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Granule
Enzymes
of Polymorphonuclear
A Phylogenetic
By P. Gregory
Comparison
Rausch
and
The functional
significance
of granule
enzymes
in polymorphonuclear
leukocytes
(PMN)
is not fully understood
because
of
the multiplicity
of the enzymes
and the
rare
occurrence
of deficiencies
in man. In
order
to select
appropriate
laboratory
animals
for
genetic
functiona!
animal
and
W
a
of enzyme
human
Neutrophils
of laboratory
fuse
studies,
comparison
PMN
Terry
phosphatase
in
the
undertaken.
alkaline
from a variety
man; the activi-
phagocytic
vacuole
or
the pH optima
lysozyme.
and
and
leukocytes
occurs
discharge
in
requirements
were
not
to explain
the
(PMN)
cytoplasmic
as
their
Differences
metal
phosphatase
cient magnitude
of this enzyme.
HEN
POLYMORPHONUCLEAR
microorganisms,
rapid
degranulation
with
G. Moore
ties of alkaline
phosphatase,
lysozyme,
myeloperoxidase,
and
$-glucuronidase
were
determined
by histochemical
and
analytical
techniques.
Marked
interspecies
differences
in enzyme
activity
were found;
many
species
were
deficient
in alkaline
phylo-
levels
was
were obtained
animals
and
Neutrophils:
hydrolases
of
of
suff)-
variations
phagocytize
granules
and
bacterici-
dal proteins.’
Investigations
of bactericidal
and
digestive
functions
of PMN
have thus centered
on the morphology
of PMN
granules
and the biochemistry
of their enzymatic
constituents.2
Studies
in rabbits
and man have identified
two
major
types
of granules,
azurophilic
and
specific.3
The
former
are
larger
and
are
synthesized
in the
promyelocyte;
they
contain
myeloperoxidase
(MPO),
lysozyme
(LZM),
cationic
antibacterial
proteins,
and a number
of acid
hydrolases including
3-glucuronidase
(/3GU).4
Specific
granules
are smaller
and are
formed
only in the
in rabbits,
alkaline
tained
That
has
myelocyte;
phosphatase
they
contain
(AKP).4’6
in specific
granules,
but in a third particle.7
PMN
enzymes
contribute
to the overall
long
been
functions
has been
study
assumed,
but
only
in the
them
functional
and the
From
the
United
Md.
Submitted
A cademy
May
States
© 1975
Vol.
Army
5. 1975;
MPO
Animal
for
Facilities
of A ccreditation
reprint
by Grune
46,
in the PMN
such
patients
the occurrence
can be performed
Medical
accepted
research
Facilities
of Sciences-National
can Association
A ddress
bactericidal
of
ability
and
of
LZM
other
and
have
and,
con-
the
cell
specific
PMN
enyzmes
the inability
to
Research
of mant”
afford
one
have
been
approach
of such deficiencies
is limited.
Institute
of
infectious
reto
is infrequent,
Diseases,
Fort
Detrick,
2170!.
in conducting
the
Laboratory
Animal
Laboratory
lactoferrin,5
AKP
is not
individually.
analyses.
However,
variety
of studies
that
Frederick,
case
(LZM),
PMN,
been identified.2
The functional
importance
of
difficult
to determine
because
of their
multiplicity
Deficiencies
of one or more
enzymes
ported
periodically;
investigations
of
Blood,
lysozyme
In human
No.
requests:
& Stratton.
6 (December),
July
1. 1975.
described
in this report,
the
and Care,
as promulgated
and
Care
of
Research
of Laboratory
P. Gregory
the
institute
Council.
of
Laboratory
The facilities
A nimal
Rausch,
investigators
adhered
by the Committee
M.D.,
Animal
are fully
to the
on the
Guide
Guide
Resources.
accredited
by
for
for
National
the
A men-
Care.
USA
MR1ID,
Frederick,
Md.
21701.
Inc.
1975
913
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914
RAUSCH
Laboratory
another
animals
in
opportunity
to
which
evaluate
enzyme
deficiencies
naturally
enzymatic
function.
