(Received for publication, October 20, 1994; and in revised form, January 16, 1995)
From the
Stimulation of macrophages induces the ``respiratory
burst'' response which is associated with the generation of
superoxide (O), a drop in cytoplasmic pH,
and a pronounced depolarization of the membrane potential. The purpose
of the present studies was to determine whether an increase in
O
was temporally related to changes in
membrane potential and transmembrane current. Release of
O
at the single cell level was
photometrically monitored during phagocytosis of immune complexes while
simultaneously measuring whole-cell current. Membrane depolarization
and the generation of a non-selective current followed an increase in
O
production with a variable lag time
which was correlated with the state of cellular maturation in culture.
In the absence of phagocytosis, the exposure of macrophages to
O
generated by a xanthine-xanthine
oxidase reaction activated a non-selective current similar to that seen
after phagocytosis. These results provide the first demonstration of
the relationship between free radical release and the ensuing
electrophysiological signaling events which are linked to particle
engulfment in phagocytic cells.
The binding of particulate as well as soluble ligands to
phagocytic membranes may result in particle phagocytosis, lysosomal
enzyme release, and generation of a respiratory burst response. The
ability to initiate a respiratory burst and thereby to produce reactive
oxygen species, e.g. superoxide anion
(O), (
)hydrogen peroxide, and
singlet oxygen is central to the cytotoxic properties of phagocytes.
Superoxide produced following the phagocytic event is concentrated
within the phagocytic vacuole and is only produced in the region of the
plasma membrane in direct contact with the particle (see review by
Segal and Abo(1993)).
Activation of the respiratory burst in
phagocytic cells has been shown to be closely associated with
stimulus-induced changes in membrane potential. Studies with HL-60
cells, for example, have demonstrated that differentiating granulocytes
gain the capacity for depolarization concomitantly with
O production (Kitagawa et al.,
1984). Moreover, when activated macrophages are cultured in
vitro, the heightened membrane depolarization and respiratory
burst reactivity are coincidentally lost (Kitagawa and Johnston, 1986).
A close correlation between the development of depolarization and
cytotoxic activities in normal myeloid cells further emphasizes that
membrane potential changes and functional reactivity are coupled
(Newburger et al., 1979). Stimulus-induced changes in membrane
potential have been reported to antecede changes in cellular oxidative
metabolism in granulocytes (Jones et al., 1981; Korchak and
Weissman, 1978) suggesting that depolarization may be an important
element in the signal transduction cascade leading from membrane
receptor ligation to an increase in NADPH oxidase activity.
In an attempt to elucidate the molecular basis of the stimulus-induced changes in membrane potential that are associated with the initiation of the respiratory burst, we examined the relationship between the free radical release, transmembrane current, and membrane potential changes following receptor-mediated phagocytosis in peripheral mononuclear cells maintained in culture. We provide evidence that superoxide release subsequent to the phagocytosis of immune complexes is associated with activation of a calcium-dependent, non-selective current, and membrane depolarization. The apparent superoxide-induced changes in membrane potential may contribute to the depression of macrophage respiratory burst capacity observed following FcR-mediated phagocytosis.
Respiratory burst activation in HMDMs was triggered and monitored photometrically through detection of superoxide using the reduced fluorochrome DCFH covalently linked to bovine serum albumin (BSA)-anti-BSA immunoglobulin immune complexes. The Fc receptor-mediated binding and phagocytosis of the fluorochrome-coupled derivative (DCFH-IC) transfers the conjugate to the phagocytic vacuole. The presence of oxygen metabolites released into the phagocytic vacuole following NADPH-oxidative activation oxidizes DCFH to fluorescent dichlorofluorescein. Flow cytometric studies on peripheral blood polymorphonuclear leukocytes have previously shown that the DCFH-IC conjugate is capable of monitoring the kinetics of the production of activated oxygen species within the phagocytic vacuoles of neutrophils (Ryan et al., 1990). To gain insight into the interrelationship between free radical release and ion channel activation in mononuclear cells during activation, whole-cell voltage clamp experiments were carried out in physiological solutions during phagocytosis of DCFH-ICs.
