TY - JOUR
T1 - Purification of functional eosinophils from human bone marrow
AU - Wong, Tina W.
AU - Jelinek, Diane F.
N1 - Funding Information:
This work was supported by the NIH Pre-doctoral Immunology Training Grant ( T32 AI07425 ). The authors would like to thank Dr. Hirohito Kita for sharing his technical and intellectual expertise and Kathleen Bartemes for her technical assistance.
PY - 2013/1/31
Y1 - 2013/1/31
N2 - Eosinophils are granulocytic leukocytes that are best known for their involvement in host immune defense and pathologic states. More recently, they have also been shown to play a role in regulation of murine plasma cell homeostasis in the bone marrow, which prompted our investigation of human bone marrow eosinophils. However, effective methods to isolate eosinophils from human bone marrow thereby allowing comparisons with circulating eosinophils have not yet been described. Herein we describe the development of a novel, cost effective protocol for the purification of eosinophils from human bone marrow that allows us to obtain bone marrow eosinophils of near 100% purity after an 8-day culture system. Furthermore, we demonstrate that bone marrow eosinophils have characteristics similar to blood eosinophils, including the expression of IL-5Rα, the presence of eosinophil-specific granules, and similar activation kinetics upon phorbol myristate acetate and high-dose IL-5 stimulation. While migratory responses toward the chemokine CXCL12 differed between purified bone marrow and freshly isolated blood eosinophils, migratory responses were similar upon comparison of bone marrow eosinophils with blood eosinophils cultured ex vivo for 8. days prior to assay. Interestingly, a concurrent upregulation of CXCR4 expression was not observed in these cultured blood eosinophils. Taken together, we have overcome the existing challenges to the study of bone marrow eosinophils through our novel strategy for cell purification and have thus enabled future investigations of these cells and their role(s) in human health and disease.
AB - Eosinophils are granulocytic leukocytes that are best known for their involvement in host immune defense and pathologic states. More recently, they have also been shown to play a role in regulation of murine plasma cell homeostasis in the bone marrow, which prompted our investigation of human bone marrow eosinophils. However, effective methods to isolate eosinophils from human bone marrow thereby allowing comparisons with circulating eosinophils have not yet been described. Herein we describe the development of a novel, cost effective protocol for the purification of eosinophils from human bone marrow that allows us to obtain bone marrow eosinophils of near 100% purity after an 8-day culture system. Furthermore, we demonstrate that bone marrow eosinophils have characteristics similar to blood eosinophils, including the expression of IL-5Rα, the presence of eosinophil-specific granules, and similar activation kinetics upon phorbol myristate acetate and high-dose IL-5 stimulation. While migratory responses toward the chemokine CXCL12 differed between purified bone marrow and freshly isolated blood eosinophils, migratory responses were similar upon comparison of bone marrow eosinophils with blood eosinophils cultured ex vivo for 8. days prior to assay. Interestingly, a concurrent upregulation of CXCR4 expression was not observed in these cultured blood eosinophils. Taken together, we have overcome the existing challenges to the study of bone marrow eosinophils through our novel strategy for cell purification and have thus enabled future investigations of these cells and their role(s) in human health and disease.
KW - Activation
KW - Bone marrow
KW - CXCR4
KW - Chemotaxis
KW - Eosinophil purification
KW - Eosinophils
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U2 - 10.1016/j.jim.2012.10.006
DO - 10.1016/j.jim.2012.10.006
M3 - Article
C2 - 23085531
AN - SCOPUS:84871454410
SN - 0022-1759
VL - 387
SP - 130
EP - 139
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
IS - 1-2
ER -