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Phagolysosomal
pH
of Human
By Petr Cech and Robert
Human
neutrophils
tides
in
two
unsealed.
The
newly
sequester
classes
pH
of
devised
sealed
zymosan
cerevisiae,
E
ARLY
STUDIES
Mandell2
albicans
yeast
probe.
Five
and
yeast
bakers
cells
measured
by
that
from
minutes
pH,
a
used
Saccharoafter
initia-
typically
indicator-impregnated
to PMN
per-
acidification,
in adherent
60 mm.
and
and
reporting
neutrophils
excitatory
1 5 mm,
6.35
after
60
mm.
by
et
of absolute
wavelength,
fluorescence
because
it
intraphagosomal
microbicidal
Sciences.
CA.
by
and
part
AI-16005.
Fellowship.
also
of
in
AI-16252
is
School
Los Angeles.
Supported
tional
ofHematology
UCLA
Oncology.
Department
Center
for
the
P.C.
Institutes
of
of
Health
is the recipient
TW-3129.
in part.
by
ofa
awarded
the
Health
Grants
Fogarty
Interna-
by the USPHS,
Swiss
Academy
of
pH
Neutrophils
were
sities-Mycology
the 24th
ington.
Research
Meeting
D.C..
supplement
Submitted
Address
Medicine.
© 1984
no. 57 ofthe
to Blood,
and
Medical
reprint
UCLA
by Grune
60(Suppl
requests
School
and
Society
The
I):61a.
accepted
to Dr.
brief
Inc.
1$01.00/0
was presented
was published
lysis,
solution,8
bovine
serum
(HBSS,
Grand
at
Washin a
Louis,
Los Angeles.
CA 90024.
for
0.26
neutro-
the vacuolar
after 2 hr.
existence
pH
of two
was
popula-
of
involved
observations
in
pH
of
METHODS
was
venous
blood
erythrocyte-free
by
in Ca2-free
Krebs-Ringer
containing
I g/liter
of glucose
7.4,
(KRP)
or Hanks’
Co.,
each
balanced
Santa
of the
mg
isomer
coupled
Clara,
method
pH
incubated
for
30
salt
and
solution
CA).
9.0,
fluoresceinated
in plastic
tubes
dissolved
and
at
preparation
NaCI,
until
(FZ)
with
washed
the
sedimentable,
in KRP
caused
for
with
1-2
mo.
by
repeated
total
residual
intensely
FZ
water.
to release
was
stored
Prior
and
The
sterilely
to use,
1-2
centrifugation
in
fluorescence
fluorescent
FZ
1 % of their
<
Ninety
added
mixing.
preparation,
useful
of
distilled
temperature
zymosan
>99.5%
of
Co.,
by minor
al. and Cochrane.9
ml of 0.5 M sodium
Ct
in 6 ml of water,
room
was
ml
Chemical
(Sigma)
in 4.5
19.5
suspended
at 4#{176}C,
remained
this
Sigma
particles
of Marshall
was
mm
resulting
I (FITC,
to zymosan
of FITC
of zymosan,
associated
normal
made
suspended
isothiocyanate,
buffer,
Storage
heparinized
technique,
Biological
milligrams
of
from
Assay
MO)
30
was
particles.
fluorescence
in
2 hr.
Washed
Department
pH
Island
modifications
mm
18. 1983.
I. Lehrer,
findings
discussed.
AND
and
albumin
Fluorescence
1982.
July
±
the intraphagosomal
of Boyum’s
hypotonic
0.9%
Univer-
of Hematology.
abstract
Robert
ofMedicine,
& Stratton.
California
(CCU-MRU)
1982.
25. 1983;
0006-4971/84/6301-001
88
Unit
of the American
in December
April
Collaborative
± 0.25
5.68
and
for systems
the aforementioned
prepared
by a modification
ml
is publication
7.38
and
Neutrophils
Briefly,
Sciences.
This
to
acidity
communicating
with
external
vacuoles).7
Because
of the impliphagosomal
heterogeneity
for
us to reexamine
carbonate
National
USPHS
supported.
and
Medicine,
the
activity,
Fluorescein
the Division
are
MATERIALS
St.
From
fell
of these
exhi-
Their
neutrophils.
phosphate
unaf-
fected
by the partial
destruction
of probe
fluorescence
by phagocytes
noted
in our system.
Segal
et al.6
adapted
this procedure
to examine
the intraphagoso-
Medicine,
mm,
reported
A
at a
was
pH
30
occurred,
so that
15 mm and 6.0-6.5
in vacuoles
(“unsealed”
of
this
human
al.5
the
after
mechanisms
alkaline,
SEM).
±
so that
implications
recently
poorly
medium
cations
with
noted
that 80%-88%
attained
a pH
Geisow
were
(mean
0.35
±
The
microbicidal
We
spectrofluorometric
pH was developed
and
to measurement
single
increased,
after
vacuoles
0.19
±
subsequently
stimulated
Poole4
sealed
7.80
tions of phagocytic
vacuoles,
“sealed
and unsealed,”
in
human
neutrophils
that
had ingested
yeast
particles
and showed
that the Candida
albicans
yeasts
are killed
particular
virtue
of this method
was the use of dual
excitatory
wave
lengths
to ascertain
a fluorescence
ratio whose
magnitude
is proportional
to pH. As will be
shown
in the present
work,
the fluorescence
ratio
is
superior
of
subsequently
neutral
after
reported
studies
but
a pH
mal pH of monolayered
human
PMNs
and reported
that phagocytic
vacuoles
were initially
alkaline,
exhibiting
a pH of 7.75
after
2 mm.
