Activation neutrophil superoxide production by concanavalin A can occur low levels of intracellular ionized calcium
Descripción
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1987 69: 762-768
Activation of neutrophil superoxide production by concanavalin A can occur at low levels of intracellular ionized calcium JC Whitin, K Takahashi and HJ Cohen
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Activation
of Neutrophil
Can
Occur
at Low By
The
effect
of concanavalin
of ionized cytes
(PMN)
indicator
was and
PMN The not
Quin2.
a
rise
in [Cam
rise
in [Cam
a
new.
higher
with attained
within
the
second
(such
production.
Thus
coupled of
the
O2
experiments A.
Quin2
could
in the
H
be
with
of granule
contents,
Other
calcium,9”#{176} and
of
the
use
respond
to
tial
to 02
A
is useful (a-MM)
(Con
ment tion
of
are
depolarized,
they
Ca
have
then
demonstrating
results also
The
in a second
that We
of a-MM.
lag
Ca present
every
data
show
that
acid,
bovine
ethyleneglycol-bis-(f3-aminoethyl
AND serum
produc-
ether)
& Stratton.
St Louis;
with
much A
by Con
could
induce
the
a Con
of
stimulus-
activation
of the
observations change
A 02
While
result
in
that
raise
] in
in [Ca,
system.
Inc.
HEPES, lysophosphatidylcholine (LPC), a/J-D-glucuronide, p-nitrophenylphosphate, and triton X- I 00 were purchased from Sigma phorbol
myristate
Brewster,
NY;
cals,
Milwaukee,
WI;
Ficoll-Paque
Fine
Chemicals, Quin2
and
acetate
cytochalasin
B at
1.0
(PMA) B from
and Dextran
Piscataway,
NJ;
and
T-500
the
ester
in dimethyl
CA.
Stock
Chemi-
from
Phar-
calcium
(Quin2
A/M)
solutions
in distilled
sulfoxide
Consoli-
fluorescent
Quin2-tetra-acetoxymethyl
mg/mL
from Aldrich
H20;
were
cytochala-
(DMSO);
PMA
at
2
mg/mL in DMSO and diluted to 20 g/mL in buffer for use; and LPC at 2 mg/mL in ethanol. Arachidonic acid was diluted to 2.5 mg/mL in 25% vol/vol ethanol and clarified with 0.1 N NaOH. Quin2 A/M was dissolved at 10 mmoh/L in DMSO. Human
peripheral
blood
PMN
were
isolated
utilizing
Dextran
T-500 and Ficoll-Paque sedimentation followed by hypotonic lysis of contaminating erythrocytes as previously described.’6”7 PMN were finally resuspended in phosphate buffered saline (PBS; 135 mmol/L NaC1, 5 mmol/L KCI, 10 mmoh/L sodium phosphate buffer, pH at 5 x l07/mL
From
that by
and
the
kept
of Ca1, time
thus
PMN activation are
cytochrome
N,N’-tetra-acetic
of below
c type
June
Supported
in part
Research Address
are
VI,
acid
Department
of
ofRochester
Submitted 1 1 198.
METHODS albumin,
a
these
generating
Midland,
7.40)
of a second
that
Con
gener-
in ice.
require-
we found is reversed
increment
increases
MATERIALS
762
times poten-
PMN by Con A can occur at levels of Ca that the levels obtained after treatment with a stimulus.
Arachidonic
of
production PMN are
of 02
addition
02
by
than
observation
A
of a large
dated
University
addition
necessity
by Grune
1987
is
correlates system.
of the
are
cytoplasts.
]
[Ca,
O2
is induced
Con
from Behring Diagnostics, La Jolla, prepared as follows: Con A at 2 mg/mL
different when Con
a diminished
indicator Quin2, rise in Ca, that
and
the
activation
indicator
a stimulant
membrane
for reactivation
in
generating about
[Ca,1
though
Quin2-loaded
coupling O
macia
ionized
stimulus: their
the
increment
Chemical,
include
A.
subsequent
activated.
secretion
02 These
in
in
even
is the
02
less
] which
induced
this
of
were of the
at [Ca, level
to range
onset
that
activation
occur
increment
(EGTA), Ficoll 70, MM, p-nitrophenyl superoxide dismutase,
to various
between example,
with a-MM, does not)5 shorter,
and
for extracellular by Con
stimulus
C
the ability of aterminate activation
by an additional
Using the trapped Ca Con A induces a sustained the
the
or phagocyto-
as
small
production
Thus.
Supporting
cytoplasts.
]
Ca elevation
a tenfold
the
at [Ca,
A can
of in the
Over
-
time
concen-
treat-
intracellular
A)
because to reversibly
treated secretion
production
remains
a very PMN
sin
for a challenge
prior
in
of 02
incremental
PMN.
doubts
These latter responses have received of their potential causal involvement
by Con A.’5 In this way the relationships PMN responses can be analyzed. For
primed
only
PMN
of PMN.
