Localization of DNA and RNA in eosinophil secretory granules.
BACKGROUND
Although the accepted paradigm is that the proteins stored in eosinophil crystalloid granules are translated from messenger RNA transcribed in the cell nucleus, recent ultrastructural evidence suggests that protein synthesis may also take place within eosinophilic granules.


METHODS
We used 2 different methods to detect the presence of DNA and RNA in eosinophil secretory granules. Using bromodeoxyuridine, a thymidine analogue, and bromouridine, a uracil analogue, we labeled the DNA and RNA in eosinophils in vivo in rabbits. Immunoelectron microscopy to localize these molecules was performed on ultrathin sections of blood and bone marrow eosinophils using monoclonal anti-bromodeoxyuridine antibody with IgG as a control. The immunogold grain density was measured in each subcellular compartment within the eosinophils and analyzed using image analysis software. A combination of DNA/CD63 immunofluorescence staining and a fluorescently labeled molecular probe that stains RNA was used to examine the presence of DNA and RNA in the secretory granules of human blood eosinophils.


RESULTS
The mean density of bromodeoxyuridine-labeled DNA and bromouridine-labeled RNA immunogold grains in the secretory granules of blood and bone marrow eosinophils were significantly higher (p < 0.0005) than cytoplasmic or background staining. We also demonstrated the existence of DNA and RNA in the CD63-positive secretory granules of human peripheral blood eosinophils by means of immunofluorescent staining and a fluorescently labeled molecular probe.


CONCLUSIONS
These results provide evidence that eosinophil granules are the site of DNA and RNA synthesis and suggest the potential for a new role(s) for eosinophil-secretory granules.
Behzad
Ali R
AR
The James Hogg iCAPTURE Centre, St. Paul's Hospital, Department of Medicine, University of British Columbia, Vancouver, B.C., Canada.
Walker
David C
DC
Abraham
Thomas
T
McDonough
John
J
Mahmudi-Azer
Salahadin
S
Chu
Fanny
F
Shaheen
Furquan
F
Hogg
James C
JC
Paré
Peter D
PD
D000818:Animals; D001854:Bone Marrow Cells / Q000737:chemistry / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D001973:Bromodeoxyuridine / Q000378:metabolism; D001976:Bromouracil / Q000031:analogs & derivatives; D004247:DNA / Q000032:analysis / Q000378:metabolism*; D004804:Eosinophils / Q000737:chemistry / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure*; D005260:Female; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D016253:Microscopy, Immunoelectron; D009930:Organic Chemicals / Q000378:metabolism; D012313:RNA / Q000032:analysis / Q000378:metabolism*; D011817:Rabbits; D022142:Secretory Vesicles / Q000378:metabolism*; D014529:Uridine / Q000031:analogs & derivatives / Q000378:metabolism
D005456:Fluorescent Dyes; D009930:Organic Chemicals; C402795:SYTOX Green; D001976:Bromouracil; D012313:RNA; D004247:DNA; D001973:Bromodeoxyuridine; C006824:5-bromouridine; D014529:Uridine