Unfortunately,
AND
MOORE
occur
offer
identifi-
cation
of suitable
models
for investigation
has been
difficult,
since
few interspecies
comparisons
have
been
published.
Although
semiquantitative
histochemical
comparisons
ofAKP
and MPO
are
biochemical
activity
or multiple
enzyme
comparisons
have not been reported.
The present
study
was
undertaken
to identify
suitable
animal
models
for assessment
of the physiologic
importance
and
Blood
was
free from
collected
washed
twice
Hank’s
balanced
were
salt
on
106
40
nucleated
per
RBC
Dextran
The
ml.
in
enzyme
37’C
for
Beckman
minute
were
linear
throughout
expressed
M
in 0.1
mm.
the
$-glucuronidase
(GU)
glucuronide
per
sensitivity
(EC
3.2.1.17)
LZM
was
were
6.8)
10 mM
calculated
the
assay
determined
the
the
molar
method
in
2
was
of Osserman
and
are
o-anisidine
per
was
minute.
with
1.0
of
of
0.25
was
absorbance
hr
mM
substrate
2.52
x
l0.
nmole/min.
Lawlor19
a
per
Results
density
coefficient
employed
in
reaction
m M
3.3
for
ice
then
read
Nanomoles
It
extinction
conditions
lysoplate
56’C
5.0).
an
was
hydrolyzed
Change
at
(pH
in
The
with
by
0.62
M 2-amino-2and
0.1 mM
MgCI2
10).
in optical
determined
the
absorbance
x
assayed
to
Since
X-lOO
1 .0 nmole/min.
peroxide.
buffer
using
and
of
as change
was
acetate
under
by the
hydrogen
expressed
activity
M
0.1
in
(1.62
diluted
removed
sonicated
of substrate
a sensitivity
1 . 1 1 . 1 .7) was
first
Triton
NaOH
Nanomoles
coefficient
(EC
results
exposure.
M
1.25
nm.16
achieved
with
and
3.2.1.31)
in
with
410
M P0
of
3.13.1)
was determined
p-nitrophenylphosphate
extinction
and
nm
were
of
suspensions
periods
and
discarded.
were
they
centrifuged
using
LZM
egg
white
as standard.
Hydrogen
fers
ion
prepared
mined
cent
(pH
(EC
minute
molar
integer.
at 460
phenolphthalein
Maximum
the
they
leukocyte
30-sec
at
NH4CI,
above.
stopped
obtained
whole
buffer
hydrolyzed
was
by
2
and
albumin;
NS,
was
and
to
Hepes-buffered
and
in
PMN
as described
isolated
reaction
using
continuously
lysis
twice
90%
of
serum
Bionetics)
washed
than
healthy
exposure
centrifugation
10 ml
processed
of0.l#{176}/,. AKP
(EC
10.2) with
15.2 mM
range
nearest
removed,
to
three
in
Litton
by
by
1% bovine
(LSM,
less
then
spectrophotometer
the
phosphate
monitored
The
was
resistant
leukocytes
with
suspended
were
lysed
removed
containing
containing
Animals
were
were
were
solution
pellet
were
the
Sonifier)
calculated
to
leukocytes
preparation
animals.
(RBC)
leukocytes
(HBSS-HEPES)
determinations,
recording
and
Isopaque-Ficol
concentration
buffer
(pH
30
DU
The
METHODS
laboratory
Erythrocytes
10 mm,
7.4
blood
W (Bronson
added
to a final
methyl-l-propanol
for
granulocyte
Any
avian
humans
(NS).
and
cellular
at 80
AND
and
pH
5 ml
se15
For
at
of
mm.14
PMN
NaCI
enzymes.
from
of phlebotomy.
solution,
top
granule
MATERIALS
at 4’C
M
in 0.15
g for
at 400
time
NH4CI
layered
bath
at the
of0.87%
of PMN
in EDTA
infection
volumes
5 x
function
by the
change
Blood
according
optima
for
in increments
inclusion
was
smears
of 0.1
calculated
for the
to the
AKP
of
were
0.2
pH
mM
from
the
with
pH
from
Mg2”,
Zn2”,
activity
determined
histochemical
methods
determined
units
or
demonstration
a series
of
8.0-10.6.