Mild
acidification
yeasts
developed
color
of 6-6.5
after
60 mm.
similar
cerevisiae),
I. Lehrer
of phagocytosis,
phil
Candida
monolayers
Bainton3
performed
(Saccharomyces
Ohkuma
biting
phagocytes
in vivo to microdyes,
suggested
that appre3-5.5) occurred.’
More
con-
Recently,
a quantitative
method
for measuring
intravacuolar
by
tion
and
of this phenomenon
in human
neuhave
led to seemingly
disparate
fed
more
vigorous
of phagosomes
of 4.5-5.0
after
par-
sealed
was
derived
that 40%-60%
of the ingested
changes
consistent
with a pH
Jacques
zymosan
procedure
on vacuolar
studies
(PMNs)
or
vacuoles:
particles,
as a pH
results.
cells
vacuoles
formed
by exposing
organisms
and indicator
ciable
acidification
(pH
temporary
trophils
yeast
phagocytic
fluorometric
fluoresceinated
myces
of
Neutrophils
particles
In a few
HBSS.
modified
FZ
FZ
were
at 37#{176}C,
washed
to contain
concentration
twice
opsonized
in 0.9%
experiments,
0.16
was
as
mM
or by spectrophotometric
of
Vol.
16 mM
63. No.
with
serum
suspended
in the
by direct
measurements,
B/cod,
and
described
instead
determined
in I ml of AB
NaC1,
text,
phosphate
hemocytometer
equivalent
1 (January).
1984:
for
30
in KRP
KRP
or
was
buffer.
counts
results.
pp. 88-95
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
PHAGOLYSOMAL
pH OF HUMAN
Phagocytosis
was
opsonized
no.
2054,
Oxnard,
for various
and
B,
was
A was
with
to lyse
clear
gel.
cells
with
an
served
Aminco
ment
Co.,
hold
the
was
stable
In some
pro-
tubes
nm,
and
exit
slits,
The
to
Corp.,
Instruto
pH
magnetic
to a
sensitivity
at 7.4
stirring
bath,
intervals
(0.16
mM)
buffered
of this
sample
employed
in our
reading
assays.
of 0.1-0.2
readings
at F” 485
PMNs
judged
in
lated
that
the
from
approximately
the
recently
not
electrode
and
to check
Standard
tion
labeled
“sealed”
heat-killed
blue
for these
or
been
method
entailed
for assaying
then
trypan
in phosphate-buffered
was
mixing
placed
50
z1 of the
examined,
contained
within
phagocyte
mixture
and
them
was
the
total
was
counted
number
recorded
without
(B).
that
FZ
from
were
standard
in
and
minimally
at pH
to
6 x
and
pH
with
removed
or
10% gelatin
viability
standard
(Fig.
a
4-10.
Half
mm
glass
50
the
remainder
combined
glass
phagocytosis.
curves
and
suspended
free,
devised
were
made
that
had
resuspending
were
ingested
PMNs
above,
in 0.1 M phosphate
PMN-FZ
or
curve
by incubating
at 37#{176}C,
as described
then
opsonized,
a standard
PMNs
latter
for 5 mm,
fixed
with
1), we also
glutaraldehyde
the
PMN-FZ
buffer,
pH
thereafter
in gelatin-stabilized
centrifug-
washed
buffers
of
7.4,
twice
various
not
stain
of
or FITC-C.
in the
fluores-
by fluorescence
FZ
with
or FITC-
50 il of 1.6%
microliters
of this
and
One
hundred
of
fluorescent
Consumption
and
opsonized
respectively)
chamber
that
contained
stirring
control.
Gilson
were
bar.
Measurements
Medical
exam-
neutrophils
sample
of stain,
particles
of the
and
FZ
(final
introduced
with
Electronics
Dual
a 1 .2-mI
electrode
FZ
of oxygen
concentrations,
into
a Clarke-YSI
PMNs
were
run
2 x
and
a small
mag-
simultaneously
consumption
Channel
I and
water-jacketed
were
Oxygraph,
as
made
with
Model
K-IC,
a
a
at 37#{176}C
for 60 mm.
were
of FITC-
coverslipped,
addition
of
measurements,
for 30 mm
RESULTS
concentra-
extracellular
opsonized
Another
FZ
external
did
et al.’#{176}”
Twenty
microscopy.
of
blue
their fluorescence
of differential
suspension
slide,
as
fluorescence
vacuoles
with
saline.
on a microscope
med by phase-epifluorescent
were
unsealed
PMNs
rou-
percentage
that
intracellular
in
blue,
it did
the
Intracellular
maintained
The principle
by Hed
was
a final
Although
opsonized
at constant
transferred
The
2).
at 4#{176}C.