PMN are while granule
Ca-repIete
response
Ca-
stimulation
of
the
A-induced
phosphatidyl inositol membrane-bound
concentration
of concanavahin
PMN responses methylmannoside
A-treated ceases,
than
with
after
aggregation, PMN
lower
by Con
of the
results
concentration.
level.
normal
to the The
addition
PMN
production Quin2
resting
Further
These 02
the
of Ca4
of [Ca,] PMN.
The
A-treated
system
of PMN
production,
potential,4’5 phosphorylation,7’8
calcium ([Ca,]).”2 much attention because activation
of 02
chemotaxis,
membrane protein
Ca. produce
I and
intracellular
addition
is a function
Quin2.
Con
ating
A is
by loading
intracellular
A. The
in a return
baseline
the
possible
activation
(PMN)
initiation
responses
changes in metabolism,6
The
extracellular to
A.
to
A
reactiva-
the
of Ca,
induced
the
in the
depleted
NEUTROPHILS
stimuli
in the
]
Con
assess
of
Con
Ca-replete
of
of
by Con and
by
to
in [Ca, of
can
UMAN
system
be
absence
PMN
sis.l_3
rise
induced
inactivation,
undertaken
the
PMN
depleted
in [Ca,4]
return
of
occurred
cells
return
level
always
of a
these
with results
production
Con
the
not
of
baseline
to
I and
but
f-depleted.
of
Calcium
PMN
to
of [Ca
A is
addition
required Ca
addition
The
PMA resting
tration
level after
acid)
to
onset
Con
original
in [Ca,
rise
the by
The
the
arachidonic
generating
for
to
A buffer.
preceded
minute
normal
markedly
induced
minutes.
activation.
were
requirement by Con
the
to the
was external
production.
increment
Con
with
A
J. Cohen
to Ca-depIeted
calcium
steady-state
]
[Ca,
as
] ]
one
of O2
in a second
tightly tion
of
cessation
stimulus
resulted 02
return
that
in the
five
(a-MM)
in the and
]
of Ca
and Harvey
ment
granulo-
of
by Concanavalin Ionized
Takahashi,
concentration
fluorescent
Production
of Intracellular
Kazuhiko
in human
addition
[Cam
The
#{247}J being
level
in
increment
a-methylmannoside resulted
The
of the
transient.
on the
1)
the
presence
production.
[Ca,
using
by the
onset
of O2
in
A)
([Cam
monitored
chelator,
resulted
enhanced
calcium
Levels
C. Whitin,
John
A (Con
intracellular
Superoxide
John
C.
Award reprint
Pediatrics
Medical
1, 1986; accepted by NIH
Whitin
The
Al
recipient
Cancer
Rochester,
September
grants
is the
and
Center,
29, 1986.
19656, of
Center,
NY.
NIH
AM
33231. New
and
CA
Investigator
HL 34543. requests
to John
C. Whitin,
PhD,
Department
of
Pediatrics, Box 777, University of Rochester Medical Center, 60! Elmwood Aye, Rochester, NY 14642. The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement “ in accordance with 18 U.S.C. §1 734 solely to indicate this fact. ©
1987
by Grune
& Stratton,
Inc.
0006-4971/87/6903-0009$3.00/0
Blood,
Vol 69,
No 3 (March),
1987:
pp 762-768
From bloodjournal.hematologylibrary.org by guest on April 12, 2012. For personal use only.
CALCIUM
IN
PMN
PMN
SUPEROXIDE
cytoplasts
described.’8 Ficoll
were
PMN
Beckman
SW-27
rotor
Ficoll
containing from
the
before
and
was
of granules
alkaline
phosphatase assayed
per
production
02
was
described.20 plus
I mmoh/L
otherwise.
of PMN
A-induced
O2
Human
was
CaCI2
and
production
20-fold. loaded
CaC12,
S mmol/L
incubated
with
37 #{176}C, after buffer
which
and
the
time
were
incubation
the
centrifuged
glucose
PMN
at
diluted
one
A/M
to I x
the
of and
with
one
time
with
unless
Quin2
A/M
Responses
in of these
the
absence PMN
were
of
Ca
monitored
as
AIM
added
to all
enhances
Con
essentially
as
bovine
1.0
serum
albumin
for ten minutes 107/mL
with
at same
20 minutes.
volume cold
the
of PBS,
cold and
At PBS, resus-
previously
were of
described.22’23
in HEPES-buffered
saline
135 mmol/L NaCI, 5 mmol/L KCI, 10 mmol/L HEPES, pH Where indicated I mmoh/L CaCl2 was also added. The fluorescence of Quin2-hoaded PMN was monitored essen-
7.40).
as described
a)1
1
J LLI I
a
previously.”2’
PMN
described
(5 x 106 in 1.0 mL
buffer)
2.2
PMN
was in the
and
the
emission
presence
obtained
in PMN
of I .0 mmol/L
Ca
and
x I07/mL
minutes
also
prepared.
presence
The
fluorescence
of 1.0 mmol/L
of
Ca
was
and compared with the standard curve. cytoplasts were loaded with chlortetracycline et al.9 PMN
were
loaded
at 37 #{176}C, after
at
with
I x
l07/mL
50 mol/L
which
they
and
as
cytoplasts
chlortetracychine
were
washed
at for
20
by centrifugation
and resuspended in PBS. Fluorescence tracings were performed at an excitation wavelength of 380 nm (slit of 6 nm) and an emmission wavelength of 560 nm (slit of 6 nm).