Fig. 1shows the time-dependent increase in membrane current following exposure of a representative cell to the fluorochrome-coupled immune complexes. Current activation was linear throughout the voltage range and was accompanied by a depolarizing shift in the zero current potential. Fig. 2shows examples of simultaneous current and microfluorimetry recordings from three macrophages at different stages of development in culture. The membrane potential was held at -60 mV and voltage ramps from -100 to 100 mV, 300 ms in duration were repetitively applied every 3 s throughout the experiment. Exposure of the cells to DCFH-ICs induced an increase in fluorescence, which represented an increase in free radical production and was associated with the generation of a current with a reversal potential of -0.9 ± 0.8 mV (n = 9). The lag time preceding the onset of the current response appeared to be correlated with the length of time cells were maintained in culture. Older cells displayed an increase in the time required to observe both free radical release as well as current activation and membrane depolarization, data which is summarized in Table 1. In the absence of stimulus, cells exhibited stable whole-cell currents and no change in fluorescence.
Figure 1: Phagocytosis of fluorescent-labeled immune complexes (DCFH-IC) induces current activation in macrophages. A, whole-cell current following step depolarizations between -110 and 100 mV at 30 mV intervals from a holding potential of -60 mV under control conditions. B, current following 5 min of exposure of the cell to a solution of solubilized DCFH-IC (15 µg/ml) used to stimulate the oxidative burst activation following phagocytosis. C, maximal current activation observed at approximately 10 min following exposure of the cell to DCFH-IC. D, associated current-voltage relationship for the currents in A-C .
Figure 2:
Response time for superoxide release as
well as membrane depolarization following phagocytosis of DCFH-IC
becomes slower the longer cells are maintained in culture. Coupled
voltage-clamp and microfluorimetry experiments showing simultaneous
changes in fluorescence, membrane potential, and membrane current in
three representative cells following exposure to 15 µg/ml DCFH-IC.
After uptake of immune complexes, reactive oxygen products are
generated, as indicated by changes in fluorescence (top panel in A-C). The accumulation of intracellular oxidants
was associated with the activation of a non-selective current and with
an accompanying membrane potential depolarization. The fluorescent
response was normalized to the peak response for each for each cell.
Variability in cell size and differences in the rate of particle uptake
precluded comparative analysis of free radial release among cells
examined. Membrane currents measured in response to 300 ms voltage
ramps from -100 to 100 mV every 3 s from a holding potential of
-60 mV are seen in the second panel in A-C. Illustrated membrane currents were obtained from
voltage ramps at six different membrane potentials between 100 and
-100 mV and shown at 40-mV intervals. Membrane potential was
determined as the zero current potential for each of the voltage ramps
over the duration of the experiment as shown in the third panel from each of the representative cells. In some cells, a transient
hyperpolarization was seen prior to depolarization. The time before
fluorescent response and the lag time between initial increase in
fluorescence and membrane depolarization was dependent on the age of
the cell in culture. A, single cell microfluorimetric
measurement of O, membrane current, and
membrane potential in a macrophage after 1 day in culture. B,
recording from a cell after 6 days in culture. C, recording
from a cell after 13 days in culture.
In order to further confirm the observation that the depolarization and conductance changes following particle ingestion are indeed due to the presence of reactive oxygen intermediates, cellular responses were measured in the presence of the specific NADPH-oxidase inhibitor diphenylene iodonium (DPI). Addition of 50 µM DPI to phorbol myristate acetate (PMA)-stimulated intact neutrophils has been shown to cause marked inhibition of superoxide generation (Cross and Jones, 1986). Similar results have also been obtained in murine peritoneal macrophages (Hancock and Jones, 1987; Stuehr et al., 1991). Digital fluorescent imaging experiments were carried out on HMDMs in the presence and absence of DPI. Cells preincubated with 5 µM DPI prior to the addition of DCFH-IC failed to give rise to an increase in fluorescence as compared to control cells, as seen in Fig. 3. Similarly, we were unable to observe current activation in cells pre-treated with DPI (5 µM) 10 min prior to exposure to DCFH-IC (Fig. 4). Peak current amplitude at maximal depolarizations of 100 mV in the presence and absence of DPI is summarized in Table 2.
Figure 3:
Oxidation of DCFH-IC requires NADPH
oxidase activity. Fluorescence intensity (490 nm) was monitored by
digital imaging, following addition of 15 µg/ml DCFH-IC. A heating
stage was used to maintain temperature at 30 °C. A and B, fluorescent images of a field of HMDMs (
400 magnification) 24 min following stimulation with DCFH-IC in
standard 140 mM NaCl bath solution (A) and in 140
mM NaCl bath containing 5 µM DPI (B). An
outline of cells in each field is overlaid on the fluorescent image. Boxes represent the areas of highest fluorescence, which were
used for data in C and D. C, change in
fluorescence intensity with time following stimulation with DCFH-IC in
each of five representative cells as seen in A. D,
change in fluorescence of five cells stimulated in the presence of DPI
as seen in B. Addition of DCFH-IC is indicated by an arrow. At 15 min following exposure of the cells to the
phagocytic stimulus, control cells exhibited a 34.2% increase in
fluorescent intensity over that observed in DPI-treated cells. The
average change in fluorescence intensity (in arbitrary units) for the
five control cells in A at 15 min was 7.6 ± 0.7
compared to 5.0 ± 0.4 measured for the DPI-treated cells in B.