The
Neutrophils
netic
concentrations
experiments,
yeasts.
incubating
the
quenched
described
C. albicans.
blue
studies.
of
4#{174}-zlsamples
volume
containing
cell
at l8Og
(v/v)
KRP
107/ml,
FZ
methylene
we employed
to distinguish
has
final
unless
and
methylene
Because
in companion
C. albicans
and
determined
completely
phagosomes
trypan
blue.
quenching
quenching
of FZ
employed,
blue
FZ,
of 0.8%
mixture
we
vacuoles.
to
section.
vacuoles
C. albicans
were
trypan
“unsealed”
of FZ,
glutaraldehyde-fixed
FZ
mixture
Oxygen
arose
used
FITC,
Results
a Teflon-coated
we calcu-
of its corrections
released
inter-
pH.
spectro-
procedure
rationale
in the
and
In addition,
in sealed
Our
and
ing the
with
viable,
of our
experiments
The
the
be presented
fluorescence
microorganisms
and
destroyed
of
heat-killed
cence
pH,
will
trypan
extracellular
particles.
a
for 30 mm
95%-lOO%
uptake
extraceilular
(Fig.
opsonized
value.
geometry
recorded
FZ
FZ in unsealed
0.8%
albicans
presence
iO
the
for
to maintain
at 37#{176}C
by a circulating
phosphate)
equal
FZ
FZ
this
in our
with
of 0.8%
remained
From
and
contained
mM
to generating
phagocytic,
in 1.25%
in our microcuvette,
to neutrophils
PMNs,
by subtracting
registered
using
opsonized
experimental
signal
FZ,
the
then
(model
for a small
Curves
485,
the conditions
described.7
quench
toxic
at P’
and
intravacuolar
cell-associated
cuvette
units
solution
5 x
examined
blank
incubated
ports
Denmark)
study
fluorescence
measure
In addition
concentrations
exclusion.
percentage
tinely
the
blue
vacuoles”
The
corrected
fluorescent
noningested
“unsealed
same
intensity
KRP-gelatin
photofluorimeter
for
were
by trypan
calculate
As the
relative
at
The
calcu-
vessel
access
to
KRP
to
and
buffer,
were
of 0. 1 N NaOH
vessel
immediately
for
used
of
gelatin-KRP
FZ
with
(0.16
addition
an
was
solution
in
U. The
us
after
with
maximum
emission
PMNs
IL)
quantities
0.02
KRP
mixed
was
M
l06
(A).
was
reaction
Copenhagen,
allowing
glutaraldehyde-fixed
so that
Rockford,
±
buffered
fluorescein
in 0.1 M NaOH
read 80% of
on the 10 scale with an excitation
(F”)
of485 nm and an
of 530 nm. The instrument
was blanked at 450 nm with a
adjusted
opsonized
bar and was maintained
and
was
It
corresponding
and
of microliter
microcuvettes
particles,
of fluorescence.
each
noted
vacuoles
Concentration
water-jacketed
(Radiometer,
external
At
in
adapted
the
electrode
addition
Owens,
C in duplicate.
instrument’s
also
pH.
in gelatinous
of
Buffer
PMNs
siliconized
Chemical,
mittent
minimally
trans-
measured
samples
and
experiments,
Pierce
water
B. The
(American
A, B, and
used.
C
and
sample
was
measurements
samples
were
Microfuge
at 37#{176}C
and
of the
reproducible
volume
removed
agglutination
pH
Sample
was
(Kimble
was
in sealed
x 100.
in a 5-mI
20013,
liberated
(A or B) were
thermostatted
and
as: (B/A)
PMN
particles
Effect
ofExternalpH
on Assay
1 % sodium
the
3200
fluorescence
particles/100
of cell-associated
and
the
an equal
as for
samples
glass
MD)
to obtain
5.5
Model
and
entrance
of
10-zl
I (Sigma).
supernatant
Stabilization
and
One-millimeter
bandpass
with
type
of KRP-gelatin,
settling
practice
A
10 Ml of 2 mM
entrap
spectrophotofluorimeter
Spring,
microcuvettes.
our
the
borosilicate
Bowman
affording
(A,
of aqueous
mixed
gel-stabilized
prevented
rpm)
coverslipped,
and
gelatin,
as microcuvettes,
Silver
matrices
volume
B was
and
volume
to 6 x 50 mm
that
lated
(30
of fluorescent
together
leukocytes
skin
FZ,
or gelatin
IL)
the
Sample
and
an equal
deoxycholate
with
percentage
samples
to determine
for 60 sec in an Eppendorf
both
ferred
mixed
saline,
an equal
10% swine
was centrifuged
rotated
measurements.
slide,
number
not cell-associated.
7.4,
containing
mixed
fluorescence
1 x 10’
(Falcon
and
tubes
20O-zl
epi-illumination
mixed
pH
in a viscous
deposit
for
and
triplicate
in phosphate-buffered
that
deoxycholate,
FZ
incubated
to a glass
fluorescence
of FZ
of KRP
were
removed
with
Sample
plastic
were
maleimide
portion
10’ PMNs
capped
that
transferred
examined
5 x
in small
at 37#{176}C.