Effect
be used
of
activation
have
recently
of
to monitor
stimuli. treated
ConA
Fig 1 . Effect of Con A and arachidonic acid on [Ca,J and 02 production. Fluorescence tracing of 5 x 10’ PMN/mL KRP that had been loaded with 50 MmoI/L Quin2 A/M as described in “Methods” (upper tracings) and continuous measurement of 02 production (lower tracings). (A) The addition of 100 sg/mI Con A (at the arrow) in the presence of 10 smol/I cytochalasin B. (B) The addition of 12.5 zg/mI arachidonic acid (at the arrow) in the presence of 10 g.tmol/L cytochalasin B.
on
changes
As many PMN can
/.
fCa1
Quin2-loaded
in [Ca1J
induced
by various
Figure IA shows that Quin2-loaded PMN, with cytochalasin B (necessary for maximum
preConA
induced 02 production), in [Ca1]. The onset
respond to Con A with an increase of the increment in [Ca1J occurs
within
precedes
1 5 seconds
and
at 30 to 50 seconds. for
PMN
(mean induced been which
±
For
under
these
there a new
nmol/L,
n
the
phenomenon
same
6)
=
conditions ±
one [Ca]
when
PMN 02
with
a-MM
sustained
in this
the
manner
treatment
response
increase
in [Ca1]
induced
In
that
followed
by a-MM
addition,
display the of changes capacity the
the
A (at
the
A-induced
and
results
are
1 B shows acid also
precedes
[Ca1]
of Con with
If
peak incre-
returns
A-stimulated
in the
of PMN
shown).
PMN,25
PMN
on fCa1 J. with 25 mmol/L
Con
Con
Treatment
if the loaded
buffering
Essentially
that
with
2A)
artifact due to the Ca-buffering the same results are obtained with Con A and are then
Quin2 course
in [Ca1], of 259 ± 19
cessation Con
of
A results
increase in [Ca1]. In addition, the fluoresto Con A appears to be biphasic. The
in a reversible
cence
decline a mean minutes.
reversed
level.
Thus
of [Ca1] the peak has
PMN treated
treatment Fig
nmol/L
value After
in [Ca14]
is completely
production.
(
[Ca1
18
±
Quin2-loaded
ofstimulated are instead
after
resting
production
arachidonic acid. Figure treated with arachidonic
transient,
in [Ca]
1 10
three
occurs
minute
to its initial,
is
is a subsequent steady-state level
zg/mL PMN
of 02 the resting
of the peak 174 nmol/L.
undergo a sustained increase onset of 02 production.
of the
onset
donors,
in approximately
treated with 12.5 that Quin2-loaded
a-MM
the
1 2 blood
SD) while that by Con A is 547
achieved reaches
ment
I
PMN
shown,”’2’24
Effect ofinactivation the Quin2-loaded PMN
A
ConA
of 4 nm),
fluorescence
in the
PMN
by Smolen
reports
(HBS;
tially
slit
pass slit of 10 nm). The [Ca1J
RESULTS
stated
l07/mL PBS. In some experiments the PMN of Ca, by incubation with varying concentrations
depleted
pass
maximum
LPC
LPC-lysed
then determined PMN and
PBS
to 5 x
pended
the
previously (KRP;
in PBScontaining
for an additional
diluted
4 #{176}C, washed
0.5%
Quin2
continued were
Quin2
and
as
routinely
its presence
at 5 x l07/mL
50 zmol/L
they
was
with
PMN
c
MgSO4)
that
(band
as the superoxide
Phosphate
zmol/L)
as we find
were
on
gradient.
membrane
cytochrome
mmol/L
function,
previously.”2’
mmol/l
1.3
B (10
of
Quin2-loaded
per cell and 4% of PMN
Krebs-Ringer
from 10 zg/mL
lysed
fl-glucuronidase
continuously
with
sions
phosphatase. of
calculated
lysed
in PBS
The content
of plasma
normalized
monitored
buffer
PMN
described
described.’9
was
in PBS
the
nm
was set at 492 nm (band
time
by measuring
Cytoplast
of alkaline
Cytochalasin
assays
were
when
Control
ofcytoplasts on
set at 339
times
one
yield
was
wavelength
of a wave-
the minimum fluorescence of LPC-lysed PMN in the presence of 10 mmol/L EGTA as described previously.2’ A standard curve of the fluorescence of known amounts ofQuin2 in Ca f-containing suspen-
20 zmol/L
three and
assessed
as a marker
reduction
The
layers.