Fe2”
in
the
without
of MPO
amino
Metal
incubation
addition
and
methyl
AKP
propanol
requirements
medium.
of metal
were
buf-
were
deterThe
per
ions.
processed
and
scored
of Kaplow.20’2’
RESULTS
Marked
interspecies
variations
in enzyme
activity
were
detected
(Table
1).
$GU
was present
in variable
amounts
in PMN
from
all species.
MPO
was
present
in all species
except
chickens
and geese.
Monkeys,
cattle,
goats,
sheep,
cats,
and hamsters
were
deficient
in LZM,
while
AKP
was undetectable
in
rhesus
monkeys,
cats, two strains
of inbred
mice,
chickens,
and geese.
Cat and
rhesus
monkey
PMN
were deficient
in both AKP
and LZM.
Histochemical
species
examined.
results
In most
correlated
species
closely
the
M P0
with
reaction
biochemical
product
activity
was
for
visualized
all
in
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ENZYMES
GRANULE
OF
POLYMORPHONUCLEAR
Table
915
NEUTROPHILS
Enzy me Activ ity in PMN From Various
1.
.
Units
No
Primates
Human
7
7.7
±
2.4
7
3.0
±
1.3
Cynomolgus
5
0.3
±
0.1
Squirrel
3t
0.4 ±
0.1
Cow
4
0.4
±
0.2
Goat
9
0.6
±
Sheep
9
Horse
3
Burro
11
<1
68 ±
114
±
11
4
5
<1
127
±
9
<1
87
±
30
±
27
±
2
4
<1
38 ±
0.1
6 ±
8
1
<1
27
±
1.1 ±
0.4
19 ±
11
3
<1
30
±
4
1.0
±
0.3
39 ±
10
1.7 ±
0.4
63
±
9
9 ±
1
16 ±
1
471
±
76
8 ±
1
536
±
201
<1
<1
5
0.7
±
0.2
13 ±
6
15 ±
3
99
±
46
lot
5.1
±
0.6
46
4
10 ±
2
95
±
34
6
2.1
±0.2
Mouse-AKR
2t
C57
2t
pig
Chicken
AKP,
nmole/min;
MPO
95±26
9±2
±
8±4
11 ±3
15±3
471
0.7
18
10
<1
1.0
20
5
3t
0.7 ± 0.1
16 ± 3
3
1.1
±
0.9
±0.1
4
Goose
OD/min
(x
0.5
10);
LZM,
±91
<1
<1
273
0
84
0
24±4
23
±
<1
14
±
<1
zg.
of pools tested.
neutrophils,
eosinophils,
however,
only
and
MPO
in
was
maximal
biochemical
required
Mg2
activated
by Zn2
was
absolute
to a lesser
present
neutrophils;
in
eosinophils
as well.
With
the exception
There
<1
0.6±0.2
Hamster
seen
86
7
±
2.9 ± 0.7
Guinea
geese,
44
38 ±
109
6
Rat
tNumber
AKP
11 ±
3
Rabbit
and
SEM
±
LZM
Dog
Lagomorpha
*flGU
PMN
Cat
Carnivora
Ayes
6
MPO
Monkey-Rhesus
Ungulata
Rodentia
3GU
Tested
Species
Species
per 5 x 10
of
for
of
and
sheep,
the
AKP
9.8-10.0.
activity,
enzyme
from
Human,
but
In
AKP,
enzymes
and
was
was
all
present
sources
sheep,
from
chickens
if present,
and
other
in
exhibited
rabbit
AKP
species
were
2).
(r
no correlation
number
in monocytes.
in eosinophils.
however,
at pH
maximum
(Table
burros,
horses
activity
extent
only
PMN
between
0.170)
=
normally
PMN
present
in
the
enzyme
content
peripheral
and
blood
of
the
each
species.
