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Association of granulocyte-macrophage colony-stimulating factor with the
crystalloid granules of human eosinophils
F Levi-Schaffer, P Lacy, NJ Severs, TM Newman, J North, B Gomperts, AB Kay and R Moqbel
Department of Allergy and Clinical Immunology, Royal Brompton Hospital,
London, UK.
We have previously shown that normal-density human peripheral blood
eosinophils transcribe and translate mRNA for granulocyte-macrophage
colony-stimulating factor (GM-CSF) and that the intracellular distribution
was granular as assessed by light microscopy immunocytochemistry. The
present study was conducted to confirm this apparent association between
GM-CSF and the crystalloid granule using a subcellular fractionation method
for human eosinophils and immunogold electron microscopy (EM). Highly
purified (> 99%, by negative selection using anti-CD16 immunomagnetic
microbeads) human peripheral blood eosinophils were obtained from four
asthmatic subjects (not taking systemic medication), homogenized and
density fractionated (5 x 10(7) cells/subject) on linear Nycodenz
gradients. Twenty-four fractions were collected from each cell preparation
and analyzed for marker enzyme activities as well as total protein. Dot
blot analysis with specific monoclonal antibodies (MoAbs) was used to
detect the eosinophil granule proteins major basic protein (MBP) and
eosinophil cationic protein (ECP). An anti-CD9 MoAb was used as an
eosinophil plasma membrane marker. Lactate dehydrogenase (LDH) was used as
a cytosolic marker. Immunoreactivity for GM-CSF was detected by a specific
enzyme-linked immunosorbent assay using a polyclonal antihuman GM-CSF
antibody and confirmed by dot blot. GM-CSF coeluted with the cellular
fractions containing granule markers (MBP, ECP, eosinophil peroxidase,
hexosaminidase, and arylsulphatase), but not those containing cytoplasm
(LDH+) or membrane (CD9+) markers. EM examination of pooled fractions
associated with the peak of GM-CSF immunoreactivity confirmed that they
contained crystalloid and small granules, but not plasma membrane. In
addition, quantification, using immunogold labeling with an anti/GM-CSF
MoAb, indicated preferential localization of gold particles over the
eosinophil granule cores of intact cells. Thus, our results indicate that
GM-CSF resides as a granule-associated, stored mediator in unstimulated
human eosinophils.
Volume 85,
Issue 9,
pp. 2579-2586,
05/01/1995
Copyright © 1995 by The American Society of Hematology
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