At intervals,
was
N-ethyl
CA),
89
by mixing
times
C)
sample
initiated
in 2 ml of KRP
FZ
NEUTROPHILS
the
Standard
Curves
FITC-labeled
zymosan
particles
(FZ)
fluoresced,
after
excitation
at either
485 or 450 nm, in direct
proportion
to their concentration
and the fluorescence
ratio (FR),
i.e., light emitted
by FZ after excitation
at
485 nm/light
emitted
following
excitation
at 450 nm,
remained
from
1.5
selected
studies.
constant
to 10 x
over a range
of FZ concentrations
iO FZ/ml
(data
not shown).
a concentration
As shown
in Fig.
pH, between
glutaraldehyde
pH
of 2.5 x i0 FZ/ml
1 FR was linearly
,
4.5 and approximately
fixation
nor opsonization
significantly
affected
the relationship
pH. The inset in Fig. 1 demonstrates
We
for our
related
to
8.0.
by
Neither
serum
between
FR and
that free FITC
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
CECH AND LEHRER
90
6
FR
4
.
6
.
2
.
C
5
cP0
FR
6
4
8H
4
3
Fig. 1 .
Effect of pH on fluorescence
of FZ in the absence
neutrophils.
The main figure shows the variation
of
fluorescence
ratio
(FR) with
pH for (#{149})
opsonized
FZ. (0)
nonopsonized
FZ. and ()
opsonized
and glutaraldehydefixed
FZ. These data points
were obtained
in three separate
experiments.
and when pooled
and analyzed
by the method
of least-mean
squares.
the values
from pH 4.0-8.0
yielded
the following
relationship;
FR - 1 .04 pH - 2.62, r - 0.96. The
inset shows
the FR/pH
relationship
of free FITC (0). assay
supernatant
(sample
C) fluorescence
(#{149}),
and assay deoxycholate-treated
(sample
A) liberated
FZ (s). The dotted
line
of the inset corresponds
to the FR/pH
relationship
of the
main figure.
of intact
2
1
4
5
pH
and
samples
FR/pH
FZ
A and
relationship
sequestered
display
different
suspension.
C of our
fluorescent
To
assays
follow
the
PMNs
containing
ingested
FZ with glutaraldehyde,
reasoning
that the subcellular
compartments
of such
fixed
nonviable
PMNs
would
manifest
the ambient
same
manifested
by the main figure.
within
neutrophil
vacuoles
might
examine
properties
this
than
possibility,
FZ
we
extracellular
hyde-fixed
in free
fixed
pH. Data
preparations
obtained
with these glutaraldeare shown
in Fig. 2. Note that
#{149}#{149}
C
6-
C
FR
5-
4-
C
3-
Fig. 2.
of pH on fluorescence
of FZ within glutaraldeneutrophils.
Sample
preparation
is described
in the text.
Analysis
ofthe
data points between
pH 4.0 and 8.5 by
the method
of least-mean
squares
yielded the relationship,
FR 0.81 pH - 1 .02. r - 0.97. The inset shows the same experimentel points used in the main portion
of the figure. in terms of their
relative
fluorescence
at a single excitatory
wavelength.
485 nm
(Fm).
The closed circles show early
incubation
times (5-20
mm)
and the open circles
show
later
times
(30-60
mm). Note the
appreciably
larger
scatter
of single wavelength
measurements,
as compared
to measurements
of fluorescence
ratio (FR).
hyde-fixed
Effect
2-
normal
1-
1
1
I
1
I
I
4
5
6
7
8
9
pH
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
PHAGOLYSOMAL
Table
Blue
pH
OF
HUMAN
1 . Comparison
Staining
of Fluorescence
Procedures
in Phagocytic
Fluorescence
Mean
and Trypan
of Percent
FITC-C.
1
33.7
36.6
42.3
35.8
40.8
40.6
3
46.8
44.3
40.5
SEM
38.7
4.0
±
relationship
the
curve
40.5
between
was
2.2
±
41.1
FR and
slightly
shifted
remained
Staining
FITC-C. albicans
albicans
pH persisted,
from
that
The
but
shown
in
Fig.
1 . In the pH range
between
6.0 and 8.0 (FR
=
3.6-5.5),
pH values
derived
from any given
value
of
FR differ by less than 0.3 pH units, when Figs. 1 and 2
are compared.
ancy
increases,
As will
At more
acid
approximating
be seen
pH values,
the discrep0.7 pH units at pH 4.
subsequently,
most
of our
between
taraldehyde-fixed
and
prepared
new
batch
of FZ.
pH
obtained
PMNs
similar
with
to
standard
Fluorescence
preliminary
curves
for
tant,
and (4) the
sealed
phagocytic
existence
vacuoles
of a class
whose
identified
fluorescent
attention.
of FZ by
of FZ fluorescence
molecules
into
exchange
relatively
freely
with
Measurements
of the distribution
by PMNs,
the superna-
of incompletely
contents
could
the external
medium.
of cell-associated
FZ
into
sealed
and
unsealed
vacuoles
were
observing
fluorescence
quenching
by trypan
FZ
were
each
identical
proportions
ingestion
of heat-killed
compared
(Table
made
blue.
by
We
of unsealed
C. albicans
and
1).
Table
2.
Factors
Factor
Fluorescence
destruction
Supernatant
Unsealed
All values
expressed
are
fluorescence
means
as a percentage
the text. Fluorescence
by <10%.
±
SEM,
of total
destruction
n
-
6. The first
cell-associated
pH,
Methods.
decrease
32.5%
was
after
60 mm.
obstacle
and supernatant
Although
(Fig.