DISCUSSION
MPO
in conjunction
exert
potent
bactericidal
with H2O2
effects,2’22
infections
occurring
PMN
have previously
without
difficulty.24
these
cells,
levels
of LZM
The
in
association
been reported
Since
other
it is interesting
functional
than
and oxidizable
and clinical
to
most
other
importance
with
MPO
deficiencies
to be deficient
in MP023
bactericidal
note
mechanisms
that
species
both
capsule
might
then
permit
in
must
chickens
and
to
of
man.8’9
Avian
but kill bacteria
be
geese
operative
in
have
higher
been
deter-
studied.
of I3GU
in
PMN
mined,
but the enzyme
may degrade
fl-glucuronic
capsular
polysaccharides
of types
II, III, and
and some
types
of Escherichia
coli, Klebsiella,
bacterial
cofactors
has been shown
reports
are now appearing
other
granules
has
acid moieties
V of Streptococcus
and Salmonella.25
bactericidal
substances
not
present
in the
pneumoniae
Lysis
of the
to take
effect.
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916
RAUSCH
Table 2. lnterspecies
PMN
Comparison
AKP Activity
activity
of pH Optima
(Values
refer
determined
and Heavy
to per cent change
at optimal
pH without
Metal
from
cation
AND
Requirements*
MOORE
for
the biochemical
addition.t)
Per Cent Change
Species
pH
Optimum
Mg2+
Zn2
Mg2
+ Zn2’
Fe 2+
Primates
Human
9.8-10.0
Cynomolgus
monkey
+5
-
+4
+9
0
+340
+270
-20
+30
0
Ungulata
Cow
0
+200
+100
-50
+120
-40
9.8-10.0
Goat
-
0
Sheep
9.0-9.2
+5
-35
-10
Horse
9.0-9.2
-30
+260
+210
-30
Burro
9.8-10.0
-5
+160
+100
-10
9.8-10.0
+5
-15
0
-15
9.8-10.0
+2
+15
+15
-10
9.8-10.0
+80
+680
+515
+65
0
Lagomorpha
Rabbit
Rodentia
Rat
Guinea
*01
pig
mM
tAKP
of metal
ion.
undetectable
in rhesus
monkeys,
cats,
mice,
chickens,
and
geese
regardless
of assay
conditions.
Sly et al.26 reported
a case of a mucopolysaccharidosis
secondary
to I3GU
deficiency.
While
no PMN
functional
studies
were reported,
increased
PMN
granulations
were noted,
and the patient
had three episodes
of pneumonia
in the first
30 mo oflife.
Both PMN
and cultured
fibroblasts
were found
to lack I3GU.
To our knowledge,
there
have been
no previous
comparisons
of LGU activity in the PMN
oflaboratory
animals.
Our finding
that it is present
in all species
and
the
it may
findings
reported
be essential
by
to PMN
Sly et al.26
suggest
that,
regardless
of mechanism,
function.
LZM
is found
in both
azurophilic
and specific
granules7
and is a constituent
of both
neutrophils
and monocytes.’9
We have
previously
reported
that
LZM
was deficient
in rhesus
monkey
PMN
but present
in rhesus
monocytes
and
macrophages.27
rabbits.29
Others
The
deficiencies
LZM
present
occurring
hydrolyzes
have
noted
report
LZM
confirms
in the
PMN
peptidoglycan
deficiencies
these
in cattle28
findings
ofmonkeys,
and
goats,
components
sheep,
of cell
and
a strain
documents
walls
of
additional
cats,
and
of
hamsters.
Gram-positive
bacteria
and occasionally
Gram-negative
organisms.2
While
the enzyme
may be
bactericidal,
it is probably
most
important
in aiding
digestion
of bacteria
already
killed
by other
mechanisms.
Thus
it is not
unexpected
that
both
rhesus3#{176}and bovine28
PMN
phagocytize
and kill bacteria
without
difficulty.
Spitznagel
et al.’#{176}
demonstrated
a bactericidal
tient
with combined
deficiencies
of LZM
and
specific
granules
themselves
were
absent,
the
defect
in PMN
from
a palactoferrin.
However,
since
the
case
is not comparable
to our
data.
