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Kinetics and mechanism of the bactericidal action of human neutrophils
against Escherichia coli
MN Hamers, AA Bot, RS Weening, HJ Sips and D Roos
A mutant strain of Escherichia coli (E. coli ML-35) was used to follow the
kinetics of phagocytosis, perforation of the bacterial cell envelope, and
inactivation of bacterial proteins by human neutrophils. This particular E.
coli mutant strain has no lactose permease, but constitutively forms the
cytoplasmic enzyme beta-galactosidase. This implies that the artificial
substrate ortho-nitrophenyl-beta-D- galactopyranoside cannot reach the
beta-galactosidase unless the bacterial cell envelope has been perforated.
Thus, the integrity of the E. coli envelope can be measured simply by the
activity of beta- galactosidase with this substrate. Indeed, ingestion of
E. coli ML-35 by human neutrophils was followed by perforation of the
bacteria (increase in beta-galactosidase activity). Subsequently, the beta-
galactosidase activity decreased due to inactivation of the enzyme. With a
simple mathematical model and a curve-fitting computer program, we have
determined the first-order rate constants for phagocytosis, perforation,
and beta-galactosidase inactivation. With 32 normal donors, we found an
interdonor variation in these rate constants of 20% to 30% (SD) and an
assay variance of 5%. The perforation process closely correlated with the
loss of colony-forming capacity of the bacteria. This new assay measures
phagocytosis and killing in a fast, simple, and accurate way; it is not
hindered by extracellular bacteria. Moreover, this method also measures the
postkilling event of inactivation of a bacterial protein, which permits a
better detection of neutrophils deficient in this function. The assay can
also be used for screening neutrophil functions without the use of a
computer program. A simple calculation suffices to detect neutrophil
abnormalities. Neutrophils from patients with chronic granulomatous disease
(CGD) showed an impaired rate of perforation and thus also of inactivation.
Neutrophils from myeloperoxidase-deficient patients or from a patient with
the Chediak-Higashi syndrome only showed a retarded inactivation of
beta-galactosidase, but normal ingestion and perforation. The role of
myeloperoxidase in the killing process is discussed. Although
myeloperoxidase does not seem to be a prerequisite for perforation, it
probably plays a role in bacterial destruction by normal cells, because the
inactivation of bacterial proteins seems strictly myeloperoxidase
dependent.
Volume 64,
Issue 3,
pp. 635-641,
09/01/1984
Copyright © 1984 by The American Society of Hematology
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