70
harvested
containing
layered
was
a
Ficoll
I 8% Ficoll
The
PMN
length
at
g in
over a prewarmed were
BSA
described.’8
unit
dismutase-inhibitable
that
used
previously
normalized
the
x
cytoplasts
analysis.
of
minutes
(wt/vol)
washed
0.5%
as previously
was
as
then
and
marker
was 2% of PMN
when
and
five
(wt/vol)
81,000
25%
Ficoll
in cytoplasts
of J-glucuronidase
content
and
I 2.5% were
71%
content was
the
glucose
counting basis
Depletion
18%
B. The
cytoplasts
5 mmol/L
a cellular
of
between
B) and
containing
at
at 33 #{176}C for 30 minutes
at 33#{176}Cin 12.5%
cytochalasin
B for
were stirred in the thermostatted (37 #{176}C) sample compartment Perkin Elmer 650- 105 spectrofluorometer. The excitation
previously
in 12.5%
centrifuged
cytochalasin
interface
(treated
as
incubated
cytochalasin
then
gradient
20 mol/L
PMN
alone
were
essentially
were
20 mol/L
PMN
discontinuous
prepared
at I x 108/mL
70 containing
37 #{176}C. These
763
PRODUCTION
PMN one minute
by Con
A is not
are
later
treated
and
with
are then
Con
loaded
While influenced
of Quin2
for FMLP-treated
are
as described the same
whether
A with
normal resting [Ca1]. in [Ca1 ] may be
phenomena
an
capacity of Quin2, since PMN are first incubated with Quin2 (data not
the by
Quin2-loaded
time the
From bloodjournal.hematologylibrary.org by guest on April 12, 2012. For personal use only.
764
WHITIN,
acid-free
A aMM
BSA
bovine
the
addition
3B).
The
serum
albumin
can
of an additional addition
of
TAKAHASHI.
also
stimulus
Con
A
[Ca]
occurs when to produce 02
vated stimuli
BSA
or control PMN are treated Quin2-loaded PMN treated by fatty producing
acid-free 02
undergo original
a reversal resting level PMN
ously
can
addition
in Fig
3A,
when
treated
with Con A. In a similar with arachidonic acid
bovine serum albumin (as shown in previous
in
[Ca,
of PMN
fCa,
J.
be reactivated of another
Con
]
acid
A followed
by a-MM,
to
02 As
is added
cease and their
by
can
be
to PMN the
the seen
previ[Ca,
increment is the same as when resting PMN are treated with arachidonic acid (Fig 1B). Figure 3A also shows that arachidonic acid can induce 02 prOduction in PMN previously treated
with
activated
with
A e
Con
original
resting
level
(Fig
ofCon
A followed
by a-MM.
arachidonic
acid
and
PMN
that
inactivated
resting to the with 78
have
been
with
fatty
increment
in
Therefore, increment
in
or reactiboth of these
system
return
by removal
of [Ca1]
to the
2).
PMN
fCa1
The
/.
cytoplasts
was
same
type
performed.
of
Control
heated to 33 #{176}C for 30 minutes cytochalasin B) and cytoplasts
]
[Cai [Ca 4).
When
Con
these
A, there
nmol/L
over
these
control
with
Con
]
[Ca1
of cytoplasts of I 5 1 ± 20
]
was
PMN A.
a very
66
in 12.5% (approxi-
small
increased the
by
with
by
in [Ca]
to 302
nmol/L
after
A,
the
A
as
treated
the
Con
a-MM
PMN
were
increment
small
was
to
[Ca1]
of
treatment
increment
Quin2-loaded
for Con A 02 This very small
induced
reversible
compared
control
contrast,
generated 02 at approximately 50% PMN (normalized per unit of alkaline
cytoplasts
these
In
extremely
Con
3 nmoh/L
±
for
cytoplasts
minutes.
Despite
induced
was nmol/L
Quin2-loaded
several
in
cytoplasts
of the rate of control phosphatase, Table
production increment
of approxiin [Ca1J of
was
both
O2
production
inhibitable
and
(data
not
shown). This would indicate that PMN cytoplasts can be activated by Con A to produce O2 at levels of [Ca1 ], which are significantly lower than even the normal resting level of PMN. The release of membrane-bound calcium (as measured
by following
cychine-loaded and cytoplasts. both dine
changes
cells) was As shown
PMN and fluorescence,
in fluorescence
of chlortetra-
also compared for in Fig 5, the addition
cytoplasts results in a loss indicative of a decrease
control PMN of Con A to of chlortetracyin membrane-
B
MM
associated time
f
calcium.
course
In contrast
of fluorescence
change
to the
Quin2
tracings,
is smaller
for
both
the PMN
Ca,’
LcoZt/\tA1
PMN
,f”’’’
e
I1
generating
A (Fig
1 . 1 x 108/mL, having the same alkaline phosphatase as control PMN) were loaded with Quin2 A/M. The
mately content
(Fig
in an production. acid, the
in the
A on cytoplast
using
I), with lag times mately 100 seconds.
