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Structural and functional heterogeneity among peroxidase-negative granules
in human neutrophils: identification of a distinct gelatinase- containing
granule subset by combined immunocytochemistry and subcellular
fractionation
L Kjeldsen, DF Bainton, H Sengelov and N Borregaard
Department of Hematology, University Hospital, Rigshospitalet, Copenhagen,
Denmark.
The existence of separate gelatinase granules in human neutrophils has been
a matter of debate in recent years. We have demonstrated that the 135-kD
form of neutrophil gelatinase is a complex of 92-kD gelatinase and a novel
25-kD protein termed neutrophil gelatinase-associated lipocalin (NGAL)
that, in addition to being complexed with part of the gelatinase, is
localized in free form in peroxidase-negative specific granules. Because
this association was not appreciated in earlier studies, we decided to
reassess the ultrastructural localization of gelatinase using specific
antibodies without immunoreactivity towards NGAL. Double-labeling
immunogold electron microscopy was performed on frozen thin sections of
human neutrophils. Twenty-four percent of all peroxidase-negative granules
were labeled with antigelatinase antibody, but not with antilactoferrin
antibody. These granules are defined as gelatinase granules. Sixteen
percent reacted with antilactoferrin antibody but not with antigelatinase
antibody. The rest (60%) reacted with both antibodies. All granules
labeling for lactoferrin are defined as specific granules. Gelatinase
granules were observed as round and oval forms of considerably smaller size
than specific granules, and were less electron dense. Isolated granules
obtained by subcellular fractionation were also examined by immunoelectron
microscopy. This demonstrated that peroxidase-negative granules comprise a
continuum from the most dense granules that contain lactoferrin but no
gelatinase to the lightest that contain gelatinase but no lactoferrin.
Thus, gelatinase granules do exist as a subpopulation of
peroxidase-negative granules and may allow for exocytosis of gelatinase
during neutrophil diapedesis without substantial mobilization of other
peroxidase- negative granules, ie, specific granules.
Volume 82,
Issue 10,
pp. 3183-3191,
11/15/1993
Copyright © 1993 by The American Society of Hematology
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