Figure 4:
Current activation is dependent upon NADPH
oxidase activity. HMDMs preincubated in DPI, a specific inhibitor of
NADPH oxidase, do not exhibit current activation in response to
DCFH-IC. Patch-clamp experiments were carried out at 30 °C in bath
solutions containing 140 mM NaCl, 5.4 mM KCl, 2
mM Ca, 2 mM Mg
,
and 10 mM HEPES, with pipette solutions containing 140 mM KCl, 1 mM EGTA, 0.2 mM Ca
, and
2 mM Mg
. A and B,
HMDMs which received no treatment prior to DCFH-IC activation. A, whole-cell current, following 100 mV pulse, before and 10
min following addition of 15 µg/ml DCFH-IC. B,
current-voltage relationship for current shown in A. C and D, whole-cell current measured in cells
preincubated for 10 min in 5 µM DPI prior to whole-cell
formation. C, whole-cell current following a 100 mV pulse
before and after addition of DCFH-IC. D, current-voltage
relationship for current shown in C.
In order to demonstrate that particle uptake was necessary to elicit a fluorescent response, cells were preincubated in 5 µM cytochalasin B, an inhibitor of phagocytosis, prior to stimulation. A representative fluorescent response for a cytochalasin-treated cell is compared to that obtained to a control cell in Fig. 5. We were unable to observe an increase in fluorescence in response to DCFH-IC in a total of six cells following pretreatment with cytochalasin, indicating that oxidation of the dye was occurring intracellularly. A similar inhibition of the fluorescent response in cells pretreated with cytochalasin B was observed in the flow cytometric studies of Ryan et al.(1990).
Figure 5: Changes in fluorescence seen during the respiratory burst are dependent on uptake of DCFH-IC particles and are due to intracellular oxidation of DCFH. Pretreatment of granulocytes with the microtubule-disrupting agent cytochalasin B has been shown to effectively inhibit phagocytosis. Two cells from the same HMDM isolation, each approximately 80 pF, in standard external solution were stimulated with 15 µg/ml DCFH-IC; fluorescence was monitored at 490 nm. Temperature was maintained at 30 °C. The trace labeled Cytochalasin B was recorded from a cell preincubated with 5 µM cytochalasin B for 25 min prior to exposure to DCFH-IC. The trace labeled Control was obtained from a stimulated cell that received no pretreatment. The decrease in fluorescence in the control cell over time may be a consequence of the pH sensitivity of the dye and attributable to the decrease in cytoplasmic pH which accompanies the oxidative burst. Upon acidification, the emission spectra of all the fluorescein dyes decrease in amplitude without a significant shift in wavelength (Tsien, 1989).
In order to determine whether the reactive oxygen species generated
during the phagocytic response were directly responsible for current
generation, we investigated the effect of O on HMDMs in the absence of phagocytosis. Exposure of cells to
O
generated in the presence of xanthine
and xanthine oxidase gave rise to changes in current and shifts in zero
current potential resembling those following phagocytosis of DCFH-ICs
as seen in Fig. 6. This result indicates that the presence of
oxygen metabolites alone is sufficient to induce current activation.
Currents induced under either condition were completely inhibited in
the presence of 1 mM La
indicating that the
non-selective currents were not simply the result of instabilities in
the electrophysiological recording or free radical-induced membrane
breakdown.
Figure 6:
Membrane current activation following
exposure to both DCFH-IC and the O generating system, xanthine-xanthine oxidase. A: upper panel, changes in membrane potential after
addition of 15 µg/ml DCFH-IC and in the presence of 1 mM La
. Membrane potential was determined as the
zero current potential during voltage ramps. Addition of DCFH-IC and 1
mM La
is indicated by arrows. Lower panel, whole-cell currents elicited by 300-ms voltage
ramps from -100 to 100 mV prior to addition to DCFH-IC, 8 min
after addition of DCFH-IC, and 10 s after addition of 1 mM La
to the cell following maximal current
activation. Capacitive current was subtracted from the records shown. B, changes in membrane potential and current following
exposure of cells to the O
generating
system, xanthine-xanthine oxidase. Upper panel, changes in
membrane potential prior to and following exposure of the cell to
xanthine oxidase in the presence of 0.1 mM xanthine and in the
presence of La
. Addition of 0.05 unit/ml xanthine
oxidase and 1 mM La
is indicated by the
arrows. Lower panel, whole-cell currents prior to activation
in the presence of xanthine; maximal current activation 4 min after
addition of xanthine oxidase, and current inhibition 4 min after
addition of La
.