In future
studies,
the LZM-deficient
species
ful for identification
of the functional
importance
It should
granulocytes,
therefore
be noted
that,
contaminating
the
values
although
our
monocytes
in Table
1 cannot
identified
of LZM.
cell preparations
could
contribute
be considered
here
contained
some
absolute.
may
be
at least
activity,
However,
use9O#{176}<,
and
since
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
GRANULE
the
ENZYMES
actual
OF
number
POLYMORPHONUCLEAR
of monocytes
NEUTROPHILS
was
low,
we
917
doubt
that
any
error
is of practi-
cal importance.
AKP
murine
cannot
PMN.3’
be demonstrated
We obtained
ciency
in avian
PMN.
previously
reported,32
chemical
cept
analyses
the
dog,
The activity
in burro
but the significance
closely
in which
In human
histochemically
similar
results
approximate
low
PMN,
levels
AKP
eosinophils
of this finding
the
activity
is variable,
data
were
and
substrate
that
occur
physiologic
during
response,
deficient
species
note
that
PMN
levels
ofAKP,
tion.27
chronic
reported
of
AKP
is known,
but
and
that
induction
absence.
infected
have
is noted
as early
as
not
the
permit
choice
vestigations
ofactivation
Interspecies
enzyme
tent or by biochemical
increases
in activity
is a
AKP-
it is interesting
monkeys
contain
12 hr following
granules.
Although
was deficient
and
any conclusions
of appropriate
regarding
experimental
to
high
inocula-
the
did
authors
did
not increase
mechanisms
and functional
variations
can be explained
differences
between
enzymes
AKP
function;
animals
for
not artifactual,
since
the
metal
ion changes.
Because
performed
for other
Another
ity in each
possible
species
pre.sent
absolute
might
in
enzyme
remained
of limited
sample
enzymes,
explanation
is related
peripheral
PMN
count
actually
To test this possibility,
Investigations
of
be
but
presumably
undetectable
volumes,
the
same
biochemical
but more
the deficiency
regardless
similar
studies
conditions
for these variations
is that
PMN
to the relative
or absolute
number
blood.
However,
regression
revealed
no correlation.
present
but
in an
specific
antibody
studies
the functional
significance
have
been
limited
because
of
deficiencies
in human
PMN.
animal
models
in which
specific
the infrequent
Identification
deficiencies
neverthefuture
in-
significance
of AKP.
either
by differences
in conwhich
require
methodologic
differences
in assay
conditions.
The AKP
data
suggest
that
ences are not sufficient
to explain
all the variations
observed;
cantly,
for those
species
deficient
in alkaline
phosphatase,
enzymes
re-
infection.
Our data
do
less, they allow
mally
versus
been
Similarly,
AKP
is reported
to increase
during
infection
in patients
with
myelogenous
leukemia34
and adult
hypophosphatasia.35
Strauss
et al.”
deficient
bactericidal
capacity
and
recurrent
infections
in a patient
whose
PMN
contained
abnormal
specific
not test for specific
granule
markers,
AKP
during
marked
Accordingly,
rhesus
ex-
of these
changes,
the function
nor
infection
have
long
been
of interest.33
If induction
it is valid to ask what
purpose
it serves
and how
compensate
for its
from
experimentally
been
bio-
in all species
deficiencies
the
has
Our
detected.
ported
in a variety
of clinical
disorders.
For a full discussion
the reader
is referred
to Kaplow’s
excellent
review.32
Neither
physiologic
simian,
or
AKP
defi-
that we noted
is unknown.
histochemical
ofenzyme
content
in feline,
cafline,’2
also demonstrated
and
inactive
of pH or
were not
apply.
enzyme
of PMN
analysis
of
Finally,
some
form
will be required.
of individual
differsignifiwas
in certain
PMN
activnoractivity
granule
species.
enzymes
occurrence
of genetic
enzyme
of a large
number
of suitable
naturally
occur
should
facilitate
the choice
of appropriate
animals
for more
detailed
studies
of PMN
function
and bactericidal
mechanisms.
Further,
these
data
emphasize
for careful
selection
of animal
models
in experiments
concerned
function
or enzymatic
changes.
enzymatic
the need
with
PMN
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
RAUSCH
918
AND
MOORE
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1975 46: 913-919
Granule enzymes of polymorphonuclear neutrophils: A phylogenetic
comparison
PG Rausch and TG Moore
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