Previously
to produce stimulus.’4”5
arachidonic
with
on
manner, followed
at one minute reports228)
of their increase (Fig 2B).
of reactivation
subsequent
results
PMN (5 x l07/mL, Ficoll/20 zmol/L
1 mm
]
activated
also
by
as Con
are either activated 1 and 3), while for
of the O2
stimulus
experiment
Fig 2. Effect of removal of stimuli on [Ca, and #{176}2 production. Fluorescence tracings of Quin2-loaded PMN and 02 production as in Fig 1 . (A) The addition of 25 mmol/I a-MM (at the second arrow) to Con A-treated PMN. (B) The addition of arachidonic acid (first arrow) followed by the addition of I .35 mg/mI fatty acid-free bovine serum albumin (BSA) (second arrow).
Effect
inactivation
of the Effect
iT-
0
PMN (Fig
COHEN
be reactivated
such
results
[Ca] and the resumption of O2 for both Con A and arachidonic
C,’
AND
-300nM
-
150
nM
ConA
VI
BSA
ConA
jconA$
I 0
0
_v
Cytoplasts
_
78
-
66 fly
nY
::
1 mm
]
Fig 3. Effect of second stimuli on [Ca, + and 02 production. Fluorescence tracings of Quin2-loaded PMN and 02 production in the presence of 10 MmoI/I cytochalasin B as in Fig 1 . (A) The addition of Con A (first arrow) followed by a-MM (second arrow) followed by arachidonic acid (third arrow). (B) The addition of arachidonic acid (first arrow) followed by BSA (second arrow) followed by Con A (third arrow).
Fig 4. Fluorescence tracings of cells loaded with 50 moI/I Quin2 AIM in KRP. The addition of 100 ag/mI Con A to 1 .1 x iO cytoplasts/mI in the presence of 10 MmoI/I cytochalasin B. For comparison. the addition of Con A to 5 x 1O control PMN/mI in the presence of 10 imol/L cytochalasin B is also shown. The resting and stimulated ICa, + + J are indicated for both cell types. Comparable results were obtained in each of three separate cytoplast preparations.
From bloodjournal.hematologylibrary.org by guest on April 12, 2012. For personal use only.
CALCIUM
IN
Table
PMN
SUPEROXIDE
P roduction
1 . 02
765
PRODUCTION
by Quin2-Ioa
ded PMN
Stimulus
Cells PMN
and Cytoplasts
PMN
ConA
8.8
The difficult
3.7
depleted
3.5
cence
PMN
PMA
PMN
Arachidonic
Cytoplasts
Con A
3.7
with
Cytoplasts
PMA
1.6
sured
Cytoplasts
Arachidonic
Cytoplasts
and
which
after
they were
production
were PMA,
moI/L
cytochalasin
the plasma
and
Shown
differ
by
tg/mL
20%)
as described
50 jzmol/L
arachidonic
marker
than
prepared
Quin2
for stimulation
by acid
are normalized alkaline
are the averages
more
comparable
with
B. Results
membrane
units.
were
determined
12.5
2.4
acid
PMN
loaded
then
jzg/mL
and
control
acid
Rates .tg/mL
determinations
of an experiment
of O2 of
two
10
of
did not
times
cytoplasts
and
more
closely
correlates
with
the
lag time
ments. using A/M
Ca
in the
depleted
absence
ofextracellular
depleted of [Cai ] , as previously and PMN.22’23 Therefore PMN 100
zmoh/L
Ca, in
after PBS
determined
as
Quin2
A/M they
usual.
The
were
intracellular
washed
as described
in the
loaded
Ca
can
with
become
reported for macrophages were incubated with 10 to
in the
which
ConA
PMN
PMN.
absence in PBS content
methods
of extracellular and
resuspended of Quin2 was
section.
ranged
[Ca] The
with
LPC
in
with
from
0.2 to 2.0 nmoh/l06
the
2),
For
these
of Ca-depleted fluorescence in
Quin2
was
PMN
that
presence
of
absence
ConA 5’
0
,n
CS)
02 C
PMN is of PMN
PBS
same
had
as the
been
mmol/L
the
fluores-
permeabihized
EGTA.
Ca4
As
concentration
of EGTA appears value is not known.
to be less
of extracellular
indicating
of [Ca1 The
the PMN
that
]
+
as shown
these
addition
were
than
PMN
PMN
again.
In
of Con
were
does
not
does 6A).
PMN resting
level
Ca allowed tion
when
in to
rises [Cai
I
and of
]
Con
A is added
that
have
been
by Con A do respond increase in [Ca1J
with (Fig
course for the increase in Quin2 fluorescence of the amount of Quin2 found within the not
shown).
At
tested,
of normal
to return
A
concentrations
finally
PMN were able repletion of [Ca,
of
returns
to
(open
to generate ] , Figure
the
02 in 7 shows
PMN to generate a ten-minute incubation
[Cai ] of these Ca to the normal resting
. When Con
all of the
[Ca1]
PMN.