2)
maintained
mentable
extracellular
for
form
3%
imposed
pH had
we
occurred.
of
also
that of FITC
“reported”
ofintravacuolar
(2)
FZ,
and
material.
(4)
free
(non-FZ-associated)
By measuring
the overall
fluorescent
from
four
FZ
signal
sources:
in unsealed
the magnitude
can calculate
vacuoles,
of any three
the signal
of its four
emanating
is illustrated
by Tables
data
calculations
referable
3.3
±
0.5
±
2.2
7.2
42.0
±
±
emission
material
uningested
components,
from
the
3 and
4, which
to a single
time
Signal
Time (mm)
30
0.9
2.7
4.3
0.4
30.0
1 1.0
1 .3
40.6
60
±
0.9
±
4.6
±
±
±
1.0
±
6.5
0.7
1 3.9
±
1.1
2.1
38.0
±
1.8
of the initially added FZ
with
blue
and opsonized
2.5
32.5
as a percentage
methylene
eluFZ
fluorescent
fluorescent
signal
approach
and
and
(3)
contain
±
60
of
from
our
assay
(1) FZ in sealed
fourth.
This
±
FZ
14% after
to degradation
pH
vacuoles,
3.5
This
initial
or FZ, so that the FR
the pH of the extracel-
overall
arises
to Fluorescent
the
Excitation
fluoresceinated
The
cuvettes
and
one
relationship
FZ preparation
FZ to a nonsedi-
PMNs.
Negligible
spontaneous
fluoresceinated
molecules
from
in cell-free
mixtures.
of the
solubilized
Calculation
28.4
fluorescence
have
FR/pH
also
approximately
closely
resembled
of this material
lular medium.
1.1
determined
an
fluorescence
at 5 mm and approximately
mm and must have been consequent
3.9
are expressed
would
intravacuolar
that
of the original
of fluorescence
from
accounted
Contributing
were
this
to recording
approximating
(Fig. 1). Release
±
factors
the “A” (deoxyin Materials
and
used absolute
values
of fluorescence
at a single excitatory
wavelength,
it did
not
confound
the
dualwavelength
FR approach
that was used.
As already
shown,
the residual
neutrophil-associated
FZ fluores-
±
cells,
point.
This
was rapid
in onset,
with
a mean
in fluorescence
of 25.3%
after
5 mm and
a major
3.5
three
extracellular
by the
quantitated
from
samples,
described
15
yeast
time
free
have
an FR characteristic
of the
7.4.
Destruction
of fluorescence
25.3
40.0
vacuoles
at each
would
5
ExtracellularFZ
PMNs
factors.
substantial,
the particles
zymosan
extracellular
Incubation
Measured
with
four
contributed
occurred
spectra
we
contribution
to the
mixtures
warranted
(1) incomplete
uptake
PMNs,
(2) destruction
(3) release
of fluorescent
virtually
when
our
Measurements
investigations,
factors
whose
of our
assay
factors
were:
noted
vacuoles
glu-
analyze
to these
although
3% of
signal
FZ by phagocytic
tion of soluble
Influencing
During
four
signal
These
and
phagocytic
data,
Factors
FR
pertaining
unassociated
cence
experimen-
tally
determined
values
for intravacuolar
pH
fell
between
pH 6 and 8, where
the two standard
curves
were
essentially
equivalent.
We elected
to use the
relationships
data
small
occurred
and
cholate-treated)
0.6
±
2 shows
Phagocytosis
of opsonized
FZ,
was incomplete.
Approximately
Neutrophils
2
±
Table
Unsealed
Quenching
FZ
a linear
that
Quenching
in Determination
Vacuoles
Subject
91
NEUTROPHILS
preparation.
heat-killed
Unsealed vacuoles,
C. albicans,
are means of the values obtained at P’ 485 and F’ 450, which
differed
as described
from
in
each other
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
92
CECH
Table
3.
Measured
Flu orescent
and Derived
Values
Used
in Cal culating
lntravacuo
Symbol(s)
A.
Measured
2.33
0.51
Supernatantfluorescence
C,C’
0.725
0.145
Unsealed
vacuoles
m
0.45
Uningested
FZ
n
0.02
pH 7.4
ratio
(sealed
Fluorescence
ratio
(unsealed
incubated
shown
measurements
with
vacuoles)
vacuoles)
opsonized
made at 450
phase-fluorescent
unsealed
albicans
consult
(n)
both
was
microscopy.
vacuoles
was
and
methylene
the text
and Table
sealed
and
measured
blue.
unsealed
d,d’
2.93
0.6 17
FR,
3.038
FR,,
4.748
listed
in part A were
of the
obtained
equations
as described
and calculations
in the text and used to
used.
The
symbol
of the formerly
ingested
particles
(d) plus
tions from formerly
uningested
FZ [n(A
determined
The
by
free
of
merely
proportion
are
shown
FITC
in Table
prime
(‘)
d in terms
3 and
of the
4 establish
to equation
measured
how
much
3,
variables.
fluorescence
FZ would
manifest
if the vacuolar
pH
unsealed
vacuoles
are presumed
to be in
equilibrium
with
it follows
(equation
(B) equals
fluorescence
from
sealed
vacuoles
(x)
+ fluorescence
from
unsealed
vacuoles
(y)
+
fluorescence
from free FITC
(C) + fluorescence
from
extracellular
FZ [n(A
C)], and may be simplified
to
defines
terms
By
made
the form shown
in equation
2 by letting
a = B
[C +
n(A
C)].
