to the enzymatic activity of trypsin degrading some possibly important proteins on the surface of cultured cells. Specifically for DC-based vaccination against tumor antigens investigators should avoid trypsin.