Ca-depleted to Con A after
with
in the
in an increase is then added
PMN
Ca followed stimulus-induced
Quin2
These response
further
Ca-depleted
(tracings
intracellular
PMN
not result If Ca
of Con A, [Cai level (Fig 6B). The
increase
contrast,
The time a function
these (Table
by depletion
A to Ca4-depleted
Ca (Fig
treated first with the characteristic 6C). was
not damaged
20
previously.23
these PMN in the presence reaches a new steady state these
mea-
of this
to generate 02 in response to Con A. However, were able to generate 02 in response to PMA
the ability of these Ca-depleted response to Con A following
I
the
10
a Caelectrode,29
absence of additional the indicated [CaJ
for 02 production. The ability of PMN to be activated by Con A at low [Ca1] was tested by the following experi-
Quin2
of Ca
of
of Quin2-loaded
unable PMN
with
results.
Studies
exact value to calculate.
In the 1
as arbitrary (which
performed
A,
amounts
reported
ofQuin2
buffer in the presence nmoh/L, but the exact
text,
Con
presence
to equivalent
phosphatase,
of duplicate
in the
A/M. 1 00
in the
the content
cells.
nmol 02/min/alkaline Phosphatase
symbols
in
k-depleted level prior
Fig
7),
both
02
in with
PMN is to activa-
the
lag
time
prior to 02 production and the rate of 02 production are close to control values. Thus these PMN can be reversibly depleted ofCa and can regain their ability to produce 02 to Con
in response Table
0n,
2.
A.
Effect
of Ca-Depletion
on Ability
of PMN
CS)
to Generate Quin2 A/M Incubation
C
Fig 5. Fluorescence tracings of chlortetracycline-Ioaded PMN and cytoplasts. PMN and cytoplasts were loaded with chlortetracycline as described in “Methods.” Tracings of fluorescence changes induced by 100 sg/mI Con A in the presence of 10 moI/L cytochalasin B were then obtained for 5 x 10 PMN/mI and 1 .1 x 107/mL KRP. A 10% change in initial fluorescence is indicated for each sample. and the absolute fluorescence scale is the same for both tracings. The two tracings are zero-offset by different amounts. Comparable results were obtained in two cytoplast preparations.
02
Stimulus
nmol
02/min/i0#{176}
-Ca
-Ca
ConA
-Ca
+Ca
ConA
3.5
±
+Ca
-Ca
ConA
0.1
±0.1
+Ca
+Ca
ConA
6.6
±0.4
-Ca
-Ca
PMA
6.7
±
-Ca
+Ca4
PMA
6.4
± 0.1
+Ca
-Ca
PMA
6.5
±
0.8
+Ca
+Ca
PMA
6.4
±
1.0
PMN
were
absence
of
measured
loaded
i
B after
concentrations Shown
with
mmol/L
of stimuli with
imol/L
or absence
comparable
values
Quin2
1 00
A/M
O2
of 1 mmol/L
incubation
were:
1.7
0.8
in the
presence
production
was
CaCI2 and
zg/mL
Con A or (mean
1 g/mL ±
or
then
1 0 tmol/L
at 37 #{176}C as indicated.
of one experiment
results.
PMN
0
as indicated.
a five-minute
are the triplicate
times
50
CaCI2
in the presence
cytochalasin
two
Assay
#{176}2
The PMA.
SD), repeated
From bloodjournal.hematologylibrary.org by guest on April 12, 2012. For personal use only.
766
WHITIN,
prior
to O2
8, when
production
was
the lag times
the
repletion
of [Ca]
the
lag
for
time
[Ca1]
measured.
are compared
02
same
production
AND
As can with
to these
of approximately
that activation occur at [Ca]
TAKAHASHI.
the
COHEN
be seen time
in Fig
tracing
Ca-depleted
always
corresponded
79 nmol/L.
This
of the 02 generating that is lower than
for
PMN, to a
would
indicate
system by Con has generally
A can been
appreciated. DISCUSSION
Several nomenon
laboratories have recently in PMN termed “priming.”
involves
the
sequential
suboptimal tion
of
PMN.
The the 02.
next
study
PMN
are
PMN
able
were
described
minutes
At
time
cm.
The
then
at 37 #{176}C in the
-
Con
by loading
were Ca
at what
with
of Ca
PMN
1 mmol/L
3
to determine
to be stimulated
depleted
above.
for two that
was designed
presence was
with
Quin2 with
added,
and
the
lag
A B.
time
/
,
/
300
,
/
U 5)
5)
C
\,o 2
200 ,/
PMN by
of a-MM
and
other
O2
can
continues.’5
resting
state
and
appear
because
lag
times
prior
tion,
these
by Con terminated
low
0)
concenat
not
PMN addition
is terminated
reversed.
The
is cells
to
“primed”
be
to O2
by
for
activated
A-induced
do not return for
control
PMN
have
reactivation
are
shorter
PMN.