Equation
3 states
that
the fluorescence
measured
in the deoxycholate
lysate (A) arises from all
calculation
vacuoles
-
-
-
Equations
4 is equivalent
contribuC)] and
-
the
extracellular
that
5)
medium
fluorescence
at pH
from
7.4,
FZ
in
unsealed
vacuoles
(y) equals
the proportion
of FZ in
unsealed
vacuoles
(m)
times
d. By substituting
this
value for y in equation
2, we derive
equation
6, which
cence
4.
Equation
defining
cell-associated
was 7.4. Since
process
is given in Table
4,
that observed
total fluores-
Table
(C).
Equations
with heat-killed
C.
Fluorescent
measure-
vacuoles
4, part C, and the algebraic
part D. Equation
1 states
0.278
0.0744
4 for details
ments at F” 485 are denoted
by A, B, or C; those at F
450 are symbolized
by the corresponding
primes.
The equations
used to calculate
the signals
arising
from
1.32
0.226
nm.
FZ
uningested
y,y’
x,x’
FZ for 60 mm, and the measurements
B. Please
in part
point of one experiment.
The five measured
values
A,
B, C, m, and n are shown
in Table
3, part
A. The
direct
15
values
Fluorescence
were
of
0.775
3.7
8,8’
the values
frequency
F’450
A,A’
All vacuoles,”
denotes
F’485
lysate
assay
B. Derived fluorescence
Unsealedvacuoles
Sealed
vacuoles
Neutrophils
lar pH
fluorescence
Total
LEHRER
values
Deoxycholate
calculate
AND
and Calculations
the fluorescence
arising
from sealed
vacuoles
in
of the measured
parameters.
following
the same
process
for measurements
at 450 nm, x’ and y’ can be obtained,
allowing
of
the
fluorescent
ratio
from
sealed
or unsealed
vacuoles
(y/y’)
by equa-
(x/x’)
tion
7. Equation
Used
to Derive
8 demonstrates
Intravacuolar
how
fluorescence
ratio
pH
C. Equations
(1)
B-x+y+C+n(A-C)
(5)
y=md
(2)
x + y
(6)
x
(3)
A
(7)
FR,
(4)
d=A-C-n(A-C)
(8)
pH
a
-
d + C + n(A-C)
-
=
a
-
=
xix’.
=
md
FR,,
(FR
+
y/y’
=
1.02)/0.81
(Fig.
2)
D. Calculations
F”485
(1) 2.33
The measured
of sealed
vacuoles.
x +
-
(2)
x+
y-
(3)
3.715
(4)
d-2.93
+
0.02(2.99)
1.545
-
d + 0.725
+ 0.02(2.99)
0.51
=
x’ +
y’
0.775
ci’
(5)
y-
(6)
x
(0.45)2.93
(7)
FR,
-
0.226/0.0744
(8)
pH,
-
(3.038
1.545
-
F’450
0.725
y +
data displayed
-
1.319
-
1.319
+
in Table
0.226
-
3.038
1.02)/0.81
3 were used
5.01
to solve the equations
fluorescence
ratio
=
V
=
(0.45)
x’
-
0.352
pH,,
for
=
(eq.
+
y’
+
0.145
+
0.02(0.63)
0.352
d’
+ 0.145
+
0.02(0.63)
0.617
-
FR,,
-
x’
0.617
-
1.32/0.278
=
0.278
=
(0.45)
0.617
=
-
0.0744
4.748
7.16
7) and consequently
to derive
the mean pH (eq. 8)
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
PHAGOLYSOMAL
is
pH
converted
OF
HUMAN
pH by
experiments
reference
such
to
generated
by
to standard
curves
as that illustrated
in
Fig. 2 (FR = 0.81 pH + 1.02).
These calculations
are illustrated
showing
vacuolar
acidification
after
neutrophils
in a single
experiment.
corresponds
FR of 3.038
the
sealed
93
NEUTROPHILS
in Table
4, part D,
60 mm by normal
The FR. of 4.748
to a pH of 7. 16 in unsealed
corresponds
to an “average”
vacuoles.
The
pH of 5.01 in
vacuoles.
Extracellular
Buffering
In addition
that
influenced
to the
the
phagocytic
and pH
four factors
fluorescent
vacuoles
effects
of
measurements.
Capacity
already
signal
containing
extracellular
In three
FZ,
we evaluated
the
of 16.9
As shown
in Fig.
3, the
pH
human
phil phagocytic
vacuoles
containing
FZ was biphasic.
During
the initial
postphagocytic
period,
the mean pH
was mildly
alkaline,
7.8 ± 0.19 (mean
± SEM,
n = 6),
after 5
preferring
become
We did not attempt
earlier
measurements,
to wait until
virtually
all added
FZ
ingested.
By 1 5 mm, the intravacuolar
pH
mm.
returned
mm,
had
the
to near
neutrality
mean
pH was 6.35
fallen
further
unsealed
vacuoles
60-mm
period.