The ionic selectivity of the free radical-induced current
was determined in experiments in which Na and
Cl
were the major permeant species and the cells
stimulated in the presence of DCFH-ICs. The cation to anion selectivity
was determined from current reversal potentials obtained when 100
mM of the NaCl in the intracellular solution was substituted
with NMDG-glutamate. Under these conditions, the cation to anion
permeability ratio (P
/P
) was
calculated to be 1.0. This value may reflect the fact that 1) the
conductance is not selective for cations over anions, i.e. the
Na
/Cl
permeability ratio is low, or
2) the conductance is partially permeant to glutamate, i.e. the Cl
/glutamate permeability ratio is low, or
3) there are a number of conductances which are simultaneously
activated during the response to free radical release.
The relative
permeability of the DCFH-IC activated conductance to several monovalent
cations was determined in experiments in which the Na in the bath solution was replaced with chloride salts of either
K
, Cs
, Li
, or NMDG.
The ionic selectivity among cations was determined from shifts in the
reversal potential as determined in three cells, each of which was
exposed sequentially to the five cations and is summarized in Table 3. The relative cation permeability
(P
/P
) of the DCFH-IC-induced current as
determined in three cells was K
(1.37) >
Cs
(1.28) > Na
=
Li
(1.00) > NMDG
(0.69) which
corresponds to the Eisenman sequence IV indicating that permeation is
likely to be weakly dominated by the dehydration energy of the
permeating species and that with the exception of NMDG, the
DCFH-IC-activated conductance showed poor selectivity among the
monovalent cations examined. While our results clearly demonstrate that
monovalent cations contribute to the free radical-induced current,
these experiments did not determine whether current flow was through
single or multiple channel types.
The sensitivity of the DCFH-IC
activated current to external Ca was examined in
experiments conducted in Ca
-free bath solutions
buffered with 1 mM EGTA. In these experiments the pipette
solution contained 10 mM BAPTA without additional
Ca
, with one exception where the standard
Ca
buffer was used. These experiments indicated that
DCFH-IC-induced current activation was significantly attenuated under
Ca
-free conditions. Peak current at 100 mV in
response to DCFH-IC in Ca
-free solutions was 248
± 52 pA (n = 3) as compared to 4804 ±
1240 pA (n = 3) obtained in
Ca
-containing solutions. The mean change in current
after addition of DCFH-IC was -183 ± 33 pA in
Ca
-free solutions and 4475 ± 1428 pA in
Ca
containing solutions (p < 0.08).
Normalized to capacitance, this corresponds to a decrease of 1.7 pA/pF
in Ca
-free solutions and an increase of 50.8 pA/pF in
Ca
-containing solutions. Capacitance values for these
cells were estimated using data in which the capacitance for HMDMs
relative to age in culture was previously determined (Nelson, 1990).
The decrease in current amplitude under Ca-free
conditions could reflect the fact that the free radical
sensitive-channels are Ca
activated or alternatively
that the activity of NADPH-oxidase is Ca
-dependent.
To differentiate between the two possibilities, fluorescence imaging
experiments were carried out on cells in the presence and absence of
external Ca
. We were unable to observe a significant
difference in the fluorescence response to DCFH-IC as a function of the
external Ca
concentration. The average increase in
fluorescence intensity in Ca
-containing solutions was
176.8 ± 20% (n = 5) as compared to an increase
of 201.4 ± 49.8% observed in nominally Ca
-free
solutions. These results are consistent with the conclusion that the
conductance increase and not the oxidase activity is Ca
sensitive.
Data supporting a link between changes in membrane
potential and cellular oxidative metabolism comes from population
studies showing that conditions which inhibit membrane depolarization
also prevent generation of respiratory burst activity (Simchowitz and
Spilberg, 1979). The importance of depolarization in the generation of
a respiratory burst response has also been emphasized in studies
involving neutrophils from chronic granulomatous disease patients.