In
a
diminished
[Cai
this
technique,
are
reported
rapid
in phagocyte
stimulus-induced
in addi-
when reactivated with permanent changes in
cator
changes
and to a true
system. Ca
to monitor
this
reversed
production
than
can of
stimulus,
Con
not
of
A to the
[Ca] might account for the priming in this Recent reports have utilized the intracellular Quin2
a rate
because
of Con
by an additional
These
PMN
previously
priming A-treated by the
depolarization
activated
at
activa-
O2
binding
production are
potential
previously
the
be reactivated
responses
degranulation
with
generate
of PMN
to inhibit
While
procedure
added
stimulus alone.30’3’ This same type of been reported using PMA and the
cell surface. O2 production therefore be prematurely a-MM.’3’5
treated
FMLP
ionophore ionomycin.23 A is useful in the study
ability
each
5)
0
0
the
example, followed
a phepriming
in a synergistic
requirement for extracellular calcium Con A.’5 This led us to speculate that
,0
0
E
as
Con
of cytochalasin
Con
stimuli,
resulting
than either has also
membrane
A to produce
incubated
PMA
far greater phenomenon
some
[Ca1
For
of
Ca
of different
concentrations,
trations
Fig 6. Fluorescence tracings of Ca-depleted PMN loaded with 50 MmoI/L Quin2 A/M in the absence of (A) The addition of 100 Lg/mL Con A to Ca-depIetod PMN in HBS. (B) The addition of 100 .tg/mI Con A to Ca-depIeted PMN in HBS followed by the addition of I mmol/L CaCI3. followed by a second addition of 100 sg/mL Con A. (C) The addition of 1 mmol/I CaCI2 to Ca-depIeted PMN in HBS. Con A is then added after stabilization of fluorescence. Cytochalasin B (10 MmoI/I) was present in all samples.
use
been reporting In general,
increases
mdi-
] in
.
Using
[Cai
]
CS)
0___
-J
E
for
PMN
stimulated
with
chemotactic
pep-
0
C ,
.
.
0
‘
,
100 .e7
I .#
I
0-- 0
a io
25 Quin2
100
50 A/M
(SM)
Fig 7. Rate of O production and lag time by Con A-treated Ca-depleted PMN in the presence of 10 zmoI/I cytochalasin B. PMN were depleted of Ca by incubation with the indicated concentrations of Quin2 A/M as described in “Methods.” #{176}2 production was measured in HBS as indicated. Closed symbols indicate PMN that had been repleted with 1 mmol/L CaCI2 for ten minutes at 37 ‘C prior to the addition of Con A. Open symbols indicate PMN that were treated with Con A for two minutes prior to the addition of 1 mmol/I CaCI. Solid lines indicate the rate of Os- production. Dashed lines indicate the lag time prior to 03 production. which for the solid symbols is calculated from the time of addition of Con A, and for the open symbols is calculated from the time of addition of Ca .
-
0 Minutes
Fig 8.
Relationship
between
lag times
for 02
production
with
ICa,1in Ca-depleted. Con A-treated PMN. PMN was depleted of Ca as described in “Methods.” Fluorescence tracings and 02 production lag times were determined in HBS. In all samples 100 zg/mI Con A was added to PMN in the presence of 10 tmol/I cytochalasin B for two minutes followed by the addition of 1 mmol/I CaCI2. The arrows for each curve indicate the lag time for 02 production for that sample. Shown is a representative experiment. For three experiments. the [Ca1] at which 02 production induced by Con A is 79 ± 20 nmol/L (mean ± SD).
From bloodjournal.hematologylibrary.org by guest on April 12, 2012. For personal use only.
CALCIUM
IN PMN
tides,”2’24
SUPEROXIDE
monocytes29
A, and
i774
Fc
sufficient
to induce
of [Ca1 induce 02 tions
of
and
macrophages
noglobulin
PMN’2’25
receptors.22
An
Ca
induce
monocytes29
PMN increase
and
[Ca1] in [Ca]
in
PMN
can
then
stimulus
such
increase
in
priming
effect
cannot
[Ca1
].
tightly
coupled
tion
of
to
as
in
result
PMN
therefore
However,
PMN
and
that
lus.28
As measured
displayed after
only
a very
plasts
with
treated
with
displayed
only
change in [Ca] able and reversible about
what
the
response
to
PMN.
small Con
Con
A
This
increase
small
in [Ca1
absence PMN level.
event
could
Quin2
Ca-depleted increase
were
granuleactiwith
did
cyto-
not
The
for PMN
the time
in
activation
ofextracellular
by
are
of fluores-
the
that
A-treated
and
not an absolute depleted of
The of [Ca1] of [Ca]
Ca-buffering
possible
to generate
to increase same
at
-
O2
unless level.
indicated
Quin2,
[Ca]
it is likely
that
It is possible that chemotactic peptides,
generating 02 higher than the
measurement.
all
in the only
and that these average value
Another
different
divergent
of
Ca
to a threshold
at the
transduction peptide stimulation
proceed
Calevels
because
possibility
mechanisms are different,
[CaJ.
is
for Con allow-
Supporting
transduction
this
mechanisms
is
that
the
recently
are suitable It has been
developed
protein kinase C [Ca].23 Thus can even
to correlate exception
fluorescent
Ca
for use in laser-activated reported that PMA-induced
the
occur
though
at
can it
occur appears
[Ca1]
at
that
increments
indicators
flow cytometry.36 activation approximately that some
are
lower
]
in [Ca,
have
of the that an is A.