It was of interest
(7.38
± 0.35,
to 5.68
was
±
7.37
of oxygen
consumption
phils (107/ml)
to twice
and
The
After
30
by 60 mm it
mean
0.03
throughout
these
kinetics
±
to compare
0.25).
±
0.26.
had
had
pH
of
this
to those
induced
by exposing
that number
of opsonized
neutroFZ in
8
I
9
1
INCUSATION
do
do
TIME
mln.
Fig.
3.
Intraphagosomal
pH of normal
human
neutrophils.
Neutrophils
from 6 healthy
subjects
were incubated
with opsonized FZ for 60 mm, and fluorescent
analysis
of intravacuolar
pH
was performed.
Solid circles represent
the pH of sealed vacuoles,
which
constituted
approximately
60% of the total.
Open circles
represent
the average
pH of sealed plus unsealed
vacuoles.
Thus,
tabolism
had
to near
resting
returned
tion
of the rate
of destruction
neutrophils
(Table
2) indicated
destruction
had already
occurred
of maximal
oxidative
to determine
the
the
to the
activity,
when
period
and the
oxidative
rates.
of
period
of
later
me-
Examina-
of fluorescence
that
77.8%
of
by 5 mm, during
metabolism.
nature
of
by
the
the
We made
destructive
the
no
DISCUSSION
The
recent
introduction
of covalently
bound,
the older
visual
techniques
of assessing
and Poole used FITC-labeled
by pinocytosis,
to demonstrate
secondary
pinolysosomes
phages
firmed
was
their
procedure
They
of murine
vacuolar
peritoneal
by
with
peritoneal
Segal
et
macro-
a carbodiimide
so that
reported
al.6
ligand,
macrophages
used
were
5.8
of 5.4
fluorescein-labeled
to assess the intravacuolar
in monolayer
culture
reported
the
initial
(pH 7.75
7.0 after
after 2 mm), with gradual
acidification
1 5 mm and pH 6.0-6.5
after 2 hr.
intraphagosomal
with
tran-
after
7 mm
±
0.2 after
aureus
neutrophils
experiences
to
induction
of
rapidly
acidi-
Staphylococcus
of human
own
conto
it could
be applied
that the phagocytic
reaching
a pH of approximately
finally
equilibrating
at a pH
mm.
interpH of
Geisow
et al.
coupling
fluorescein
siently
alkaline
(pH
7.75)
2 mm
after
phagocytosis.
Thereafter,
the vacuoles
1 5-20
pH.
dextran,
that the
474
cerevisiae
modified
the
phagolysosomes.5
vacuoles
in murine
approximately
results,
and
Saccharomyces
fled,
and
fluo-
pH probes
that can be measured
quantitatively
to overcome
some of the limitations
inherent
Ohkuma
nalized
Our
z
107/min.
corresponded
burst”
occurred
in
neutro-
1 .2 nmole/
alkalosis
“respiratory
acidification
rescent
promised
of sealed
values
for 02 consumption
were as follows:
unstim-
maximal
vacuolar
process.
Neutrophils
±
intravacuolar
the signals
were independent
of the buffer
concentration (16 mM or 0.16 mM KRP)
present
during
the
phase
of ingestion
or measurement
(data
not shown).
We obtained
similar
results
when
HBSS
replaced
KRP in other experiments
(not shown).
ofHuman
on
found
rate
period
attempt
pH
capacity
we
electrode.
The
neutrophils/min)
ulated,
1.1 ± 0.1; 5 mm, 8.7 ± 0.9; 15 mm, 6.0 ± 1.0;
30 mm, 3.9 ± 0.8; and 60 mm, 2.4 ± 0.8 (mean
±
SEM,
n = 3). Maximal
oxygen
consumption
occurred
between
I .2 and 3.2 mm after addition
of FZ and had a
these
that
Intravacuolar
buffering
experiments,
enumerated
arising
from
an oxygen
(nmole/107
pH
fluorescent
pH
and
to be alkaline
to pH
probe
meth-
odology
suggest
that while
fluorescent
particles
may
provide
elegant
pH probes,
they must be used with an
understanding
of the biologic
complexities
of phagocytes.
We identified
several
important
factors
that
contributed
to the fluorescent
signal
emerging
from
suspensions
of normal
neutrophils
and
fluorescent
zymosan
(FZ).
These
included:
destruction
of fluorescent molecules
by postphagocytic
leukocytic
events,
solubilization
of fluoresceinated
and their release
into the external
molecules
medium,
from
FZ
incomplete
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
94
CECH
uptake
of FZ by neutrophils
(minor),
and the existence
of two classes
of phagocytic
vacuoles,
sealed
and
unsealed.
Because
we determined
a fluorescent
ratio
(FR)
that correlated
with pH, rather
than
absolute
values
of fluorescence
destruction
of 25%-33%
phagocytic
intensity
in our
of the overall
neutrophils
did
not
rity of our measurements.
measure
the other
factors
experimental
arising
from
system,
vacuole
We
al.,6
results
sealed
measurements,
fluorescence
compromise
We devised
quantitatively,
allowed
phagocytic
the
procedures
and
derivation
of
vacuoles,
which,
comprised
approximately
60%
population.
found,
in confirmation
of the report
that
sealed
alkalosis,
initiation
vacuoles
initially
manifesting
a pH
of phagocytosis.
by
integ-
of
the
demonstrated
a well
(azurophil)
activity
in
FR
cell-free
et
a mild
5 mm after
i.e., attain-
ment of a pH below 7.0, first appeared
between
1 5 and
30 mm after ingestion,
concomitant
with the waning
of
respiratory
burst activity.
sealed
vacuoles
was 5.68
By 60 mm,
± 0.26.
