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Published online before print November 8, 2007
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*Institute for Clinical Transfusion Medicine and Immunogenetics Ulm, University of Ulm, Red Cross Blood Center, BaWü-Hessen, Germany; and University of Pittsburgh, Departments of Surgery and Bioengineering, Pittsburgh, Pennsylvania, USA
@ To whom correspondence should be addressed. E-mail: lotzemt{at}upmc.edu.
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Abstract |
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There are increased eosinophils in tumors, and they are generally associated with a good prognosis, whereas their presence in rejecting allografts is largely seen as a harbinger of poor outcome. The biologic role of eosinophils in their pathogenesis is more poorly understood than in allergy and asthma. Myeloid conventional dendritic cells (DCs) and conversely, plasmacytoid DCs are similarly associated with a good prognosis in cancer patients. We hypothesize that eosinophils, similar to NK cells, could mature DCs, and that could be responsible for regulating immunity in the setting of necrosis-associated chronic inflammation as occurs in cancer and transplant rejection. We have demonstrated that CpG DNA promotes eosinophil-induced DC maturation. As such, a greater linkage than had previously been considered between innate immune cells such as eosinophils and the adaptive immune response can be considered. Granulocytes were isolated from normal human whole blood by density gradient centrifugation followed by ammonium chloride-potassium lysis of the remaining red cells. Eosinophils were negatively separated using magnetic beads. Immature DCs were generated from CD-14 positively separated monocytes, which were cultured for 6 days with GM-CSF and IL-4. CpG ODN 2395 (CpG-C) as a pathogen-associated molecular pattern surrogate was used to induce eosinophil-based DC maturation. Transwells were used to assess cell–cell interaction between eosinophils and DCs. Eosinophil survival was assessed by flow cytometry; cells, which did not stain with Sytox-Orange, were considered viable. In the presence of CpG-C, eosinophils induced DC maturation. Similar results were obtained when eosinophils were pretreated with CpG for 4 h, washed, and cocultured afterwards with DCs. Eosinophil-induced maturation of DCs directly correlated with the eosinophil:DC ratio. Transwell studies showed that the direct cell–cell interaction between eosinophils and DCs enhances maturation. CpGs did not adversely affect eosinophil survival; thus, we could exclude the possibility that DC maturation was caused by sensing eosinophil cell death. While eosinophil-derived neurotoxin did not contribute to the described effect, DCs took up and internalized major basic protein (MBP), which was released from CpG-stimulated eosinophils, revealed by confocal imaging and flow cytometry. Thus, the DC maturational-inducing effect of eosinophils may be a result of released MBP from eosinophils. CpG-activated eosinophils mature conventional DCs. The role of viral or bacterial products or potentially, host-derived DNA as eosinophil activators with consequent DC maturation should be considered in more detail in the inflammatory settings in which eosinophils have been observed.
Key Words: dendritic cells • asthma • tumor immunology • CpG • DAMPs
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