PMN resting
been
shown
with activation of PMN by all stimuli with the of PMA.37 In contrast, it has been reported that
granule secretion with either saponin
by
levels
Levels
of Cat38’39
PMN that or digitonin
have been is induced
of [Ca]
of this
permeabihized by micromolar magnitude
stimulus-induced
occur
of 10
than
in
increases
]
in [Ca,
are
are producwhether
activating
events
(or promoters of activation) of O2 production. It may be that changes in membrane-associated calcium are activating events in PMN and result in an increase in [Ca]. Only identification vated
of the
or inactivated
PMN
components
by calcium
that
will clarify
are this
either
acti-
issue.
by loading Ca.
ofCa
of
ACKNOWLEDGMENT
These
an the
We discussions
are
indebted on the
to Drs role
Sean
Scully
and
free
calcium
of intracellular
George
Segel
for
in phagocytes.
to these
to the original resting to resting levels was action
of
found
with
to increasing
unable
were were
resting
they
in
O2 or undergo with Con A in
addition
were
of intracellular
type
of
needed
normal
obtained
repleted
began
PMN that
the results,
changes
of [Ca]
by [Ca]
to
whether are
changes
by Con
extracellular
not generate treatment
Ca.
level
O2
than
were
been
allowed
range
in this
]
not required for the activation ofCon A-induced O2 tion by either PMN or cytoplasts. It is still unclear
cytoplasts
of PMN
been
that the Ca-dependent A and chemotactic ing
O2
PMN were being activated. of Sklar and Oades, using
levels,
by
course
than
possibility
in the activation
in the return of the return the
a tenfold
of the studies
of normal about
[Cai
to our
absolutely
production
responses
to monitor difficult to
with a role in the activation system. It is also possible
did after
[Ca]
slower
the in Con
PMN
over
PMN nmol/L
small
cytoplasts was inhibitthe result raised questions
chlortetracychine calcium
much
absence
O2
of had
were
had
Since
using
gener-
while in another generate 02 but
[Ca].33
[CaJ
question
lower
experiments
PMN
production 79 nmoh/
report that demonstrates different sensitivity of Con A and chemotactic peptide stimulation to pretreatment of PMN with pertussis toxin.35 These questions will be best answered
minutes
report,
that
Our
possibility
stimu-
several
C5a
in
that
raises
the
]
are
in contrast
a fraction of the PMN had [Ca]
cytoplasts
previous
in [Ca],24 FMLP did
increase
be reversibly in
resulted The rate
influenced
over
In one
is needed
frame consistent 02 generating
PMN
is
every
using
of these
fragment
calcium
important
with
]
reactiva-
of another
[Ca]
A.
of [CaJ
membrane-bound
increment
in
in chlortetracychine-loaded PMN and cytowith Con A were similar, and the time course changes of Quin2-loaded cytoplasts in
control
the
by
occur
the
which
a tenfold
have also recently reported that Cagenerate 02 in response to chemotactic
rates
PMN
[Ca4]
elevations
[Ca]
depleted depleted
the
[Ca1
A. Over
the resting
raises
that
increasing
[Ca
A followed
and
addition
Quin2,
quantitate,9”#{176} it is noteworthy
in a time cytoplast
to
second
cytoplasts. Cytoplasts 02 can be inactivated
Con A. Although results using changes in membrane-associated cence changes plasts treated of fluorescence
that
obtained
in Con A-treated by a-MM, but
level
a
that
of experiment
an increase treated with
a very
Con
in
complement
age O2 or display report cytophasts
in
because
inactivation,
type
with using
treatment
with
E
of a second
resulting
increments
reactivated
addition
do confirm
depleted and enucleated PMN vated with Con A to produce and
However,
results
time PMN are activated. We performed the same
a-MM
at [Ca1]
Ca].34
manner return and inactivated
than
This
peptides
to permanent
activation,
is lower
transient
in
] at
[Cat
to Con
[Cai J levels above the normal resting for activation of PMN by Con A.
do
increase
of the
in response
PMN.
and Oades PMN can
be due
these to the
which
Ca-replete
Sklar depleted
is important
treated
the determination
begins
of PMN by Con A similar to that seen
in this activated
acid,
This
but
allows
of intracellular Quin2 concentration, 02 began at the same [Ca,] (approximately
L
does not concentra[Ca]
This production
range always
is not
This sustained a-MM is added to
by the
arachidonic
seen
the
treated J, These
be reactivated
[Ca14].
a-MM
activation in [Ca]
02
elevation
ionomycin chemotactic
by FMLP.”2’24 be reversed when
Con A-treated PMN. PMN the original resting [Ca1
[Ca]
in
Quin2.
Con
for immu-
since
increase
contrast
induced can
in
by PMN,
a maximal
with
higands
increase
ionophore because
and
not induce 02 production. Our data show that the results in a sustained increase in
with
production
with the production”
FMLP
stimulated
stimulated
02
767
PRODUCTION
intracellular
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