These cells lack the ability to produce
O, and they additionally fail to undergo
significant depolarization upon surface stimulation (Seligmann and
Gallin, 1980; Whitin et al., 1980). Furthermore, studies on
lipopolysaccharide-activated macrophages (Kitagawa and Johnston, 1985)
revealed that these cells concomitantly develop an increased capacity
for depolarization and respiratory burst responsivity, suggesting that
cell depolarization does not occur artifactually as a result of
membrane perturbation, but may be an important mechanism of signal
transduction.
Superoxide generation is not limited to phagocytes and
bactericidal activity. The consequences of free radical generation
might be generalized and expected to regulate variety of cellular
functions. Transformed as well as normal human B lymphocytes
efficiently release O when stimulated by
surface immunoglobulin cross-linking or phorbol esters (Dorseuil et
al., 1992; Maly et al., 1988; Volkman et al.,
1984). Moreover, it has been suggested that reactive oxygen
intermediates may regulate genes involved in inflammatory and immune
responses serving as signaling molecules in T lymphocytes controlling
activation of the transcription factor NF
B (Schreck et
al., 1991).
Whitin et al.(1980) have reported that
phorbol ester-stimulated granulocytes undergo membrane depolarization
under anaerobic conditions where no O is
produced, suggesting that membrane potential and superoxide production
are not coupled. Phorbol esters known to activate protein kinase C, as
well as purified protein kinase C have been shown to regulate the
activation of both anion and cation channels (Berger et al.,
1993; Charpentier et al., 1993; Fournier et al.,
1993; Li et al., 1993; Oike et al., 1993; Tabcharani et al., 1991). Therefore, in phorbol ester-stimulated cells,
it is not unreasonable to expect that changes in membrane potential
might be observed as a consequence of protein kinase C-mediated ion
channel phosphorylation independent of O
generation. The electrophysiological response of the cells
undergoing phagocytosis in our studies might well be different from the
response in cells stimulated with phorbol esters.
The time course of
phorbol ester-induced depolarization has been shown to be a sensitive
function of the fluorescent probe used to measure membrane potential in
flow cytometric studies of phorbol ester-stimulated neutrophils
(Seligmann and Gallin, 1983). Seligmann and Gallin(1983) were able to
demonstrate that the fluorescence of the carbocyanine dye
di-S-C(5) used as the membrane potential probe is destroyed
by reactive oxygen products produced by neutrophils following
stimulation, giving rise to an apparent insensitivity of the cellular
response to the effects of scavengers or inhibitors of various reactive
oxygen species. This fact coupled with the toxicity of the carbocyanine
dyes at high concentrations (Seligmann and Gallin, 1983) would lead to
the erroneous conclusion in phorbol ester-stimulated cells monitored
with carbocyanine dyes that membrane potential and
O
are not coupled. In our studies, we
have determined membrane potential directly, thereby circumventing the
problems encountered with dye toxicity and sensitivity to free radical
destruction.
The ability of mononuclear phagocytes to respond to
respiratory burst activators is also related to the stage of cellular
differentiation. Resident tissue macrophages produce only low levels of
superoxide anion (Biggar et al., 1976; DeChatelet et
al., 1975). Monocytes allowed to differentiate in culture resemble
tissue macrophages as evidenced by a progressive loss in respiratory
burst activity (Musson et al., 1982; Seim, 1982). Our
observation that the time course of current activation following the
generation of free radicals in HMDMs is dependent upon age in culture
may be the consequence of the reduction in the
O generating capacity of the cells with
differentiation and suggests that a critical concentration of free
radical is needed to induce an electrophysiological response. The large
lag time between oxidant production and current change observed in the
older cells may alternatively be due to 1) a decrease in the oxidant
sensitivity of the ionic channel(s) generating the current, 2) a
decrease in the expression of the oxidant sensitive channel(s) or 3) a
decrease in the expression and/or oxidant sensitivity of a regulatory
protein which controls channel opening.
This study demonstrates that
oxygen metabolites generated during the phagocytic response, as well as
exogenous O, activate a non-selective
membrane current which induces membrane depolarization and is
inhibitable by La
. Data from this investigation
provide evidence that membrane depolarization does not signal
activation of the superoxide generating system but rather is a
consequence of its activation. It has been generally observed that
following Fc receptor-mediated phagocytosis there is a general
depression of macrophage respiratory burst capacity as well as
tumoricidal activity (Commins et al., 1990; Loegering and
Schwacha, 1991; Schwacha et al., 1993). Thus, membrane
depolarization which accompanies the initial phagocytic event may be
one of a number of signals which down-regulate the granulocyte response
to subsequent stimuli.