This
attenuation
interest
antimicrobial
primary
crobial
by Segal
of7.80
± 0.19,
Acidification,
develops
after
of considerable
dase-mediated
oxidase,
total
the mean
pH of
value resembles
LEHRER
The evident
temporal
dissociation
betweeen
respiratory burst
activity,
maximal
quite
early
in the postphagocytic
response,
and vacuolar
acidification,
which
to
our
in our
the
AND
or cessation
of the burst,
in considering
myeloperoximechanisms.
Myeloper-
characterized
model
component
granules,
vitro
at
systems.’
oxidase
requires
and halide
ions
of neutrophil
displays
optimal
pH 4.5-5.0
in
Inactive
by itself,
the simultaneous
to effect
microbial
is
antimivarious
myeloper-
presence
killing.
of H202
However,
our data,
and that of Segal
et al., suggest
that H202
production
by intact
neturophils
would
be maximal
when the phagocytic
vacuoles
have a pH between
7.0
and 7.8. Although
human
neutrophils
acidified
their
vacuoles
to an
H202-generating
indirectly
“average”
oxidative
as enhanced
its earlier
maximal
pH
of 5.7 after
metabolism
02 consumption)
rate
by this
1 hr. their
(measured
was
time.
<10%
Thus,
we
of
con-
the equilibrium
pH values
reported
for murine
peritoneal macrophages
containing
Saccharomyces
cerevisiae (5.4 ± 0.2), but is lower than the values
reported
dude
that myeloperoxidase
operates
in intact
phagocytic
vacuoles
quite
far from
the conditions
that
support
its optimal
antimicrobial
activity
in model
systems.
Possibly,
this partially
accounts
for the dis-
by Segal
et
fluoresceinated
crepancy
between
the
such
cell-free
models
Although
layers
al.
for human
Staphylococcus
technical
factors,
or different
crepancies,
et al. were
such
particles,
it should
uncorrected
neutrophils
aureus
(pH
as the
might
containing
6.0-6.5).
use
explain
of mono-
clinical
these
neutrophils.
The coincidence
of vacuolar
alkalinization
and
piratory
burst
activity
may well be a consequence
dis-
be noted
that the values
of Segal
for any effects
of uningested
bacteria,
unsealed
vacuoles,
and released
FITC.
To the
extent
that
any of these
factors
were
present,
they
would
tend to mask a lower pH signal
from phagocytic
vacuoles
with a signal whose
FR reflected
the extracellular milieu.
Several
aspects
of neutrophil
influenced
by intravacuolar
pH.
are neutral
to slightly
favor
the early
action
brane
enzymes
with
behavior
That
sealed
may
be
vacuoles
active
at
and various
this pH
neutral
proteases.’5’6
One neutral
protease,
a chymotrypsinlike enzyme,
has been
reported
to have
antibacteriaV7”8 and antifungal
activity’9
in vitro. It is noteworthy that the protein’s
bactericidal
activity
against
S.
aureus
or Streptococcus
faecalis
only at or above pH 7.0, conditions
for at least 1 5 mm after ingestion
as in our study,
or Staphylococcus
sequelae2#{176}25 attending
the alkalinizing
oxide
anions
the respiratory
its absence
effect of the dismutation
produced
by NAD(P)H
burst.26
This reaction
from
in
intact
resof
of the superoxidase
during
may be written:
2 02
+ 2 H
H202 + 02, or alternatively:
2 02
+
2 H20
H202
+ 02 + 2 0H.
Observations
that
neutrophils
from subjects
with chronic
granulomatous
disease,
deficient
in 02
production,
acidify
their
vacuoles
more promptly
than normal36’26
and lack the
initial
phase of alkalosis6
are consistent
with this view.
It is possible
that
such
premature
acidification
.-
-+
alkaline
for 1 5-30
mm should
of granule
and vacuolar
memconcordant
pH optima.
Among
the neutrophil
enzymes
optimally
range
are NAD(P)H
oxidase’2’4
potency
of myeloperoxidase
and the relatively
infrequent
is exhibited
in vitro
now shown
to exist
of zymosan
particles,
aureus.6
deprives
chronic
the antibacterial
cationic
protein
granulomatous
disease
neutrophils
activities
of their
chymotrypsin-like
by rapidly
achieving
a pH below
of
7.0.
Evidence
that
vacuolar
alkalinization
by permeant
buffers
promotes
staphylocidal
activity
by chronic
granulomatous
disease
neutrophils
has been advanced
by Segal
et al.6 but
requires
confirmation.
ACKNOWLEDGMENT
We
thank
Lenore
E. Cohen
for excellent
technical
assistance.
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1984 63: 88-95
Phagolysosomal pH of human neutrophils
P Cech and RI Lehrer
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