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Abstract
We have developed an alternative method to study the degranulation in electropermeabilized
human neutrophils by measuring the up-regulation of the specific membrane markers
CD63 (residing in the azurophilic granules of resting neutrophils) and CD67 (present
in specific granules). The expression of these marker proteins was measured by the
binding of specific antibodies to paraformaldehyde-fixed cells and subsequent flow
cytometry. We first investigated whether the changes in CD63 and CD67 expression after
stimulation of intact cells were comparable with earlier measurements of neutrophil
degranulation, in which the release of soluble marker proteins was measured. These
experiments indicated that this new method compares favourably with earlier studies,
both with respect to kinetics and stimulus dependency. Subsequently, we applied this
method (which does not include centrifugation of the cells) to study degranulation
in electropermeabilized neutrophils. In permeabilized neutrophils, a clear up-regulation
of the specific granule marker CD67 was observed upon incubation with a free Ca2+
concentration of 1 microM, a value of the cytosolic free Ca2+ concentration occurring
in formylmethionyl-leucyl-phenylalanine (FMLP)-activated neutrophils. The azurophilic
granule marker CD63 required GTP-gamma-S besides 1 microM Ca2+ for a significant up-regulation.
Hence, our study indicates a different requirement for intracellular signals of the
two main types of granules in human neutrophils.